Expert Intelligence for Better Decisions. MicroRNA: An Insight to mirna-based Microarrays, Diagnostics and Therapeutics

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1 Expert Intelligence for Better Decisions MicroRNA: An Insight to mirna-based Microarrays, Diagnostics and Therapeutics

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4 MicroRNA: An Insight to mirna-based Microarrays, Diagnostics and Therapeutics Published in May 2014 by Cambridge Healthtech Institute iii

5 Insight Pharma Reports is a division of Cambridge Healthtech Institute, a world leader in life science information and analysis through conferences, research reports, and targeted publications. Insight Pharma Reports focus on pharmaceutical R&D the technologies, the companies, the markets, and the strategic business impacts. They regularly feature interviews with key opinion leaders; surveys of the activities, views, and plans of individuals in industry and nonprofit research; and substantive assessments of technologies and markets. Managers at the top 50 pharma companies, the top 100 biopharma companies, and the top 50 vendors of tools and services rely on Insight Pharma Reports as a trusted source of balanced and timely information. Publisher: Science Writer: Design Director: Production Director: Customer Service: Corporate Subscriptions: Lisa Scimemi , lscimemi@healthtech.com Chelsea Macary , cmacary@healthtech.com Thomas Norton , tnorton@healthtech.com Ann Marie Handy , ahandy@healthtech.com Adriana Randall , arandall@healthtech.com David Cunningham , cunningham@healthtech.com iv

6 MicroRNA: An Insight to mirna-based Microarrays, Diagnostics and Therapeutics For more information about published Insight Pharma Reports, visit or call Rose LaRaia at A Cambridge Healthtech Institute publication 2014 by Cambridge Healthtech Institute (CHI). This report cannot be duplicated without prior written permission from CHI. Every effort is made to ensure the accuracy of the information presented in Insight Pharma Reports. Much of this information comes from public sources or directly from company representatives. We do not assume any liability for the accuracy or completeness of this information or for the opinions presented. Cambridge Healthtech Institute, 250 First Avenue, Suite 300, Needham, MA Phone: n Fax: n v

7 Executive Summary Executive Summary MicroRNA: An Insight to mirna-based Microarrays, Diagnostics and Therapeutics is a new report to Insight Pharma Reports. microrna (mirna) is a relatively new space in heavy pursuit of research because of its versatility and stability, properties that are unique to mirnas compared to other RNA components. Characterized by its non-coding nature, yet its ability to affect genetic expression, these features make mirna a novel candidate for use as biomarkers for a variety of diseases. This prompted several companies to pursue mirna-based microarrays and diagnostics for the advancement in therapeutic development. Furthermore, there are a number of academic laboratories not only validating mirnas, but also identifying them in a number of diseases and examining their molecular actions in the presence of other molecules. This report is broken up into five parts. After covering brief background information, the second and third parts of this report expand upon microarray analysis, oligonucleotide synthesis, and diagnostics in development. Several companies featured in these sections include: Affymetrix, Exiqon, Diamir, Rosetta Genomics, and Eisai. Chapters within these sections feature company backgrounds and highlight proprietary technologies and platforms. Additionally, several advantages and limitations are also discussed. Even more significant, these chapters are accompanied by interviews with company representatives exclusively conducted for this report. The fourth part of this report explores the academic research and community perspectives. Although not developing diagnostics per se, academia is diligently investigating several aspects of mirnas including their use as biomarkers for cancer, neurodegenerative diseases, and nephropathy. Universities interviewed in this space include Stony Brook University School of Medicine, Grand Valley State University, and Van Andel Research Institute. Furthermore, Dr. Argyropoulos, a Medical Affairs Consultant, provides insight to this rapidly growing field and highlights advances in his research. Also included in these sections are interviews with leading experts from these universities and institutions. Finally, the report concludes with an in depth analysis of survey results. With 152 researchers, these results provide an overall perspective of the research community, capturing an accurate representation of research demographics, challenges afloat in the industry, and market outlook of mirna. vi

