DETERMINATION OF STRATEGIC PRIORITIES FOR A MICROBIOME COMPANY THROUGH ANALYSIS OF TECHNICAL CAPABILITIES AND CURRENT MARKET LANDSCAPES
by BRANDON EDWARD ANDREW
Submitted in fulfillment of the requirements for the degree of Master of Science
Thesis Advisors: Dr. Christopher Cullis, Dr. Emmitt Jolly, Dr. Neema Mayhugh
Department of Biology CASE WESTERN RESERVE UNIVERSITY
May 2020
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
Brandon Edward Andrew
candidate for the degree of Master of Science.
Committee Member
Christopher Cullis
Committee Member
Emmitt Jolly
Committee Member
Neema Mayhugh
Date of Defense
April 2, 2020
*We also certify that written approval has been
obtained for any proprietary material contained
therein.
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Table of Contents
I. Technical and Scientific Background I.I Human Microbiome and Large-Scale Research Initiatives…………………..…….1 I.II Some notes on the design of current studies in microbiome-related research……………………………………………………………………….………..6 I.III Metagenomics and Sequencing Methods for Microbiome Characterization……………………………………………………………...….……..7 I.IV 16S and 18S rRNA Sequencing ………………………..………………………...9 I.V Whole Genome Sequencing: Coverage, Read Length, and Depth……………….12 I.VI Gut Microbiome and the Fungal Connection: Dr. Ghannoum’s Research ……...15 I.VII Crohn’s Disease, Ulcerative Colitis, and Irritable Bowel Disease ……………..20
II. Business Development Strategy and Commercialization Roadmap II.I Biohm’s Current Business Model ……………………………..……………………22 II.II Medical Foods Overview………………………………….…..…………………....23 II.III Medical Food FDA Regulations and Approval Process …………..………………25 II.IV Medical Food Market Size, Competitors, and Strategic Considerations….…….....27 II.V Direct-to-Consumer Test Kits Overview: Market and Competitor Case Studies…..30 II.VI Direct-to-Consumer Kit Regulations………………………..…………………..…32 II.VII Direct-to-Consumer Kits Strategic Considerations…………..………………..….33 II.VIII Therapeutic Pipeline Strategic Considerations ………………………………….36 II.IX Therapeutic Pipeline FDA Regulations……………………….………………...…38 II.X Intellectual Property Relevant to Biohm………………………..………………..…38
III. Strategic Conclusions for Biohm’s Business Development ……………..…....…40
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List of Figures
1. Number of microbiome-related publications since 2000………………………………….7
2.Organisms sequenced and Phylogenetic Context from Human Microbiome Project……..9
3.Microbiome research funding related to HMP vs. non-HMP from 2007-2016………….11
4.Schematic detailing metagenomics analysis workflows and pipelines…………………..15
5.Gene map for E. coli 16S ribosomal subunit with variable/hypervariable regions……...16
6.Sample workflow for a whole genome shotgun sequencing experiment from sample prep
through data preparation for bioinformatic analysis……………………………………..19
7.Taxonomic distribution and abundance of fungi in biofilms and image of fungal invasion
of colon sample…………………………………………………………………………..23
8.Formation of biofilms in colon tissue by C. tropicalis, E. coli, C. albicans, and S.
marcescens……………………………………………………………………………….25
9.Imaging of colon tissue demonstrating pathological differences in ulcerative colitis and
Crohn’s disease………………………………………………………………………..…28
4
List of Tables
1.Largest medical food manufacturers with indication pipelines………………………..35
2.Companies developing a data-driven platform supplemented by at-home test kits…...41
3.Therapeutic pipelines and indications for microbiome pharmaceutical companies…..43
4.Relevant patents for Biohm and Mahmoud Ghannoum………………………………45
5
Acknowledgements Marissa – You have always been my light in the dark and the foundation from which I can grow. Thank you for listening to everything that comes out of my head during this process. Thank you for your eternal patience and unquestioned love. I would not be able to be who I am without you to be my guide star.
Mom and Dad – I am who I am, and I am on the journey I am because of the model you provided. Thank you for teaching me radical independence and authenticity.
Dr. Jolly – You gave me an opportunity to believe in myself when I couldn’t do that for myself. Thank you for being an incredible mentor and teaching me the value of “thinking about and doing science.” Your faith in me won’t go unrewarded.
Neema – You chose to mentor me and advise me and let me grow in your incredible shadow. I appreciate how our relationship has blossomed and how you have given me the space to be authentic in my work. I look forward to the future in our work.
Dr. Cullis – Thank you for guiding me through the racecourse of this program. I have always appreciated your insight and encouragement.
Emmaline – You have become my closest friend and confidant over the years. From Posner’s lab to Bioenterprise, I am so appreciative to be able to grow and evolve alongside you. You have given me courage and support in what feels like Sisyphean work. Thank you.
To those not mentioned here, I appreciate you, too. Whether it was sharing a beer or trying to listen and understand what I do, thank you. You will never know how much you mean to me.
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Determination of Strategic Priorities for a Microbiome Company Through Analysis of Technical Capabilities and Current Market Landscapes
Abstract by:
BRANDON EDWARD ANDREW
The “mycobiome,” coined by the CWRU investigator Dr. Mahmoud Ghannoum, refers to the composition of both bacterial and fungal communities in the human gut microbiome and has been the focus of disease-state correlations investigated by his lab. Dr. Ghannoum started the company, Biohm, to sell direct-to-consumer at-home microbiome sequencing kits and probiotics that aim to balance the gut biofilm that contributes to the dysbiosis- associated conditions. Biohm has expressed an interest in developing new business strategies to leverage their intellectual and technical strengths. This thesis is composed of two parts:
The first section is a scientific and technical investigation of the micro- and myco-biome, sequencing techniques and strategies (16S, ITS, WGS, and Shotgun Metagenomic Sequencing) that play a role in the characterization and identification of fungal and bacterial colonies in the gut. These strategies aim to overcome challenges in characterizing and quantifying microbiota composition. Next, this sequencing data can form a robust database of patient data that plays a role in disease identification, and this thesis identifies some of the bioinformatic analyses to achieve this goal. The section concludes with how insights derived from patient data can be used in the optimization of cohort design in clinical trials for various diseases.
The second section investigates three different business models that Biohm has expressed interest in exploring for future development: (1) medical foods; (2) a therapeutic pipeline; and (3) a data-licensing and discovery platform for drug development. A detailed analysis of the market dynamics, competitive landscape, regulatory issues, and other nascent concerns was performed for each potential vertical as a foundation to develop future business strategy for Biohm. The thesis is concluded on a holistic analysis of the scientific and technical assets and business opportunities and strategies necessary for Biohm to successfully implement new business development. The final section serves as a final assessment of the holistic analysis and provides considerations regarding sustainable business growth for Biohm or any other microbiome-based biotech startup.
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I. Technical and Scientific Background I.I Human Microbiome and Large-Scale Research Initiatives
The human microbiome has been a focus of biologists for many years but has become increasingly focused upon within the last 10-15 years. Figure 1 below from a
Silicon Valley Bank annual report1 shows that the number of microbiome publications went from about 500 collectively in 2010 to over 10,000 collectively by the year 2016.
