Cambridge Healthtech Institute’s 8th Annual
Engineering Genes, Vectors, Constructs and Clones
Upstream Decisions Lead to Downstream Success
January 18-19, 2016

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 8th Annual Engineering Genes, Vectors, Constructs and Clones 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.

Final Agenda

MONDAY, JANUARY 18

7:30 am Conference Registration and Morning Coffee

GENOME ENGINEERING

9:00 Chairperson’s Opening Remarks

Mark Welch, Ph.D., Vice President, Research and Development, DNA 2.0

9:50 High-Throughput Engineering of CHO Cells Using CRISPR-Cas9

Bjørn Voldborg, MSc, Director, CHO Cell Line Development, Novo Nordisk Foundation Center for Biosustainability (CFB), DTU Biosustain, Technical University of Denmark

We are establishing a high-throughput genome engineering pipeline using CRISPR-Cas9. The pipeline is being used in our efforts to generate a panel of genomically engineered CHO cells with improved properties for the production of recombinant therapeutic proteins. The setup of the pipeline and potential future applications will be presented

10:20 Coffee Break

10:45 Expediting Protein Biomanufacturing through the UCOE Gene Expression Platform

Michael Antoniou, Ph.D., Reader, Molecular Genetics, Medical & Molecular Genetics, King’s College London

Ubiquitous chromatin opening elements (UCOEs) derived from housekeeping gene loci are compact and easy to manipulate genetic regulatory elements, which provide highly reproducible and stable expression irrespective of transgene integration site within the host cell genome.

11:15 The Emerging Era of Creating Designer Microbes - Recent Advancements in Cloning and Manipulating Natural and Synthetic Chromosomes in Yeast

Bogumil J. Karas, Ph.D., Adjunct Scientist, Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute; Founder & CSO, Designer Microbes Inc.

The development of microbes suitable for industrial use often requires engineering of multiple sites throughout the chromosome, but techniques for genome engineering are severely limited outside of model organisms such as E. coli and yeast. To overcome this problem, we have developed novel technologies at the JCVI which allow cloning of whole chromosomes as centromeric plasmids in yeast, where they can be manipulated and transplanted inside selected microbial cells.

11:45 SINEUPs: A New Class of Antisense Long Non-Coding RNAs that Specifically Activate Translation of Targeted Proteins

Silvia Zucchelli, Ph.D., Assistant Professor, University of Eastern Piedmont, UPO; CSO, TransSINE Technologies

SINEUPs represent a new functional class of natural and synthetic antisense long non-coding RNAs that UP-regulate translation of partially overlapping sense mRNAs through the activity of an inverted SINEB2 element. Given their modular structure, SINEUPs can be designed to increase protein synthesis of potentially any gene of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.

12:15 pm Manufacturing of Recombinant Biopharmaceuticals by FOLDTEC® - a Recent Case Study

Andreas Anton, Ph.D., Director, BioProcess Development, Wacker Biotech GmbH

Wacker Biotech is showcasing its novel refolding technology for bioengineered pharmaceutical proteins. With the new technology biopharmaceuticals that tend to aggregate can be efficiently produced in their soluble-active form in high yields. The proprietary process utilizes optimized bacterial strains and a patented, antibiotic-free expression system. WACKER can now cost-efficiently and reliably produce pharmaceutical proteins that are prone to aggregation, and thus difficult to manufacture, in high yields and utmost purity for its customers.

12:45 Session Break

1:00 Luncheon Presentation I: Engineering Biological Systems from Genes to Genomes

Mark Welch, Ph.D., Vice President, Research and Development, DNA 2.0

Recent developments in the synthetic biology toolbox allow comprehensive engineering of biological components and systems. We describe applications of the expanding toolbox where machine learning technologies are leveraged to engineer protein production and function for a range of target proteins and hosts.

1:30 Luncheon Presentation II (Sponsorship Opportunity Available)

VECTOR DESIGN

2:00 Chairperson’s Remarks

Andrea Throop, Ph.D., Production Manager, Center for Personalized Diagnostics, Biodesign Institute, Arizona State University

2:05 Expression Vector and Gene Engineering: Approaches to Improve Recombinant Protein Production in CHO Cells

Janice Tan, Ph.D., Research Scientist, Bioprocessing Technology Institute, A*STAR

The increasing demands for recombinant biologics produced in CHO cells highlights the need to improve efficiency and yield without compromising quality of these biologics. Two approaches were explored by our lab to achieve these objectives: (i) engineering selection stringency in the expression vector resulted in faster generation of stable cell pools with high titers and (ii) overexpression of CHO heat shock proteins improved performance of CHO cells in fed-batch bioreactors.

2:35 Selecting the Optimal Vector for High-Throughput Cloning and Protein Arrays

Andrea Throop, Ph.D., Production Manager, Center for Personalized Diagnostics, Biodesign Institute, Arizona State University

Large-scale experiments requiring protein expression from thousands of genes require an efficient method for cloning the genes into protein expression vectors. The choice of vector and cloning scheme is critical in obtaining reliable and consistent downstream experimental results. This talk discusses the selection and molecular characteristics of vectors utilized for high-throughput cloning and protein arrays.