8 Table of Table of Executive Summary vi Part I: Background and Introduction 14 CHAPTER 1 What is mirna? 15 Transcription 15 Gene Expression 15 Part II: Oligonucleotide Technologies 16 CHAPTER 2 mirna Creation and Application 17 Engineering Oligonucleotides 17 Photolithographic Synthesis 17 Engineering mirna Antagonists: OMe, MOE, and LNA 18 Other Engineering Methods: mirna Mimicry 18 CHAPTER 3 Affymetrix: mirna Microarrays and Photolithographic Technology 20 Company Background 20 GeneChip mirna 4.0 Array 21 Photolithographic Process and Assay Features 21 Specificity and Sensitivity 21 Applications 22 Areas of Improvements and Future Expectations 23 vii

9 Table of Interview with Dr. Johanne McGregor 25 Company Background 25 GeneChip mirna 4.0 Array 26 Targets and Applications 27 Technology 27 Challenges, Benefits, Improvements, and Future Expectations 29 CHAPTER 4 Exiqon: Improving mirna Detection with LNA Technology 30 Research Background 30 The Biology of mirnas as Biomarkers 30 Locked Nucleic Acid (LNA) Technology 31 mircury LNA qpcr Platform: Advantages and Market Outlook 32 Interview with Dr. Henrik M. Pfundheller 33 Research Background 33 Locked Nucleic Acid (LNA) Technology 33 microrna qpcr Platform 34 Part III: mirna as Diagnostics 36 CHAPTER 5 mirna Use as Diagnostic Biomarkers 37 mirna Diagnostic Applications 38 CHAPTER 6 Rosetta Genomics 39 Research and Company Background 39 microrna Diagnostic Platforms 39 Advancements in Healthcare 41 Areas of Improvement and Limitations 41 viii

10 Table of Interview with Ken Berlin 43 Company Background 43 microrna Diagnostic Platforms 43 Platform Specifications 44 Advancements in Healthcare 45 Improvements and Limitations 46 CHAPTER 7 DiamiR: mirna Neuro-Diagnostics 47 Company Background 47 Universal Screening Test and Specifications 47 Advantages and Future Outlook 48 Interview with Kira Sheinerman 49 Company Background 49 Platforms and Specifications 50 Advantages, Limitations, and Future Expectations 51 CHAPTER 8 Gensignia: mirna Lung Cancer Diagnostics 52 Company Background 52 mirna Signature Classifier (MSC) Lung Cancer Test 52 Advancements in Healthcare 53 Interview with Dr. Elisa Romeo 54 Company Background 54 MSC Lung Cancer Test 54 Advancements in Healthcare 55 CHAPTER 9 Eisai: mirna Research and Diagnostics for Alzheimer s Disease 56 Company Background 56 ix

11 Table of mirnas As Circulating Biomarkers 56 Eisai s mirna Research in Alzheimer s Disease 57 Competitive Strategies 57 Improvements and Limitations 58 Future Expectations 58 Interview with Dr. Pavan Kumar 59 Company Background 59 mirna Research 59 Competitive Strategies 60 Advancements 60 Improvements 61 Future Expectations 62 Part IV: mirna Research in Academia: Targets, Diagnostics, and End User Perspectives 63 CHAPTER 10 mirnas in Academia 64 CHAPTER 11 Stony Brook University School of Medicine:miRNA in Colorectal Cancer 65 mirna in Colorectal Cancer 65 p53 and mirna in Colon Cancer 66 Epithelial-to-Mesenchymal Transition (EMT) 66 Diagnostic and Therapeutic Applications 67 Challenges Encountered and Areas of Improvement 67 Interview with Dr. Jingfang Ju 68 mirna Research 68 mirna Properties and Technologies 68 p53 and mirna 68 x