Several explanations for this exponential growth in research are explored here and include, but are not limited to: (1) large-scale, massively funded research initiatives such as the Human Microbiome Project (HMP), Integrative Human Microbiome Project
(iHMP) and Metagenomics of the Human Intestinal Track (MetaHIT); (2) improvements in research tools and approaches like the accessibility of next generation sequencing, metagenomics, and bioinformatic tools; and (3) the increasing awareness of the connection between the microbiome and human diseases such as gastrointestinal diseases like Crohn’s Disease and IBD, as well as cognitive disorders like Autism Spectrum
Disorder, depression, and anxiety.
Figure 1: Number of microbiome-related publications since 2000.
1 Silicon Valley Bank. 2017 Annual Report. San Francisco, CA: SVB, 2017. Accessed February 15, 2020. 8
A microbiome refers to an entire ecosystem comprising all the microorganisms living together in a particular environment, as well their constitutive genes and interactions with the environment. This collection of organisms includes bacteria, fungi, viruses, phages, archaea, and other microbial eukaryotes.2 The human microbiome then is a localized ecosystem of microbiota that exist on or near a human, and there is significant diversity depending upon that existent location. The human microbiome has huge amounts of genetic diversity and material when compared to the native genome of its host.3 Interestingly, organisms in separate locations on a human may be stable over long periods of time and can be considered endemic, but they also may be transient and change over the course of an individual’s life. Figure 2 below shows the diversity and distribution of organisms discovered and sequenced during the Human Microbiome
Project.
2 Cox, Michael J, Cookson, William OCM, Moffatt, Miriam F. “Sequencing the human microbiome in health and disease.” Human Molecular Genetics 2013; 22: R88-R94. 3 The Human Microbiome Jumpstart Reference Strains Consortium. “A catalog of reference genomes from the human microbiome.” Science 2010; 328: 994-999. 9
Figure 2: Organisms sequenced during the Human Microbiome Project are highlighted in blue. This figure details the overall distribution of sequenced strains around tree of life.
Microbiome-associated diseases can find their etiologies in both traditional pathogen infection at the cellular level and in more complex integrated network pathways. That is, disease origin may be an emergent property of dysfunction in regulatory gene networks or metabolic networks within hosts. A holistic, systems approach to studying the gut microbiome has recently begun to elucidate how functional metagenomics better explains host-microbe relationships and disease etiology.
One of the first large-scale microbiome research initiatives was the Human
Microbiome Project (HMP) supported and launched by the US National Institutes of
Health in 2007. It occurred in two phases with the second beginning in 2014, called the
Integrative Human Microbiome Project(iHMP). The first phase relied on 16S sequencing and metagenomic analysis to provide deep, culture-independent sequencing and
10 characterization of the entire healthy human microbiomes (not only the gut). Phase 1 had five distinct goals4:
1) Develop a reference set of microbial genome sequences and perform preliminary characterization of the human microbiome 2) Explore the relationship between disease and changes in the human microbiome 3) Develop new technologies and tools for computational analysis 4) Establish a resource repository 5) Study the ethical, legal, and social implications of human microbiome research
Phase II of the project, the iHMP, began immediately and set forth to apply integrative -omics techniques to the immense datasets produced in Phase I. Additionally, the primary goal of the second phase was to create longitudinal datasets to understand the biological properties of hosts and their microbiomes from three different cohorts. This phase employed many of the tools and techniques developed during the HMP and included the use of new whole metagenome sequencing, whole genome sequencing, metatranscriptomics, and other large integrated datasets.
Success of these projects can be measured in the number of publications, number of new technologies produced, amount of data produced, or, more nascently, the greater depth of knowledge and appreciation the role of the human microbiome plays. However, the NIH report5 detailing the amount of funding that has resulted alongside the NIH funding into the HMP and iHMP is more revealing to the lasting impacts these initiatives have on research. Figure 3 shows the amount of funding for microbiome research related to the HMP vs non-HMP from 2007 to 2016.6 This shows a significant amount of non-
4 Human Microbiome Project / Program Initiatives. The NIH Common Fund. Retrieved March 12, 2020. 5 NIH Human Microbiome Portfolio Analysis Team. “A review of 10 years of human microbiome research activities at the US National Institutes of Health, Fiscal Years 2007-2016. Microbiome 2019; 7:31 6 Ibid 11
HMP investment being poured into microbiome as a result of the interest and growth in the human microbiome.
Figure 3: Microbiome research funding related to the HMP vs. non-HMP from 2007 to 2016.
Other than large reference databases, a major technological leap to come from these large research initiatives is the number of tools and technologies that allow deep research to be performed. From whole metagenomic sequencing to analytical techniques beyond 16S and 18S rRNA sequencing, this massive increase in funding has fueled a lot of our understanding of the etiologies and pathologies of GI-related diseases and provided the groundwork for many industry-led pharmaceutical companies to begin to develop microbiome-related therapies for these diseases. Most importantly, the publicly accessible databases that have allowed for platform construction and therapeutic pipeline construction are the most valuable of consequences to occur from these public research initiatives.
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I.II Some notes on the design of current studies in microbiome-related research
The iHMP provides a lot of the framework necessary for long-term, longitudinal studies in how changes in a healthy microbiome may lead to a disease state. However, many published articles are not following similar study designs, and some time should be committed to explaining the differences and strengths each may provide. In short, the types of studies covered here are not comprehensive, but are the most relevant to the access of public reference databases and microbiome-related study designs. The
Microbiome Quality Control Project was established to quantify these variabilities and provide quality controls for the field, as well as provide standardization for study design.
The most common types of studies in microbiome research covered are: (1) prospective cross-sectional cohort studies; (2) case-control studies; and (3) longitudinal retrospective studies.78 Each of these may provide different conclusions, but they have their own idiosyncrasies and issues. While not covered here, the fundamental issue with performing large-scale research across multiple labs and multiple research centers is the systematic and relevant variation in measurements and reporting of controls.
Prospective cross-sectional are appropriate for cases where longitudinal sampling is not feasible. These are probably as close to optimal as design as we’ve had so far in these studies.9 However, for cases where the disease is neuro-based, the latency and idiopathic nature of these diseases make controls difficult to establish. This is a similar issue with case-control studies, where there is no standard control for the human gut
7 Goodrich, Julia K., Di Rienzi, Sara C, et al. “Conducting a Microbiome Study.” Cell 2014; 158(2): 250- 262. 8 Sinha, Rashmi, Abnet, Christian, et al. “The microbiome quality control project: baseline study design and future directions.” Genome Biology 2015; 16:276. 9 Ibid 13 microbiome.10 We should select from the same population in these studies, but this has so far not been the case.
