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Tackling Transfection Tasks - Niel McKenna


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Genetic Engineering News article on transfection of DNA Vaccines. Several DNA Vaccines experts, including Althea's Magda Marquet, share their perspectives on DNA vaccine delivery and the future of …

Genetic Engineering News article on transfection of DNA Vaccines. Several DNA Vaccines experts, including Althea's Magda Marquet, share their perspectives on DNA vaccine delivery and the future of theis great technology.

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  • 1. Volume 33, number 1 Tools | Technologies | Trends January 1, 2013 CHRONIC LYMPHOCYTIC LEUKEMIA HEPATOCELLULAR CARCINOMA ALZHEIMERS OR ALZHEIMERS EPITHELIAL*MESENCHYMAL ACUTE MEYLOID LEUKEMIA COMPANION DIAGNOSTIC DEAF OR HEARING LOSS HODGKINS LYMPHOMA COLORECTAL CANCER Biobusiness BURKITT LYMPHOMA ADENOCARCINOMA HCV OR HEPATITIS C B-CELL LYMPHOMA BLADDER CANCER GASTRIC CANCER HEPATIC CANCER HEMATOPOIETIC INFLAMMATION HEMATOPOIESIS BONE MARROW COLON CANCER GLIOBLASTOMA HEART DISEASE ANGIOGENESIS LUNG CANCER Breast Cancer MELANOMA METASTASIS BIOMARKER LEUKEMIA EXOSOME DIABETES EMTMARKER14-3-3ALPHA-FETOPROTEINBLADDER TUMOR ANITGEN 0 0 1 25 170 0 30 0 0 4 17 0 0 1 1 34 1 4 37 13 8 4 4 68 0 39 47 0 0 0 0 2 0 0 8 9 0 8 12 0 Cancer 0 0 0 3 0 1 22 38 0 1 2 0 2 0 5 4 0 0 110 6 0 0 1 1 6 2 15 0 0 2 0 8 20 0 4 2 0 0 10 0 10 2849 0 0 0 0 11 17 0 19 13 0 28 31 0 6 9 0 14 18 4 0 BiomarkerB-TYPE NATRIURETIC PEPTIDE 0 0 0 4 0 0 235 4 6 0 0 0 0 0 0 46 0 0 0 0 0 4 0 1 0 0 0 0 31 0 9 0 0CA 125 0 14 3 0 9 2 7 85 7 56 1 2 2 12 0 0 1 0 0 1 9 0 2 0 5 1 0 6 1 12 5 41 3 53CA 15_3 0 20 0 0 0 2 21 1 435 0 0 1 6 0 0 0 0 0 0 2 0 0 0 2 0 0 1 0 0 1 7 1 33 HotspotCA 19-9 1 18 1 0 3 0 29 39 4 15 0 0 15 14 7 0 0 9 0 0 0 10 3 0 5 0 4 0 0 22 0 5 3 27 2 64CD98 0 1 0 1 0 0 0 0 4 0 0 0 0 0 0 1 0 0 0 0 1 0 2 2 0 0 0 0 5 2 5 0 1CEA 0 805 1 15 1 58 152 48 627 0 1 3 13 12 6 6 0 3 27 0 1 4 4 59 5 7 0 17 2 6 88 0 31 14 58 8 51 449 AnalysisFASCIN 0 22 1 4 2 3 6 2 11 0 0 2 6 0 2 0 2 3 1 3 5 0 0 3 0 0 4 0 3 1 5 5 20HER2_NEU 1 264 1 78 1 53 241 47 54 4 5 0 2 21 46 4 1 7 14 13 7 153 30 3 0 14 0 0 19 0 20 33 231 44 287IGF-11 0 32 9 10 0 2 14 14 13 7 0 0 30 27 0 0 92 0 0 0 10 6 9 3 11 0 1 80 0 14 21 23 6 14LEPTIN 3 24 30 71 2 1 119 62 19 5 0 1 32 39 0 1 12 79 1 2 1 12 5 47 15 22 1 0 25 0 52 7 43 18 16 23OSTEOPONTIN 2 26 4 55 2 1 10 1 2 12 10 7 0 0 9 23 0 0 49 0 6 0 18 9 14 4 31 1 1 65 0 154 19 43 43 19 4PROLACTIN 2 55 34 54 1 1 56 24 668 1 0 2 9 0 36 19 5 3 2 1 1 3 11 6 33 6 0 7 0 38 36 7 3 28 11PSA 1 56 9 9 51 1 30 299 39 99 0 1 6 14 0 4 40 9 6 2 3 2 6 1 4 2 0 6 0 10 2 23 26 6 2 13THYROGLOBULIN 0 35 4 3 1 1 10 12 9 1 0 2 2 0 14 116 0 2 0 0 0 35 0 1 1 0 5 0 10 12 5 7 10 0TROPONIN I 1 0 0 15 1 0 2 10 44 14 0 0 0 1 0 0 20 1 0 0 0 0 2 0 7 0 0 0 0 60 4 1 1 0 Drug Discovery Novel Aldevron’s Genetic Immunization and Strategies for Antibody (GIA™) technology is used Cell-Based to test new DNA Assays vaccine techniques in animal models. 14 OMICS Genomic Tackling Transfection Tasks Neil McKenna, Ph.D. Analysis Drives Biomarker The discovery in the early 1990s that administration in mice of plasmid DNA encoding Discovery both viral and nonviral antigens induced antibody responses held out the prospect of achieving broad immunogenicity using DNA vaccines without the safety issues associated 26 with a replicating pathogen. Despite this promise, the efficacy of first-generation DNA vaccines against HIV, HPV, and hepatitis was compromised by low an- Bioprocessing tibody titers and sporadic immune cell responses. In addition, DNA vaccines face broader challenges encountered by other classes of vaccines such as manufacturing, scale-up, and purification, as well as vaccine resistance. A number of companies are developing processes and technical platforms designed see page 22 Twisting and Turning for Lab Automation Sparks Creativity Better Protein Expression 30 Kate Marusina, Ph.D. Translational Just two decades ago we were only beginning to recog- Medicine Necessary nize the potential of automated technologies to enhance Liaisons: throughput in drug discovery research. Today, it is difficult CROs Gain to imagine a modern laboratory without robotic equipment. Clout & “We see continuous trends