12 Table of Epithelial-to-Mesenchymal Transition (EMT) 69 Diagnostic and Therapeutic Applications 69 Challenges and Areas of Improvement 70 Future Applications 70 CHAPTER 12 Grand Valley State University: mirna in Parkinson s Disease 71 Circulating mirna as a Biomarker for Parkinson s Disease 71 Cerebral Spinal Fluid vs. Plasma 72 Advantages, Limitations, and Areas of Improvement 72 Interview with Dr. Sok Kean Khoo 74 Research Background 74 Competition 74 Methods and Properties 75 Areas of Improvement and Challenges 76 CHAPTER 13 Independent mirna Research: 77 The Role of mirnas in Nephrology 77 Research Background 77 mirna in Type 1 Diabetes and Diabetic Kidney Disease 77 Research Limitations, Challenges, and Areas of Improvement 79 Future Outlook, Applications and Growth 80 Interview with Dr. Christos Argyropoulos 82 Research Background 82 Challenges, Benefits and Areas of Improvement 84 Future Outlook and Industry Impact 85 xi

13 Table of CHAPTER 14 Van Andel Research Institute: 87 mirna Discoveries and Technology Advancement 87 Institution Background 87 Research Background 87 mirna Properties and Discoveries 88 mirna Tissue Slide-Based Assays 88 Advantages of mirna Tissue Slide-Based Assays 89 Challenges and Limitations of mirna Tissue Slide-Based Assays 90 Areas of Improvement 91 mirna Market Outlook 91 Interview with Dr. Lorenzo Sempere 92 Research Background 92 mirna Properties 92 Diagnostics and Therapeutics 93 Limitations and Advantages 94 Future Expectations 95 Part V: mirna Highlights, Challenges, and Advances in R&D 96 CHAPTER 15 Survey Analysis 97 References 101 About CHI 104 xii

14 Table of TABLES Table 3.1: GeneChip mirna Microarray Version Comparisons 22 Table 3.2: Instrument Compatibility of GeneChip mirna 4.0 Array 23 Table 3.3: Platform Specifications 24 Table 6.1: Rosetta Genomics Testing Specifications 42 Table 7.1: Universal Screening Test Specifications 49 Table 8.1: mirna Signature Classifier Lung Cancer Test Specifications 53 FIGURES Figure 6.1: Product Pipeline and Market Forecast 40 Figure Survey Demographics: Classification of Researchers 98 Figure Research Areas: Pursuing mirna in Drug Discovery or Assay Development 98 Figure Drug Discovery: Drug Target Status 98 Figure Drug Discovery: The Challenges and Difficulties in Studying mirna 99 Figure Drug Discovery: Who is Studying What? 99 Figure Drug Discovery: mirna Mechanisms of Interest 99 Figure Drug Discovery: mirna Effects of Interest 99 Figure Assay Development: Technological Status 100 Figure Assay Development: Competitive Advantage 100 Figure Assay Development: Challenges Encountered 100 Figure mirna Therapeutic Market Outlook 100 xiii

15 What is mirna? CHAPTER 1 What is mirna? MicroRNAs (mirnas) are a very popular area of interest. They are short sequences (roughly nucleotides) of RNA that are produced via specialized RNA transcripts coded in the genome. 1 These sequences downregulate gene expression by attaching themselves to messenger RNAs (mrnas), which, depending on the extent of base pairing, either leads to destruction of the mrna or prevents them from being translated into proteins. 1,2 Ever since their discovery in 1993 by Victor Ambros, Rosalind Lee, and Rhonda Feinbaum, mirnas have been pursued in several areas including diagnostics and therapeutics. 3 In order to understand the role of mirna in genetics, this section will briefly discuss the process of transcription and gene expression. Transcription After DNA synthesis, DNA is transcribed into small segments of RNA, known as messenger RNA (mrna), through the combined work of many proteins. These segments are further analyzed by even more proteins, which facilitate an action called splicing. This process occurs to differentiate introns from exons. Introns, or junk DNA, are DNA not used for translation purposes. Exons contain segments of genetic information which may be eventually translated into proteins. However, there are a lot of mechanisms which affect genetic expression including histone acetyltransferases, methylation, RNA stability and many others. One of the most notable players in genetic expression is microrna. Gene Expression After DNA is transcribed into mrna it is translated into proteins, which express various phenotypic and genotypic characteristics. microrna plays a significant role in genetic expression as it binds to the 3 -UTR (untranslated region) of their target mrnas. This destabilizes the mrna and represses its protein production, inducing translational silencing. 4 A similar RNA subtype is sirna (small interfering RNAs). However, instead of suppressing genetic expression, this type of RNA actually leads mrna down a path of destruction; any information is degraded and never has the opportunity to be translated. 2 Based on findings, researchers have discovered that the key difference between suppressing genetic expression and destroying any translatable material revolves around the binding of the molecule. In sirnas, a perfect match with the target mrna marks the duplex for destruction by endonucleases. But most mirnas do not match the target sequence exactly, and are unable to distinguish tiny variations in the recognition site. 2 -Steven Buckingham 15