Ideally, we would move away from large cohort studies and move into conducting prospective, exploratory studies that focus on the more emergent properties of microbiome systems. For example, more recent studies have focused on elucidating properties of molecular interaction networks and how “dark” gene properties give rise to unknown metabolic properties of microbiome communities.11,12 In these studies, many of the unknown properties of microbial genes are interrogated for their potentially rich sets of data. These studies could provide the basis for discovery of novel metabolic compounds that have potentially therapeutic potential but are beyond the scope of this paper.13
I.III Metagenomics and Sequencing Methods for Microbiome Characterization
Sampling and identification of the microbial composition of the microbiome traditionally relied on traditional culturing methods in the lab. However, this method is only successful with bacteria and fungi that are culturable outside of the ecological environment where they were sampled. Isolated culturing of microbiome communities will then miss a significant portion of the community composition because it is estimated that 50-60%14 of microbial organisms are unculturable and cannot be observed in
10 Ibid 11 Ling, Yiwei, Watanabe, Yu, and Okuda, Shujiro. “The human gut microbiome is structured to optimize molecular interaction networks. Computational and Structural Biology Biotechnology Journal. 2019; 17:1040-1046. 12 Kurokawa K, Itoh T, Kuwahara T, Oshima K, Toh H, Toyoda A, et al. “Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes.” DNA Res 2007;14:169–81. 13 Levy R, Borenstein E. “Metabolic modeling of species interaction in the human microbiome elucidates community-level assembly rules.” Proceedings of the National Academy Sciences USA. 2013; 110:12804– 9. 14 Browne, Hillary P, Forster, Samuel C, et al. “Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation.” Nature 2016; 533:543-546. 14 isolation. Analysis of microbiome communities initially relied of culturing environmental samples in isolated cultures. The diversity of a community was evident in isolated culture, but a significant proportion of the community was left undiscovered and uncharacterized.
In the early 1990’s, ecological sampling of sea water led to the realization that that a vast majority of organisms are unable to be cultured in lab using traditional methods.15 New approaches had to be developed to address this gap in coverage, and, as a result, the field of metagenomics began to grow. Covered in this section are: 16S, 18S rRNA sequencing, whole genome shotgun sequencing, and sequencing technologies necessary for constructing and annotating new genomes de novo from the “unculturable” organisms sampled in the microbiome.
Metagenomics is the discipline and set of techniques that allow scientists to begin to fill in the full picture of an environmental sample such as the human gut microbiome.
The continued development of better next generation sequencing and advancing bioinformatic capabilities have allowed scientists to begin identifying and classifying new organisms, as well as their abundance and relation to other identified microbes.
Additionally, metagenomics includes techniques and experimental approaches that aim to fully interrogate the scope of a sample’s microbial community and its interactions. This is especially important for the human gut microbiome as the connections between disease and the gut become more elucidated. Using metagenomic approaches allows researchers to avoid a faulty, incomplete foundation for translational studies of the microbiome. Metagenomics has largely flourished due to the advancement
15 Hugenholtz, Philip, Goebel, Brett M., Pace, Norman R. “Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. Journal of Bacteriology 1998; 180(18): 4765-4774. 15 and affordability of next-generation sequencing and the growth and development of better computing power and bioinformatic tools. Figure 416 shows the overall workflow of different metagenomic analytical approaches.
FIGURE 4: A schematic detailing the workflow for different sequencing and analytic techniques used in the study of the microbiome. 16S rRNA and Whole-Genome sequencing include additional steps.
I.IV 16S and 18S rRNA sequencing
The first metagenomic sequencing of environmental samples began with using the
16S rRNA approach. This approach works off the idea that the gene for the 16S ribosomal subunit is high conserved across many phyla and be used as a target gene for identification. The 16S gene is composed of highly conserved regions and hypervariable regions. The hypervariability is genus and species specific which serves as the basis for phylogenetic construction. Figure 5 below shows a layout of the 16S gene in E. coli to
16 Cox, Michael J, Cookson, William OCM, Moffatt, Miriam F. “Sequencing the human microbiome in health and disease.” Human Molecular Genetics 2013; 22: R88-R94. 16 demonstrate the conserved and hypervariable regions that are targeted and used for phylogenetics of environmental samples.17
Figure 5: ~1.5kb of the 16S rRNA gene for E. coli. There are 9 variable regions in the gene which provide the basis for 16S as a phylogenetic marker.
16S rRNA sequencing relies on the clustering of sequences by related identity and counting the representative identity number of each cluster. However, due to multiple copies of the 16S gene in some bacteria, the relative abundance of an organism may be overrepresented (up to 15 copies in E. coli). Additionally, bacteria and archaea do not map directly to traditional taxonomies like eukaryotes, so, to address these issues, similar sequences or clusters of similar sequences are referred to as “Operational Taxonomic
Units” (OTUs).18,19
OTUs are assembled in a table with relative abundances for each organisms’ genera and then these tables are used for downstream analyses.20 While these can be used for abundances, hierarchical organization of phylogenies is also possible. One issue is the percent threshold of similarity needed to identify a sequence to the genus or species level.
Generally, 97% sequence identity is sufficient for the species level and 95% is sufficient
17 Ibid. 18 Ibid. 19 Thomas, Torsten, Gilbert, Jack, and Folker, Meyer. “Metagenomics – a guide from sampling to data analysis.” Microbial Informatics and Experimentation 2012; 2:3-12. 20 Blaxter, Mark, Mann, Jenna, Chapman Tom, et al. “Defining operational taxonomic units using DNA barcode data. Philosophical Transactions of the Royal Society 2005; 360: 1935-1943. 17 for genus. These sequence identities are compared to reference databases that have been collated via the Human Microbiome Project and other initiatives over the last 15 years.
However, even with these reference databases, many bacteria and archaea identified can only be classified down to the family or genus level, and species level can be difficult for all prokaryotes due to the nascent and shifting nature of species-level definition for these organisms. Even so, 16S is best used for abundance and not necessarily classification down to species level which can have consequences for determining cause of disease related to an organism.
While 16S provides a good footing for the analysis of prokaryotic organisms, eukaryotic organism identification and quantification relies on the 18S subunit.21 The 18S is a cytosolic homologue of the 16S ribosomal rRNA and provides the basis for similar gene barcoding used in the 16S method. Its main differences rely on different universal primer design and identification of eukaryotic organisms in environmental samples.
Databases such as The Ribosomal Database Project, HMP shared databases, and
GreenGenes provide the basis for quality assessment and optimization of sequence alignments and play a crucial role in the development of phylogenetic compositions of microbiome samples.
Alongside the 16S and 18S sequencing, reliance on the internal transcribed spacer
(ITS) is commonly used in the identification of organisms in a sample. 22 Often, ITS is used in place of 18S sequencing for fungal species due to a higher degree of
21 Meyer, Achim, Todt, Christaine, Mikkelsen, Nina T, and Bernhard, Lieb. “Fast evolving 18S rRNA sequences from Solenogastres (Mollusca) resist standard PCR amplification and give new insights into mollusk substitution rate heterogeneity.” BMC Evolutionary Biology 2010; 10:70. 22 Schoch, C.L., Seifert, K.A., Huhndorf, S., Robert, V., Spouge, J.L., Levesque, C.A., Chen, W., Bolchacova, E., Voigt, K., Crous, P.W., et al. "Nuclear Ribosomal Internal Transcribed Spacer (ITS) Region as a Universal DNA Barcode Marker for Fungi". PNAS. 109;16: 6241–6246.
18 hypervariability in fungi. The ITS in prokaryotes is a single sequence that is located between 16S and 23S rRNA genes and has less variability than it does in eukaryotes, especially fungus. It is more variable in fungi due to there being two ITS regions, as well as experiencing a higher rate of crossover events in these organisms. It provides a better look into species-level identification than other sequencing technologies for environmental samples such as microbiome samples.
I.V Whole Genome Sequencing: Coverage, Read Length, and Depth
While 16S and 18S sequencing provide a good basis for understanding the abundance and identification of organisms, metagenomics and whole genome sequencing are much more relevant in terms of providing insight to functional dynamics of a sampled community. 16S and 18S rely on normal PCR and primer design for the amplification of a targeted gene, but whole genome sequencing relies on a method of shotgun sequencing.