3:05 Genome-Wide RNAi Screen for Improved Functional Expression of Neurotensin Receptor and Other Proteins

Joseph Shiloach, Ph.D., Head, Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health

Genome-wide RNA interference screen is emerging as a powerful methodology for deducing gene functions in various diseases. We applied this technology to generate genome-wide profile of genes related to recombinant protein expression process from HEK293 cells. We utilized human microRNA library of 875 microRNA and siRNA library targeting 21,000 genes. By implementing high-throughput screening, we identified miRNAs/siRNAs that significantly increased expression of different recombinant proteins.

3:35 Selected Oral Poster Presentation: NHEJ-Mediated DNA Cloning and Manipulations in Yeast and Mammalian Cells

Rinji Akada, Ph.D., Professor, Department of Applied Molecular Bioscience, School of Medicine, Yamaguchi University

DNA cloning is commonly performed in E. coli, though it is still time-consuming work. In eukaryotic organisms, DNA double-strand break can be repaired by non-homologous end joining (NHEJ), suggesting that introduced non-homologous DNA ends will join in a cell by NHEJ. Therefore, we developed NHEJ-mediated DNA cloning method in yeast and mammalian cells. The DNA manipulations with only PCR fragments will change recombinant DNA technology from E. coli to PCR.

3:50 Refreshment Break in the Exhibit Hall with Poster Viewing

4:30 Direct Expression of PCR-Amplified Genes in Mammalian Cells - Linear DNA Technology Using Terminator Primer and Lipofection Enhancer Reagents

Mikiko Nakamura, Ph.D., Research Fellow, Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University

We found two reagents that synergistically enhance mammalian cell transfection with lipofection reagents. The enhancers allowed PCR-amplified DNA as a source for gene transfection in 96-well cell cultures. In addition, transcriptional terminators were minimized to the length designable as oligonucleotide primers, which we called “terminator primer”. The PCR-mediated gene manipulations in mammalian cells will transform gene expression by allowing for extremely simple and high-throughput experiments with small-scale cell cultures.

5:00 Modification of GENSAT BAC with Lambda-Red Recombineering System for Transgenic Animals or Cell Lines

JrGang Cheng, Ph.D., Associate Professor, The Neuroscience Center, University of North Carolina at Chapel Hill

Based on BAC transgenic mouse platform, GENSAT Brain Atlas provides an invaluable resource for studying gene expression and cellular migration in vivo. In order to use a specific GENSAT BAC with the desired expression profile and expand its implications, the modification of eGFP to a different transgene is greatly beneficial. Modified GENSAT BAC can not only be utilized in making transgenic animals but also in transfecting cell lines.

6:30-7:45 Welcome Reception in the Exhibit Hall with Poster Viewing

7:45 Close of Day

TUESDAY, JANUARY 19

8:00 am Conference Registration and Morning Coffee

ENHANCING EXPRESSION SYSTEMS

9:00 Chairperson’s Remarks

James D. Love, Ph.D., Director, Technology Development & Research Assistant Professor, Biochemistry, Albert Einstein College of Medicine

9:05 A Systematic Approach to Engineering Antibody and Integral Membrane Protein Expression

James D. Love, Ph.D., Director, Technology Development & Research Assistant Professor, Biochemistry, Albert Einstein College of Medicine

A systematic engineering approach that combined machine learning methods with gene synthesis to explore vector element and codon optimization determinants of protein/antibody expression was investigated. Expression elements explored include secretion signals, transposases, viral amplifiers and RNA export signals in addition to novel combinations of enhancer, promoter, intron, polyadenylation signal elements. Systematic use of a panel of transient transfection vectors enabled rapid expression success of a series of high-value targets.

9:35 Selected Oral Poster Presentation: Expression of a Novel Pre-Miniproinsulin Analogue Gene in Escherichia coli

Ahmed Abdel Aleem Abolliel, MSc, Research Scientist, Faculty of Pharmacy, Microbiology Department, Cairo University

A pre-miniproinsulin analogue was designed. Homology modeling of the designed protein was carried out. The designed gene was synthesized using DNA synthesis technology then cloned into pET-24a(+) and propagated in E. coli strain JM109. Expression was successful in two E.coli strains. SDS-PAGE analysis was carried to check protein size. Protein Rapid screening and purification was carried by Ni-NTA technology. The identity of the expressed protein was verified through a western blot.

9:50 Coffee Break in the Exhibit Hall with Poster Viewing

11:00 Development of Effective Expression Systems for the Production of Glycosyltransferases Used in the Glycoengineering of Biotherapeutics

James Meador, Senior Scientist, Protein Expression and Purification Group, Research Department, Momenta Pharmaceuticals, Inc.

We have developed a highly efficient process to fully sialylate the Fc glycans of immunoglobulins that involves using two human glycosyltransferases. We needed to produce the two enzymes at sufficient quality and quantity to make such a process economical. We discuss the various expression systems screened and ultimately used to produce the highly purified enzymes at >100 mg/liter levels from HEK293 cells.

⊲ Featured Presentation
11:30 One for All

Anton Glieder, Ph.D., Professor, Molecular Biotechnology, Graz University of Technology

Since optimal genetic constructs for high-level gene expression remain target-dependent and unpredictable, feedback from fermentation scientists supports the design and construction of improved second-generation production strains. Alternatively, the design of new production strains employing differently regulated synthetic bidirectional promoters with additional copies of target genes allows construction of strains permitting use of different cultivation and production strategies to maximize yields for each target without additional steps back to strain development.

12:00 pm Sponsored Presentation (Opportunity Available)

12:30 Session Break

12:45 Luncheon Presentation (Sponsorship Opportunity Available) or Enjoy Lunch on Your Own

1:45 Close of Conference