16 mirna Identification and Creation CHAPTER 2 mirna Creation and Application Engineering Oligonucleotides micrornas offer a novel approach to diagnostic and therapeutic applications. However, in order to achieve these robust applications, mirnas first need to be detected and analyzed. Due to poor detection methods, several companies have been taking different approaches to improve mirna sensitivity and specificity. One of the ways to solve this issue is by synthesizing oligonucleotides with preemptively optimized properties. Affymetrix is one company working on perfecting microarray technologies. They are working on enhancing the detection of mirnas across a number of research areas by using a photolithographic process to synthesize oligonucleotides. Another company highlighted in this section is Exiqon, which features locked nucleic acid as its primary method for enhancing mirna detection and therapeutic application. This section will discuss several methods to synthesize oligonucleotides. Photolithographic Synthesis Photolithographic synthesis seems to be most commonly utilized by Affymetrix for their GeneChip technology. 17 This application is a lightdirected oligonucleotide synthesis; more specifically the methodology employs MeNPOC photo-activatable nucleoside monomers. 26 With an ability to print individual 10µm 2 probe features at a density of 10 6 probes/ cm 2, this method facilitates the widespread use in the hybridization-based detection and analysis of mutations and polymorphisms. 26 Ultraviolet (UV) masking, in combination with light-directed chemical synthesis, selectively synthesizes probes located directly on the surface of the array. 17 These probes contain a protecting group on their free end that is activated once in contact with the UV light. 17 A photolithographic mask directs the UV light in such a way that exposure to specific nucleotides can be controlled. 17 It works by deprotecting, and therefore activating, a series of hydroxyl groups. 17 This effect initiates coupling with incoming protected nucleotides that attach to the activated sites. 17 Although each probe on the chip requires four masks per round of synthesis, this method features increased sensitivity and specificity that enable discrimination of single mismatched base pairs