Whole genome sequencing is the foundation for a comprehensive analysis of an organism’s function, and this leap in analytical ability is due to the lowering costs of next-generation sequencing, increasing availability of genome databases with annotations, and rapid development of new bioinformatic tools for advanced analysis.23
This approach is one of the most powerful in studying the microbiome because it allows for a deep interrogation of pathogenicity in a complicated and constantly changing environment.
Metagenomic shotgun sequence analysis (MSSA) is one of the most popular approached, but still has some limitations that will be covered here. MSSA is accomplished by sequencing all the genomes of all the organisms present in a sample,
23 Cox, Michael J, Cookson, William OCM, Moffatt, Miriam F. “Sequencing the human microbiome in health and disease.” Human Molecular Genetics 2013; 22: R88-R94. 19 and even the sequences of unculturable organisms are covered by this technique. One of the limitations of this approach is the idea of sequencing depth, and this is mostly limited by the cost of this sequencing technique.24 Coverage of a sequence is typically equated to the total number of bases generated divided by the size of the target genome. This should not be taken to mean that these are targeted sequencing approached, like 16S/18S, but rather the targeted sequences are what is in the sequenced sample. A typical workflow for whole metagenomic shotgun sequencing can be seen below in Figure 6.25
24 Depth and coverage here are somewhat interchangeable, but in this thesis will be used to mean the number of times each sequence is “covered” by a sequencing technology. For example, 10X coverage means that all sequences were covered/sequenced 10 separate times. 25 Zaheer, Rahat, Noyes, Noelle, Ortega Polo, Rodrigo, et al. “Impact of sequencing depth on the characterization of the microbiome and resistome.” Scientific Reports 2018; 8:5890.
20
Figure 6: Sample workflow for a whole genome shotgun sequencing experiment from sample prep through data preparation for bioinformatic analysis.
High-throughput sequencing of microbiome samples is the foundation for performing metagenetics. Originally, pyrosequencing was used, and this sequencing is where the sequential additional of all four nucleotide bases is incorporated into a synthesized molecule by a DNA polymerase. This polymerization releases pyrophosphate and is converted in two enzymatic reactions to produce light. A camera detects the photons of light and relays that signal to a computer for translation into a specific base in the sequence.26 The data quality in this method is often complicated by the production of artificial replicated sequences and requires a good quality assurance control on the analytical end to sort these replicates out. Pyrosequencing for shotgun-sequencing produces an average read length of 600-800bp which is a good length to produce minor loss in sequence compilation and annotation.27
Another approach for sequencing of metagenomic experiments is the use of the
Illumina HiSeq2000. This technology provides superior coverage and read depth which is important for the identification of low abundance organisms. 16S and 18S have been the primary method for determining identity and abundance, but they are limited in the depth of their coverage and may miss the low abundance organisms. Illumina HiSeq2000 is capable of producing hundreds of millions of reads per channel in the assay chip.28 The workflow seen in Figure 6 is a study design for this instrument.
26 Thomas, Torsten, Gilbert, Jack, and Folker, Meyer. “Metagenomics – a guide from sampling to data analysis.” Microbial Informatics and Experimentation 2012; 2:3-12. 27 Ibid. 28 https://www.illumina.com/documents/products/datasheets/datasheet_hiseq2000.pdf. Accessed March 10, 2020. 21
The Illumina has a sequencing technique called terminator-based sequencing by synthesis, which is way adaptors are required to be attached to the ends of random sample
DNA fragments. This enables a massively parallel sequencing of fragments. A fluorescent-label is imagined as each dNTP is synthesized into a new stand and then cleaved to allow for the next base to be added.29 A computer and a camera detect and interpret the resulting fluorescence as a DNA sequence. Read lengths are short and range from 35-300 bp which requires a very large coverage, but significantly limits the amount of errors in the process.
Finally, more recent technologies can have read lengths as long as the entire DNA molecule. Single-molecule sequencing is still being case tested in microbiome research but is beginning to prove extremely valuable in coverage and cost. Ion Torrent detects proton release during DNA polymerization and include read lengths on the order of thousands of bp. PacBio also has a single molecule sequencing technology with real-time data output. While these new sequencing technologies can provide superior read length with limited coverages (compensated by increased sample size), they can be prone to errors that would hinder data quality and bioinformatic analysis such as annotation and discovery of functional analysis.
I.VI Gut Microbiome and the Fungal Connection: Dr. Ghannoum’s Research
Dr. Mahmoud Ghannoum’s research has largely focused on the function role that fungi have played in the health of the human gut. His research has demonstrated that fungi like Candida tropicalis play an important role alongside the bacterial microbiome in regulating and maintaining the health of the human gut. Previously, the role and
29 Meyer M, Kircher M. "Illumina sequencing library preparation for highly multiplexed target capture and sequencing". Cold Spring Harbor Protocols 2010: 5448. 22 presence of fungi in the gut was either unknown or overlooked as having a similar impact as the bacterial communities. While Dr. Ghannoum’s career has largely focused on the creation of biofilms in the gut, other researchers have provided groundwork in our understanding of the role fungi play in the microbiome.
Illiev, et al30. investigated the scope and extent that the mycobiome exists within the gut, and two key insights arose from this study. This study identified 10 distinct species of fungus that played a role in the microbiome. While C. albicans was the most abundant, other notable genus such Saccharomyces, Aspergillus, Fusarium, and
Cryptococcus as being among the most abundant fungi. Some of these fungi are well- known pathogens and can cause serious illness, but within the gut, their pathogenicity is reduced by either the immune system or other community members. Illiev also demonstrated that the previously held belief that bacteria were the main contributors to dysbiosis in the gut was incorrect. Rather, it was the fungal communities that played the main role in contributing to the aggravation and inflammation of the gut.31
30 Iliev ID, Funari VA, Taylor KD, Nguyen Q, Reyes CN, Strom SP, et al. “Interactions between commensal fungi and the C-type lectin receptor dectin-1 influencecolitis.” Science 2012;336:1314–7. 31 Ibid 23
FIGURE 7: (A) The taxonomic distribution and (B) relative abundance of fungi genera in colitis mice models. (C) shows a DAPI and sDEC-1 stained colon sample with fungal invasion of tissue.32
Illiev showed that there is a link between fungal colonies and a polymorphism in the gene (Clec7a) for Dectin-1 that contributes to the inflammation associated with UC.33
Figure 7 above shows the range of fungal species and their relative abundance in mice colitis models, as well as a DAPI and sDEC-1 probe stained histology sample showing fungal invasion of colon tissue. Dectin-1 acts as a major part of the innate immune system in mice and humans as a sensor for fungi. When this gene had a deleterious polymorphism, an increased susceptibility to gut epithelial inflammation was observed in the presence of pathogenic mice. This observation led Illiev to discover and characterize the ten most prevalent fungi in the gut, as well as their ability to exaggerate the gut inflammation present in GI disorders. This study was pivotal in providing the
32 Ibid 33 Ibid 24 groundwork for understanding the role that fungal colonies play in the disease state of condition like UC and CD.