toward increased reliability of robots, partially driven by the Responsibility Primary mouse embryonic fibroblast strain introduction of new types of motors,” reports Malcolm Crook, Ph.D., CTO, Process Analysis DBA/2J cells imaged using Olympus 40X/0.9NA & Automation (PAA). “Another trend is toward smaller targeted systems, that still flexibly COMPATIBILITY 36 SENSING air objective lens on the BD Pathway 435 accommodate peripherals as needed.” High Content Imager. Cells were labeled PAA will be presenting the “ harmony,” its user interface for software inte- with Hoechst 33342 (blue), YoYo-1 (green), gration, at the Society for Laboratory Automation ONE TOUCH TEACHING MitoTracker Orange (red), and Cytochrome C – SCREENING AlexaFluor 647 (cyan). RELIABILITY Hamner Institute and Screening (SLAS) meeting later this month. see page 16 CAPABILITY MULTISPAN HIGH THROUGHPUT OPTIMIZATION CAPABILITY HOW EXTRAORDINARY HAPPENS IntroducIng the Agilent encore MultispAn liquid HAndling systeM CONFIDENCE MODULARITY FLEXIBILITY SPEED CONTROL ACCURACY
  • 2. OMICSTransfection Continued from page 1to circumvent these issues, with particular across the cell membrane to avoid degrada-emphasis on improving the efficiency of de- tion by endonucleases.livery and uptake of vaccines by target cells, Inovio’s approach in this area includesreferred to as transfection, which can be con- informatically assisted optimization of thefounded by differences in tissue and cell type coding sequence to expand immunogenici-accessibility between individuals, in addition ty across multiple targets and boost expres-to other factors. sion levels of immunogens in target cells, Many of these companies were present and to improve the delivery of the plasmidat last month’s DNA Vaccines conference in DNA into host cells to minimize exposureSan Diego. The meeting was sponsored by to extracellular nucleases. The developmentInternational Society of DNA Vaccines and of one of Inovio’s candidate high-gradeorganized by BioConferences International, cervical dysplasia vaccines, VGX-3100,a Mary Ann Liebert company. encoding the HPV16 and HPV18 E6/E7 “Vaccines have saved more lives than antigens, incorporated a combination ofany other invention in human history”, ex- these features to increase vaccine antigenplained J. Joseph Kim, Ph.D., president and immune potency.CEO of Inovio Pharmaceuticals. “Conven- “The VGX-3100 vaccine was developedtional vaccines have been successfully used using our SynCon platform and includesagainst the lower hanging fruit in terms of highly efficient leader and Kozak sequenc-disease, and more complex diseases such es,” said Dr. Kim. “We also introduced anas cancer represent both opportunities and endoproteolytic cleavage site to improvechallenges for DNA vaccines. protein folding and cytotoxic T lymphocyte According to Dr. Kim, one of the biggest processing.” Vaccine delivery using electroporation (>10–100x enhancement in immune responses). Inoviohurdles for DNA vaccines has been induc- In addition, Inovio has developed an elec-ing sufficiently high immune responses for troporation-based delivery system that, ac-effective vaccination. “There are two issues cording to Dr. Kim, allows for more efficient siently realign into a more porous state,” he Looking to the future, “Electroporation isat stake,” he said. “The first is to make plas- and safe targeting of the antigenic sequence explained, adding that electroporation is the not a static technology,” continued Dr. Kim.mids more ‘people-friendly’ to optimize their to the target cell. “A low voltage electrical most efficient and safe mode of delivery of “We are developing new devices, referredexpression once in the cell, and the second field is applied at the site of vaccine injec- nucleic acids, requiring no additional chemi- to as surface electroporators, that sit on theis to optimize the rapid transfer of the DNA tion, causing the cell membranes to tran- cals, preservatives, or adjuvants. See Transfection on page 24 NEWS Genomics & Proteomics > CLC bio Contributes processes—how, when, and why are cer- expression using flow cytometry and mul- complete BioCyc Pathway Database Col- Expertise to STATegra tain genes switched off or expressed?” tiplex assays, solidifying the connection lection. The integrated package will