17 mirna Identification and Creation Engineering mirna Antagonists: OMe, MOE, and LNA Locked nucleic acid (LNA) is a technique that has been used for mirna antagonists, which are the most widely used approach to regulate mirna levels in vivo. 22 Using inhibitors is similar to other inhibitory therapies that target a single genetic product (i.e. small molecular inhibitors or sirnas). 21 Furthermore, the chemical make-up of these therapeutics are so pristine that, when bound to RNA, binding is often irreversible. This makes the antagonist incredibly stable; often it is unable to be processed by RISC (RNA-induced silencing complex) and/or degraded. 21 Although this therapy seems ideal and provides great potential for being applied to multiple diseases, there is always the concern that the antagonist will bind to the incorrect RNA, which would create great concern in those potential side effects. 21 mirna antagonists are modified antisense oligonucleotides that contain the full or partial complementary reverse sequence of a mature mirna, and are generated to inhibit endogenous mirnas that show a gain-of-function in diseased tissues. 21,22 These highly chemically-modified mirna passenger strands are often investigated for use in cancer therapies. The anti-mir, or antagamir, binds with high affinity to the appropriate active mirna strand. 21 This is one of the key requirements to developing the chemistry of an mirna antagonist. Other requirements include: cell permeability, stability in vivo, possessing high specificity and affinity for binding, and resistance to rapid excretion. 22 In order to achieve these properties, several chemical modifications have been used to engineer the antagonists. These include: 2 -O-methyl-group (OMe)-modified oligonucleotides and locked nucleic acid (LNA)-modified oligonucleotides as the most common. 22 Another less common method is 2 -O-methoxyethyl (MOE) modified oligonucleotides. The difference between these three types of chemical modifications is the way in which the RNA is engineered. OMe modifications happen to be one of the oldest modifications known to date. It is also the simplest. 23 The methyl group not only contributes limited nuclease resistance but also improves binding affinity to RNA compared to unmodified sequences. 23 MOE modifications, on the other hand, contain higher affinity and specificity to RNA than OMe modifications. Because of this, they have been historically used to redirect mrna splicing and inhibit protein translation. Using this application in mirna research has also demonstrated effective therapeutic benefits, primarily through inhibition. 23 The last method that has been known to be used is locked nucleic acid modification. While the latter two methods were a matter of adding a methyl or methoxyethyl group to the appropriate oxygen in the ribose ring, LNA features a distinctive bond between the 2 -O-oxygen and the 4 -carbon atom via a methylene linker. This bridges the gap between these two atoms, literally locking the ribose structure in place. 23,24,25 The main advantage to this modification is that LNA creates the thermodynamically strongest duplex formation with complementary RNA known to researchers. 23 Although LNAs have been used as northern probes to detect mirnas, their heightened sensitivity and selectivity makes them excellent candidates for mirna antagonists. 23 Other Engineering Methods: mirna Mimicry Although not specifically featured in this report, another method on the rise worth noting is mirna mimicry. mirna mimicry caters to creating a replacement, optimally-functioning mirna that the body cannot distinguish from the naturally, and often mutated, produced mirna. It is a different way of utilizing mirna therapeutics. Instead of inhibiting mirnas that show a gain-of-function, mimicry is meant 18

18 mirna Identification and Creation to address the mirnas that exhibit a loss-of-function. 21 It is considered to be a replacement therapy, aiming to re-introduce mirnas into dysfunctioning cells that would otherwise have normally expressed the mirna in question. 21 By reintroducing these synthetic mirnas back into the patient s systems, they reactivate normal pathways indigenous to the life of a cell. Researchers have demonstrated that mirna replacement therapy (by mimicking tumor suppressor mirnas) has been linked to oncogenic pathway stimulation, apoptosis, and ultimate eradication of tumor cells. 21 This type of mirna has stirred a lot of excitement in the industry because it targets a different function of mirnas, exploiting new opportunities for tumor suppressors. 21 Furthermore, these therapeutics can be delivered systemically using technologies similar to the ones used to deliver sirnas and, because normal cells already express the mirna in question, adverse events are unlikely to occur

19 Survey Analysis as assays as the greatest challenge in developing therapeutics. Another obstacle in therapeutic development is the fact that 58% of mirna assays are still in their underdeveloped states, while only 16% are launched. This leaves researchers with only a handful of technologies to work with, limiting their ability to advance mirna therapeutics. Furthermore, the survey also addresses challenges that are present in assay development. According to researchers, the most challenging aspect is validation (noted by 48% of researchers), which inadvertently affects the clinical status of drug discovery and development. However, on a positive note, 42% of researchers in assay development claimed the greatest advantage to their assay over their competitors is due to higher accuracy and sensitivity. This paints a brighter future for mirna therapeutic advancement. Despite these many challenges that are sometimes dealt with on a daily basis, the mirna industry still holds a solid reputation and has the potential to significantly impact healthcare as an effective diagnostic and therapeutic. Figure Survey Demographics: Classification of of Researchers What best describes your organization? Figure Research Areas: Pursuing mirna in in Discovery Drug Discovery or Assay or Development Which therapeutic area are you working in? Other 25% Assay development 36% Figure Drug Target Discovery: Status Drug Target Status What is the status of your most current drug target? Non applicable 18% Drug Discovery 27% Drug development 12% Other 9% Hospital clinic 3% Government research (including measurement institutes) 28% Biotechnology 28% On the market 2% Phase III clinical development 5% Phase II clinical development 5% Still in development/ mouse models 61% Academic research 48% Pharmaceutical 7% Phase I clinical development 9% 98