Hoffman et al. demonstrated the effect that diet has on the composition and regulation of the micro- and mycobiome.34 Hoffman and Ghannoum found that there is a negative association between C. albicans and C. tropicalis and the bacteriodes found in an inflamed gut. Fungi were found to correlate with relative levels of bacteria, but not the other way around. A diet high in carbohydrates had high, positively associated levels with Candid species, but these same species had a low, negatively associated correlation with saturated fatty acids. Additionally, Aspergillus species negatively correlated with short chain fatty acids (fiber-rich foods). These findings provide important insight into the role that the mycobiome plays with diet and chronic inflammatory GI diseases.
In 2017, a study by Hager and Ghannoum further investigated the role that the mycobiome plays in gastrointestinal inflammatory diseases Crohn’s and IBD.35 They discovered that, in particular, three organisms play a cooperative role in the formation of biofilms in individuals with inflammatory diseases such as CD, UC, and IBD. The three organisms with the highest levels of cooperation are Candida tropicalis, Escherichia coli, and Serratia marcescens. In Figure 8, Ghannoum and Hager imaged the role these three organisms play in the formation of biofilms in the distal colons of individuals with CD.
34 Hoffmann C, Dollive S, Grunberg S, Chen J, Li H, Wu GD, et al. “Archaea and fungi of the human gut microbiome: correlation with diet and bacterial residents.” PLoS ONE 2013; 8:e66019. 35 Hager, C. and Ghannoum, M. “The mycobiome: Role in health and disease, and as a potential probiotic target in gastrointestinal disease.” Digestive and Liver Disease 2017; 49:1171-1176. 25
Figure 8: These images show the formation of biofilms all with the presence of C. tropicalis and varying levels of C. albicans, E. coli, and S. marcescens. It demonstrates the central role C. tropicalis plays in the formation of gut biofilms.
Advantages for forming and inhabiting a mutualistic relationship in a biofilm matrix varies depending on the organism. C. tropicalis and C. albicans specifically benefit from gaining pathogenicity via the induction of hyphal formation and increased secretion of extracellular enzymes like aspartic proteinase.36 The cooperative relationship in biofilms is advantageous for these fungi as they provide better invasion of the host’s system, as well as defense from the host’s defenses. Likewise, bacteria also benefit from the construction of a biofilm in cooperation with fungi. Ghannoum and Hager found that colonies of E. coli and S. marcescens play a significant cooperative role in producing many of the extracellular proteins necessary for the construction of the biofilm matrix.
Biohm’s primary technology is a novel, patent-pending composition of a probiotic to target the dysbiosis caused by biofilms built up in an inflamed colon. Biohm sells
36 Ibid. 26 direct-to-consumer testing kits and probiotic supplements that target the biofilms discussed previously. Dr. Ghannoum’s research does provide some evidence that use of certain probiotics is effective at combating the build-up of potentially dysbiosis-causing biofilms in the human gut.37 The composition of a novel probiotic allowed Dr.
Ghannoum and his team to determine the fungal-bacterial correlative role in the inflammatory process. They were able to identify novel probiotic species that antagonized the biofilms formed, and with the addition of amylase and other digestive enzymes were able to effectively reduce the pathogenic properties of the biofilms formed in the inflamed mice colons.
I.VII Crohn’s Disease, Ulcerative Colitis, and Irritable Bowel Disease
This section provides a brief overview of the most common gastrointestinal diseases related to dysbiosis in the gut microbiome, Crohn’s Disease (CD), Ulcerative
Colitis (UC), and Irritable Bowel Disease (IBD). Our understanding of gastrointestinal diseases like CD, UC, and IBD has been significantly supplemented by the increase in microbiome research. Importantly, microbiome research and its connection to gastrointestinal diseases has largely driven the therapeutic pipelines of microbiome- related biotech startups.
Crohn’s Disease (CD) is a disease mediated by a multitude of factors including host genotype, dysregulated immune response, and the intestinal microbiota, and since members of a family share genetics, environment, diet and bacterial microbiota and are
37 Hager, Christopher L, Isham, Nancy, Schrom, Kory P. Chandra, Jyotsna, McCormick, Thomas, Miyagi, Masaru, Ghannoum, Mahmoud. “Effects of a novel probiotic combination on pathogenic bacterial-fungal polymicrobial biofilms.” American Society for Microbiology 2019 (2); e0038-19.
27 more similar to each other than they are to unrelated individuals.38 CD affects about 3 in every 1,000 people in North America and Europe, and incidence rates have been increasing since the 1970’s.39 In CD, individuals experience multiple symptoms that extend beyond inflammation in the colon including: inflammation and sores of the upper respiratory tract (mouth), small-bowel obstruction, fistulas and perineal disease, transmural mucositis, and granulomas.40 CD has been demonstrated to have a microbiome connection, and Dr. Ghannoum has demonstrated a connection between biofilm and increased inflammation in CD patients.41
Ulcerative colitis (UC) is also an inflammatory bowel disease that is indicated by chronic inflammation of the colon, ulcers, abdominal pain, and frequent diarrhea or constipation.42 Similar to Crohn’s, UC is differentiated by waning or acute symptoms that are localized to the colon, whereas CD can affect the entire GI tract (mouth to anus). UC affects about every 20 in 100,000 people in Europe and North America, and a total of
1,000,000 people in total are affected by both diseases combined. UC has similarly been shown to have a microbiome connection consistent with Dr. Ghannoum’s research on chronic inflammation and the biofilms created in dysbiosis.43 Figure 944 below illustrates the pathology expected in patients with the diseases.
38 Baumgart, Daniel, Sandborn, William. “Inflammatory bowel disease: clinical aspects and established and evolving therapies. The Lancet 2007; 9573: 1641-1657. 39 Ibid 40 Ibid. 41 Rodriguez-Palacios, A, Harding, A, Menghini P, et al. “The artificial sweetener Splenda promotes gut protebacteria, dysbiosis, and myeloperoxidase reactivity in Crohn’s disease-like ileitis.” Inflammatory Bowel Disease 2018; 24(5): 1005-1020. 42 Baumgart, Daniel, Sandborn, William. “Inflammatory bowel disease: clinical aspects and established and evolving therapies. The Lancet 2007; 9573: 1641-1657. 43 Hager, C. and Ghannoum, M. “The mycobiome: Role in health and disease, and as a potential probiotic target in gastrointestinal disease.” Digestive and Liver Disease 2017; 49:1171-1176. 44 Ibid. 28
Figure 9: Imaging showing the different pathologies in UC and CD. UC has the presence of polyps and spontaneous hemorrhaging and CD is noted by the chronic inflammation and loss of notable vasculature.
II. Business Development Strategy and Commercialization Roadmap II.I Biohm’s Current Business Model
The following sections aim to summarize and clarify Biohm’s current business model and to prepare a roadmap for expansion into one or more (of three) potential verticals. It should be noted that Biohm is already generating revenue from an existing business model but desires to leverage their assets and intellectual capital to further develop the company’s value. Therefore, this section will provide a strategic playbook and commercialization roadmap for each of the potential verticals where Biohm could succeed. Included in each of the subsections will be an addressable market size, competitive analysis, regulatory considerations (if necessary), and strategic considerations.
Biohm currently operates a business model that is based on selling a direct-to- consumer testing kit for the microbiome, as well as a line with various pro- and pre- biotics supplements. The supplements range in price from $39.99-$99.99 for consumers.