allow CLC bio is the latest company to par- states Michael Lappe, Ph.D., senior bioin- between the genotype and phenotype of scientists to access, combine, visualize, and ticipate in the European FP7 project, STAT- formatics scientist at CLC bio. “We aim to interest. These types of connections pro- analyze biological datasets across multiple egra. It will receive $1.3 million out of the leverage a framework which makes this vide confidence to preclinical and clinical “omics” experiments (genomics, transcrip- total $7.8 million budget for its efforts. The extremely complex network biology un- decision-makers regarding drug safety and tomics, proteomics, and metabolomics). STATegra project aims to develop an inte- derstandable. Ultimately our tools will fa- efficacy, says Christina Shasserre, vp of dis- The new software package will enable re- gral analysis platform for different omics cilitate the translation of massive amounts covery and development. searchers to display biological pathway data data that is capable of providing a more ef- of data into useful insights that can be ap- generated by Agilent’s suite of instruments, ficient use of genomics technologies. This plied in clinical settings.” > Agilent and SRI Combine software, and reagents. Agilent products project will generate and integrate data Within the overall project, methods Technologies for New Offering include microarrays and next-generation se- obtained in proteomic, metabolomics, and for the validation and design of follow-up Agilent Technologies and SRI Interna- quencing technology, as well as LC/MS, GC/ epigenomic experiments. studies are implemented. Utilizing and val- tional signed a licensing agreement to of- MS, ICP/MS, and NMR systems. “One of the big challenges today is go- idating existing datasets, STATegra is also fer laboratory research customers a pack- ing past the raw sequence data and getting going to feed its experimental results back age that combines Agilent’s latest release > Cancer Genome Consortium to grips with the complexity of epigenetic into the public domain. of GeneSpring GX, GeneSpring NGS, Mass Expands China Research Profiler Professional, and Pathway Archi- The International Cancer Genome Con- > EMD Millipore Institutes tect version 12.5 with SRI International’s sortium is expanding its research in China, Personalized Medicine Services where new projects seeking genomic factors EMD Millipore’s discovery and devel- in four cancer types common to the world’s opment solutions business is now offer- most populous nation will be launched. ing genomic biomarker services. Com- The projects—focusing on colorectal, pany officials say the purpose is to aid esophageal, liver, and nasopharyngeal drug companies in targeting patient sub- cancers—join the consortium’s two-year- groups for improved clinical outcomes, old and still-ongoing Chinese project fo- understanding drug safety and efficacy, cused on gastric cancer, which made new and characterizing tumors. Full-scale version of methionine metabolism data from sequenced tumors public. Researchers at EMD Millipore recently pathway for Saccharomyces C., using BioCyc The new projects are expected to take content and rendered visually in “simple” mode profiled transcription in gefitinib-treated less time than the typical four years, since Rendering of the nucleosome (DNA blue, in MPP/Pathway Architect 12.5; also shows histone proteins green) highlighting positions non-small cell lung cancer cells by qRT-PCR. average, normalized differential abundances the consortium began collecting tissue of known epigenetic marks in spherical The genotypic and mRNA expression data for four conditions in the HeatStrips for some samples from patients with the diseases representation. CLC Bio were then cross-validated with protein of the metabolites. over the past two years. n22 | January 1, 2013 | | Genetic Engineering & Biotechnology News