The “Biohm Gut Test” kit for $219.99 which includes the gut test, written actionable recommendations, and a 30-minute nutrition phone consultation. (This last feature is
29 optional and if a consumer opts-out, the kit cost drops to $179.99.) The written report contains 4 sections: (1) an overall gut score comparing your microbiome diversity against other microbiomes; (2) a comparison of the levels of 6 major bacterial communities and 4 fungal communities against normal, healthy levels; (3) a strain-level analysis which details the strains present in the consumer’s microbiome; and (4) actionable recommendations from registered nutritionists to address any potential issues with the individual’s microbiome.
Biohm’s microbiome platform positions the company with an opportunity to develop three separate verticals, or business models, to further their value. The first vertical addresses medical foods and consumer supplements. The second vertical is selling at-home testing kits for sequencing an individual’s microbiome, as well as the development of a discovery platform to be licensed to strategic partners. Finally, the third vertical is a therapeutic pipeline developed by leveraging Biohm’s existing database of microbiome data.
III.II Medical Foods Overview
Medical foods should not be confused with nutraceutical products or supplements that can be commonly found in health food stores or at pharmacy chains like Walgreens or CVS. Medical foods are regulated separately through the FDA under guidelines established in the 1988 Orphan Drug Act.45 As defined in the Orphan Drug Act, medical foods are “formulated to be consumed or administered enterally under the supervision of a physician and which is intended for the specific dietary management of a disease or a condition[…]” While nutraceuticals or supplements may address certain conditions or
45 FDA Medical Food Definition – Orphan Drug Act of 1988. Accessed February 25,2020. 30 health issues, medical foods should be understood as specifically formulated for diseases or disorders that require evidence-based nutritional needs.
Medical foods are traditionally placed within the context of personalized medicine, but this distinction does not apply here. Rather, medical foods should be understood as consumer supplements or personalized nutrition developed from the results of an individual’s personal microbiome composition. Ideally, a medical food would be developed to treat a person’s gut micro/mycobiome dysbiosis. Previously discussed evidence supports the hypothesis that a dysbiosis or imbalance in a person’s gut can contribute and lead to a variety of conditions and diseases. Specifically, diseases such as
Crohn’s, Ulcerative Colitis, and Irritable Bowel Syndrome have been shown to have a connection to gut dysbiosis. Interestingly, other hypotheses suggest that conditions and diseases such as depression, anxiety, and autism spectrum disorder have, in part, a connect to the gut microbiome.
While a profile for each consumer would occur on a case-by-case basis from at- home testing kits, the development of a medical food or personalized supplement would not need to occur on a case-by-case basis. Rather, the consumer’s profile would be grouped into other similar profiles, and the development of medical foods would be similarly be grouped. The grouping for individual’s and their correlating medical food would be based on a root cause of dysbiosis. For example, if an individual is shown to have an overabundance of a particular bacterial genus or a lack of a fungus, then a range of supplements would be made to address the specific issue. This model is similar to the current business strategy for Biohm but would necessitate an expansion in both the ability to process samples and production of personalized supplements.
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III.III Medical Food FDA Regulations and Approval Process
Medical foods do fall under the supervision and approval of the FDA, but they do not have the same regulatory pathway seen with medical devices or drugs. Medical foods were defined in the 1988 Orphan Drug Act, and are narrowly constrained by the FDA, as
" a food which is formulated to be consumed or administered enterally [orally] under the supervision of a physician and which is intended for the dietary management of a specific disease..." This definition provides a slight distinction from traditional nutritional supplements or special diets in that medical foods are designed to provide nutrition to a patient who could not otherwise, either in diet or lifestyle, provide the necessary treatment for a disease. Additionally, the condition that medical foods be monitored and provided under the supervision of a physician is distinct from other supplements. Medical foods are then are a major treatment component in the management of a disease, much like a drug.
Medical foods are understood to be analogous to drugs in the sense that they are major components of a treatment plan for a disease. However, medical foods are not considered drugs under FDA regulations, and, therefore, are not subjected to the same regulatory and approval processes of a drug. Medical foods are not required to go through the FDA approval process or attain any pre-market approval. Medical foods are also exempt from many of the labeling requirements for health or nutrient content claims that were established by the Nutrition Labeling and Education Act of 1990. Thus, the regulatory pathway and road map to commercialization is mostly unburdened.
Although medical foods face relatively few regulatory barriers, the FDA does list five criteria that must be met in order for a product to be a medical food. The following
32 criteria that clarify the legal definition of a medical food can be found in FDA’s regulations at 21 CFR 101.9(j)(8).46 A medical food is exempt from the nutrition labeling requirements of 21 CFR 101.9 only if:
“a. It is a specially formulated and processed product (as opposed to a naturally occurring foodstuff used in its natural state) for the partial or exclusive feeding of a patient by means of oral intake or enteral feeding by tube, meaning a tube or catheter that delivers nutrients beyond the oral cavity directly into the stomach or small intestine; b. It is intended for the dietary management of a patient who, because of therapeutic or chronic medical needs, has limited or impaired capacity to ingest, digest, absorb, or metabolize ordinary foodstuffs or certain nutrients, or who has other special medically determined nutrient requirements, the dietary management of which cannot be achieved by the modification of the normal diet alone; c. It provides nutritional support specifically modified for the management of the unique nutrient needs that result from the specific disease or condition, as determined by medical evaluation; d. It is intended to be used under medical supervision; and e. It is intended only for a patient receiving active and ongoing medical supervision wherein the patient requires medical care on a recurring basis
for, among other things, instructions on the use of the medical food.”47 Biohm already manufactures and sells probiotic supplements direct to consumers. These probiotics are designed to address the dysbiosis discussed previously. However, Biohm’s products do not currently fall within the definition of a medical food, and if Biohm
46 “Frequently Asked Questions About Medical Foods; Second Edition – A Guidance for Industry.” U.S. Department of Health and Human Services Food and Drug Administration Center for Food Safety and Applied Nutrition May 2016. Accessed Feb 10, 2020. 47 Ibid. Accessed February 10, 2020. 33 wishes to pursue this vertical, they would need to address the additional criteria and concerns mentioned here.
III.IV Medical Food Market Size, Competitors, and Strategic Considerations
According to BCC reports, the US medical foods market was valued at $17.27B in 2018 and is expected to grow to $29.54B in 2026.48 This is a reported CAGR of 6.9%.
Two primary factors driving this growth are an increasing geriatric population with increasingly specific nutritional needs and the increase in prevalence in metabolic and digestive disorders. There is some cross-over in the reporting and segmentation of populations contributing to growth. For example, nutritional deficiency disorders are more prevalent in geriatric populations than others, which leads to this population being a key contributor to the market’s success.
Primary indications that contribute to the size and growth of medical foods markets are diabetes, chronic kidney disorders like hepatic encephalopathy, intractable epilepsy, and orphan nutritional diseases such as phenylketonuria (PKU) and tyrosinemia
(TYR). However, not every patient with a specific indication will require the use of a medical food, and some cases may be treated and managed using lifestyle or dietary adjustments without the use of a medical food. Regardless, these are large markets with increasing number of incidences and prevalence each year. Notably, these are often chronic conditions that require lifelong management and make the market for medical foods large and sustainable.
In Table 1, a few of the largest competitors are listed, as well as the indications on which they focus their pipeline. including Nestlé, Alfasigma USA, Inc, and Metagenetics,
48 Kumar, Aneesh. “Probiotic Market.” BCC Market Research 2018. Report Code: FOD035F. 34
Inc. Of the competition in this market, significant attention should be given to Ajinomoto
Cambrooke (formerly Cambrooke Therapeutics) as developers of nutritional therapeutics for a variety of conditions. Cambrooke has multiple product lines that are broadly categorized into areas like “Metabolic,” “Specialty- Renal/Protein,” and “Ketogenic.”49
The company has used a biotechnology developed at the University of Wisconsin –
Madison to isolate and purify in high concentrations glycomacropeptide (GMP) as the basis for many of its foods that address metabolic disorders such as PKU and TYR. In addition to their substantial market presence in childhood or infant metabolic disorders,
Cambrooke also develops medical foods for those with renal protein disorders and ketogenic formulas for patients with intractable epilepsy.
49 https://www.cambrooke.com/about/#.XkmhaZNKjGI. Accessed February 12, 2020. 35
Company Name Medical Foods for Current Pipeline Indications, if Indications applicable Nestlé Health Science50 Many, grouped by disease Metabolic Disorders, type: gastrointestinal, Pediatric Renal Disease, aging/cognitive health, Ketogenic Diet, critical care and surgery, Gastrointestinal Disorders, pediatrics, chronic medical and Cognitive Disorders conditions, obesity, food allergy, and malnutrition Alfasigma USA51 Irritable Bowel Syndrome, Mild Cognitive Ulcerative Colitis, Impairment, Prenatal Depression (Deplin), Vitamins (l-methylfolate) Diabetic Nerve Damage, Male Infertility Metagentics, Inc.52 Compromised Gut Gastrointestinal-related Function, Inflammatory Cognitive Disorders Bowel Disease, Sarcopenia, Glucose Response, Dyslipidemia, Endothelial Function Ajinomoto - Cambrooke Table 1: Competitors in the medical food industry with indications and pipelines.
Biohm has many of the necessary collateral to succeed in the medical foods market, including: a large database of microbiome sequences, already generates revenue in the direct-to-consumer probiotic market, and medical team expertise. Strategic recommendations would include continuing development and sales of probiotic supplements, but not the raising of capital around the development of a medical food pipeline. The medical foods market would be difficult to enter without novel approaches to a disease requiring a medical food. The identification of novel microbiome-associated metabolic and nutritional disorders would be necessary to differentiate Biohm in the market, and this condition is not currently present with the company.
50 https://www.nestlehealthscience.us/nutritional-therapies. Accessed February 13, 2020. 51 https://www.alfasigmausa.com/. Accessed February 13, 2020. 52 https://www.metagenics.com/static/medical_foods/1.html. Accessed February 13, 2020.
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Probiotic supplements and their place in the medical food industry remains uncertain. Probiotics have a very large market of $36.6B in 2016 and growing at a rate of
7.8% CAGR to $57.7B in 2022.53 This would be a good market for Biohm to continue existing with their current business model. Probiotics are considered food supplements by the FDA and would not require any clinical evidence to support their development.
Finally, many of the larger companies with medical food portfolios would have little motivation to put large amounts of funding or time into developing probiotics. Even if they did pursue some sort of FDA approval, clinical trials and placebo-controlled studies are long and expensive.54
III.V Direct-to-Consumer Kits Overview: Market and Competitor Case Studies
As scientific and industry momentum increase, the accessibility of direct-to- consumer personalized medicine increases to larger markets. This section will cover what the direct-to-consumer genetic testing market looks like, as well as provide a case study into uBiome, a microbiome testing company currently under investigation by the FBI. At- home genetic testing kits started gaining widespread popularity in the early 2010’s as companies like 23andMe and Ancestry.com began kits to test an individual’s genetic ancestry. The scope of testing rapidly grew to include claims about an individual’s genetic risks, gut health, and a person’s wine preference. This last one is obviously less evidence-based, but it reveals the direction and opportunity in this market. According to an article published in Health Affairs, researchers found that as of 2017 there were about
53 Kumar, Aneesh. “Probiotic Market.” BCC Market Research 2018. Report Code: FOD035F. 54 Sator RB. Efficacu of robiotics for the management of inflammatory bowel disease. Gastroenterol Heaptology. 2011; 7:606-8. 37
75,000 genetic tests on the market, as well as there being 10 new tests entering the market daily.55
The direct-to-consumer testing market is structurally fragmented with significant variation in business models but is rapidly growing. According to a BCC report, the US market for direct-to-consumer genetic testing was $648M in 2017 and is expected to grow to $6.36B by 2028, a 22.84% CAGR.56 Even with this rapid growth, there are several items that need addressing by companies to grow at this expected rate including improving the reliability of test interpretation, improved data security and consumer privacy protection, and general uncertainty regarding the regulation of direct-to-consumer tests. This last item is especially pertinent within the context of uBiome’s FBI investigation.
uBiome began as a citizen science group that distributed microbiome testing kits.
They became an incorporated company in 2012 and began selling direct-to-consumer microbiome testing kits and providing the resulting health analysis back to the consumer.
An interesting point, uBiome was the first microbiome testing company to gain private insurance approval for reimbursement. This drove significant numbers of sales, and uBiome was poised to become the next 23andMe as they were approaching a $500M valuation as recently as 2017. However, in April 2019, the FBI raided uBiome and began an investigation into insurance fraud. It was found that uBiome had been double or triple
55 Phillips, Kathryn A, Deverka, Patricia A., et. al. Genetic Test Availability and Spending: Where Are We Now? Where Are We Going? Health Affairs 37, No. 5 (2018):710-716. 56 “Next-generation Sequencing: Emerging Clinical Applications and Markets. https://www-bccresearch- com.eu1.proxy.openathens.net/market-research/biotechnology/next-generation-sequencing-markets- report.html. Accessed March 3, 2020. 38 billing insurance companies for a consumer’s test and additional add-on tests that the consumer did not request.
The investigation is on-going, and uBiome filed for Chapter 11 bankruptcy in
October 2019. The main point to take away from this (very brief) case is that uBiome had an opportunity to be a leader in the microbiome testing industry. They have a good story coming from a citizen science background, and they were growing at a rate that had venture capital firms very excited. However, their fraudulent practices have them worth nothing now, and the founders of the company are facing jail time. The take-away is that
Biohm may have a good opportunity to fill this gap in the market. The process of gaining private insurance reimbursement is complicated, but the niche of covering micro- and mycobiome health analysis would be an ideal situation for Biohm.
III.VI Direct-to-Consumer Kit Regulations
The FDA considers direct-to-consumer genetic tests to be medical devices and requiring regulatory approval prior to being marketed but only when they provide medical recommendations rather than health survey reports. This is a key distinction that became prominent within 23andMe.
Two important pieces of legislature need to be considered before pursuing at- home microbiome testing and data licensing as a viable vertical. The first is the Genetic
Information and Nondiscrimination Act (GINA) of 2008.57 This law has two important implications for the use of a person’s data in either licensing to an external company, selling data information to an insurance or healthcare institution, or pursuing internal development of a therapeutic pipeline or technology. The first implication establishes that
57 https://www.genome.gov/about-genomics/policy-issues/Genetic-Discrimination. Accessed February 19, 2020. 39 genetic test information falls under HIPAA Privacy Rules and must be kept private and non-distinguishable from other’s data. The second implication establishes that an individual may not be discriminated against by either insurers or employers on the basis of their genetic information. This second implication plays a more important role when pursuing the development of an FDA-approved at-home microbiome test.