  • 3. OMICSTransfection Continued from page 22surface of the skin, and a piezo-electric-based to produce large amounts of ultrapure DNA posomal complex to be processed aseptically and extremely small DNA for vaccination,system that doesn’t even require contact with vaccines. and filled into vials,” she explained. the minicircle,” said Dr. Schleef.the skin.” “Aldevron is also working with compa- Martin Schleef, Ph.D., CEO of the Plas- According to Dr. Schleef, PlasmidFactory nies like Sekris Biomedical to develop new midFactory, echoed Chambers’ concerns has obtained all relevant patents for this Immune Escape host strains that enable scientists to fine-tune over the high standard of purity required technology and supplies researchers in gene Annie DeGroot, M.D., CEO and CSO the methylation patterns of the plastids they of DNA for delivery in humans, and cited a therapy and DNA vaccination worldwideof EpiVax, highlighted immune escape as an use for immunization,” he added. recent study (Woodell et al., J. Gene Med), with this safe, nonviral vector. PlasmidFacto-important issue in the optimizing the amount Finally, Aldevron’s genetic immunization which demonstrated that chromosomal bac- ry is also tackling the issue of scaling of plas-of information that DNA vaccines deliver. and antibody service allows our clients to terial DNA, which is typically present in kit- mid preparations to the amounts requiredImmune escape is thought to arise when an try multiple delivery technologies like Ichor’s grade plasmid DNA preparations, is a sig- for more demanding applications, e.g., largeinfectious agent or, in the case of cancer, a tu- TriGrid electroporation system and Pharma- nificant contaminant leading to low efficacy clinical trials.mor, acquires a genetic profile distinct from jet’s needle-free delivery system to find the and severe toxic effects. “We are developing technologies forthat toward which a DNA vaccine was ini- optimal way to administer their vaccines,” “We have developed a manufacturing ultra-large scale production of plasmidtially targeted, allowing it to escape the im- he noted. technology to avoid such contamination,” DNA (e.g., kg scale) by use of fed-batch andmune pressure of the vaccine. said Dr. Schleef. In addition, he highlighted large scale lysis to obtain pure ccc-plasmid- “In response to the problem of immune Complex Formulations plasmid topology as an important parameter DNA,” he said.escape, our approach at EpiVax has been to Since cells prohibit entry to large mol- in determining transfection efficiency. Cova-combine multiple key antigens or specific ecules like DNA, unique delivery and for- lently closed circular (ccc), or supercoiled, Critical Factorepitopes within these antigens, in a single re- mulation technologies must be fine-tuned DNA adopts a compact form due to internal As both Chambers and Dr. Schleef havecombinant vaccine,” noted Dr. DeGroot. to optimize the transfection and delivery of tensions in the DNA molecule that is optimal pointed out, the purity of a plasmid prepa- “We use in silico methods to optimize a DNA vaccines. for transfection efficiency. ration is a critical determinant of successfulgene sequence for improved vaccine expres- “Technologies for delivering DNA vac- “Ideally the proportion of ccc-DNA in vaccine transfection or delivery.sion,” added Lenny Moise, Ph.D., director of cines include liposomes, polymers, or elec- the preparation should be 95% or higher,” “If you don’t start with pure DNA, youvaccine research at the company. “They in- troporation,” noted Magda Marquet, Ph.D., said Dr. Schleef, “and we use a manufactur- can forget about reproducible and effectiveclude codon optimization and gene analysis, co-founder and co-chairperson of Althea ing technology to obtain pure preparations transfection,” said Bill Kuhlman, vp Northincluding 5´ end secondary structure, cryptic Technologies. “However, these methods are of this form.” America for BIA Separations. “Removal ofsplice sites, human genome homology, bac- often cumbersome for patients to receive, PlasmidFactory has also developed a capil- endotoxins and genomic DNA is common-terial promoters, eukaryotic promoters, in- and present difficulties in manufacturing and lary gel electrophoresis-based analytical tool ly achievable at small scale, but producingverted repeats, palindromes, tandem repeats, scale up.” to quantify the major plasmid topologies in a DNA vaccines at commercial scale requiresand nucleosome positioning.” Althea is pursuing complex formulations, given plasmid preparation, namely ccc-DNA, process steps that can efficiently produce tens Similar to Inovio, Epivax’ transfection such as polymers and liposomes, to over- oc (open circular)-DNA, and linear DNA. of grams of highly pure supercoiled plasmidmethod is to rely on electroporation to en- come the obstacle of DNA vaccine delivery In addition to fine-tuning the conformation DNA per run.”hance uptake to improve vaccine immuno- by delivering DNA directly to cells. Some of DNA for transfection, stripping plasmids BIA Separations specializes in separa-genicity. “There are about 30 Phase I and II formulations have challenging requirements. of genes encoding resistance to antibiotics or tions, with particular emphasis on monolith-clinical trials using electroporation of DNA Dr. Marquet cited one case in which asep- other selection markers is an important step ic HPLC columns. Rather than traditionalvaccines, more than any other delivery tech- tic processing was required throughout the for preparing DNA for transfection. bead-based separation columns, monolithicnology. It represents the most promising formulation since product could not be ster- “We have designed strategies to remove columns are composed of an organic or in-DNA vaccine delivery method with a path to ile filtered prior to filling. “Althea successful- such genes from the bacterial backbone of organic substrate and multiple highly perme-the clinic,” said Dr. Moise. ly scaled up the process and brought in GMP the plasmids used, in addition to the origin able and porous channels that afford a large formulation equipment that allowed the li- of replication, resulting in a simply circular surface area to the stationary phase. Range of Technologies “We’ve achieved homogeneity of greater Aldevron was the first company to pro- CIMmultus columns than 97% supercoiled DNA with greaterduce a DNA vaccine used outside an ex- for DNA plasmid than 99% removal of host DNA, protein,perimental setting, a West Nile Virus DNA purification at all and RNA at development through industrialvaccine that has been used to protect endan- scales,” Kuhlman pointed out. BIA Separa- scalesgered species. tions has recently introduced large-scale “There are many reasons why so few BIA Separations disposable Monolith columns allowing 48cells are transfected by DNA vaccines,” said grams of super coiled DNA to be purifiedMichael Chambers, president and CEO of in a single run, while maintaining the purityAldevron. “One potential hurdle related to and recovery seen at small scale.DNA vaccine manufacturing includes re- In addition to manufacturing and deliv-sidual E. coli impurities like colanic acid,” ery, another important consideration forreferring to an exopolysaccharide common DNA vaccines, like any other candidate ther-to many enterobacteria. apeutic, is safety. The potential integration of “These can cause toxicity and overall DNA vaccines into the host cell genome is ofnegative effects that hurt transfection effi- Althea reports that concern due to the possibility of insertionalciency,” he pointed out. In addition to im- its plasmid DNA mutagenesis resulting in the inactivation ofpurities in the vaccine formulation, plasmid manufacturing tumor suppressor genes or the activation ofmethylation patterns can also play a role in oncogenes in the host genome. processes have beenlow transfection and expression. Other hur- According to Dr. Marquet, “DNA vac-dles include issues related to vector size, re- successfully scaled cines have been proven to be safe in multiplegional micro environments around the DNA up to the 1,000 L studies, with no integration of the DNA intoinjection site, and the inherent toxicology of level. Plasmid DNA the host chromosomes ever being observed.”most compaction/transfection reagents. produced in Althea’s Her assessment is reinforced by Dr. Kim “Aldevron has developed or in-licensed 1,000 L fermentor from Inovio Pharmaceuticals. “We haveadvanced manufacturing and purification vaccinated or treated over 600 patients can yield millionssystems to increase in vivo transfection ef- with good safety and tolerability profiles,”ficiencies,” said Chambers, citing the use of vaccine doses, he said. “Adverse events are typically mildof Nature Technology’s HyperGro plasmid according to the to moderate, and any injection sites resolvefermentation technology that enables them company. without sequelae.” 24 | January 1, 2013 | | Genetic Engineering & Biotechnology News