In general, a direct-to-consumer DNA or microbiome test does not need FDA approval. The information being relayed back to the consumer is that of a health report or ancestry tree and would not fall under FDA regulations. However, 23andMe began to attract regulatory attention when it began to provide consumers with additional analytics related to diseases and other medical issues. GINA does not apply to direct-to-consumer kits, but any licensing of that data or use of it to develop a platform would require anonymization of all data.
II.VII Direct-to-Consumer Kits Strategic Considerations
When considering the commercialization pathway and roadmap for at-home microbiome testing kits and data licensing, the recent past has provided two very important and strong case studies to take into consideration. The first case study is concerned with 23andMe’s market and FDA approval to provide genetic analysis results to consumers from their at-home test kit. This case provides the first precedent for regulatory pathway for direct-to-consumer kits and providing medically actionable results. The second case study is concerned a company called uBiome, a now defunct company from San Francisco. uBiome began as a crowdfunded, citizen science project, and was a venture capital darling for several years. However, in April 2019, the company was raided by the FBI and had charges of insurance fraud and privacy violations filed
40 against them. This is an especially important case study with several lessons any biotech firm dealing with personal data should be aware of when beginning.
At-home testing of the microbiome could prove to be a viable business model but is not sufficient to capture the entire market of possibility faced by Biohm. To fully articulate the opportunity and capture a significant market share, a horizontal integration into data analytics and licensing has shown to be a viable business model. There exist several examples where startups provide additional value to the market by developing drug discovery or therapeutic programs on top of the direct-to-consumer kits they provide. This model assumes that the data collected from consumers is sufficiently rich in depth and fidelity. The data serves as the basis for conditioning of machine learning models whereby drug discovery or modulatory targets can be assessed. To drive value in this horizontal integration, platform-based companies must form strong industry partnerships with large industry players willing to license the platform and partially pay in future royalties on commercialized products.
Table 2 displays companies that base their primary business models on leveraging data into a platform. Included in dataset are the most relevant competitors in this market:
Seres Therapeutics, Kaleido Biosciences, Finch Therapeutics, Freenome, and Human
Longevity, Inc. Each of these companies bases their business model on the development of a platform that is then licensed to strategic partners or forms the basis for the development of a therapeutic pipeline. As seen in the above analysis, significant venture capital has been put into this business model especially in the last few years (2016-2019).
Of these competitors, two serve as good comparable models for Biohm.
41
Finch Therapeutics is a platform-based, microbiome therapeutics company in
Rockville, MD. Their "Human-First Discovery Platform" was developed with data from human microbiota transplantation studies. This platform uses machine learning-based algorithms to determine causal roles between microbial populations and patient outcomes. This platform has informed 2 therapeutic approaches and has resulted in 4 open-label clinical studies with industry partners. The first approach is Finch’s "Full-
Spectrum Microbiota Products," which are therapies that contain a diverse community of microbiota from screened donors and delivered via capsules (CP101 is lead candidate for
C. difficile). The second approach is "Rationally-Selected Microbiota," which are products designed to contain selected microbial strains that are grown in pure culture and engage targeted mechanisms (partnership with Takeda on FIN-524 for ulcerative colitis).
Finally, Finch in internally developing a therapeutic called FIN-211 and is an investigational oral microbiome therapeutic being developed to re-establish a normal microbiome composition and function in individuals with Autism Spectrum Disorder.
Company Fundraising Amount (USD) Year
Round
Finch Therapeutics Series C $53.5M 2019
Seres Therapeutics IPO $134M ($681M post-money) 2015
Kaleido Biosciences IPO $75M ($444.5M post-money) 2019
Freenome Series B $155M 2019
Human Longevity Series B $200M 2017
Second Genome Series B $51M 2016-17
Insitro Series A $100M 2019
Vivante Health Series A1 $5.8M 2020
Table 2: Top microbiome platform companies with most recent funding round
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II.VIII Therapeutic Pipeline Strategic Considerations
The microbiome naturally serves as a basis for drug discovery and therapeutic targets. As discussed previously, researchers have developed better understanding of the connections between digestive, metabolic, and immune-related diseases and an individual’s microbiome. Here, an analysis of some of the more commonly approached indications will provide the basis for providing recommendations to Biohm for their therapeutic development strategy. The most prevalent indications addressed are
Clostridium difficile (C. diff) infections, damage and dysbiosis caused by antibiotic use, and gastrointestinal diseases like Crohn’s and ulcerative colitis. As more research on the connection between neurodegenerative and cognitive disorders and the microbiome comes out, you will see an increase in therapeutic pipelines focusing on these disorders.
Microbiome research has largely consisted of inventories, toolboxes of reference data sets that serve to catalyze the development of medical diagnostics and interventions.58 Therapeutic pipelines across the industry have shown to be heavily focused on gastrointestinal diseases, metabolic diseases like obesity, and bacteria or antibiotic related conditions (i.e. C. difficile). The concentration of companies, as well as venture capital dollars, focusing on diseases like IBD, ulcerative colitis, and other GI disorders should deter new entrants into the microbiome therapeutic space. However, there are new disease related areas that are beginning to accumulate the necessary research footing to be considered potential candidates in therapeutic development.
The microbiome industry is concentrated around companies developing therapeutic pipelines. Table 3 below contains a sampling of therapeutic development in
58 Proctor, Lita. “What’s next for the human microbiome?” Nature 2019 (569): 623-625. 43 the microbiome industry. The table shows that there is a high concentration of companies developing therapeutic pipelines in the gastrointestinal and infectious disease (mainly C. difficile infection). Several companies have emerged as leaders in developing microbiome therapeutics as they have robust pipelines in multiple indication areas. It is important to note that companies like Kaleido Bioscience, Finch Therapeutics, Seres
Therapeutics, and Evelo not only have robust pipelines, but large industry partnerships with companies like Johnson & Johnson and Nestlé. These partnerships are significant to note as they strengthen the position of the smaller startup by providing capital and resources that normally would be inaccessible. Another interesting feature of these same companies is that they developed their pipelines on the foundation of a strong discovery platform. These platforms laid the groundwork for target discovery in the disease areas the startups have grown.
Preclinical Phase I Phase II/III
Multiple Thx -Kaleido Biosciences (Multi-drug -Scioto (Metabolic -Kaleido (Urea Cycle Pipelines resistance, Hepatic encephalopathy, diseases diabetes and Disorders) tumor response) obesity) -Rhythm Metabolics -Rhythm Metabolics (Prader-Will (POMC/LEPR Deficiency syndrome) Obesity, Bardet-Biedl Syndrome, Alström Syndrome) GI/Infectious Disease -Second Genome (IBD, IND enabled) -Scioto (C. diff) -Finch (Recurrent C. diff, -Seres (rationally designed ulcerative -Inception (IBD) Ulcerative Colitis) colitis candidate) -Finch (Crohn’s -Scioto (Infant gut Disease) dysbiosis) -Seres Therapeutics (IBD, ulcerative colitis) Cognitive Disorders -Finch (ASD) -Axial Biotherapeutics (ASD) -Scioto (ASD) Cancer/Autoimmune -Evelo (MSS Colorectal, Triple-negative breast cancer, and Anti-PD-1 Relapsed) Dermatology -Evelo (Psoriasis, atopic dermatitis) Table 3: Therapeutic pipelines of various companies with indications.
New areas with high potential that Biohm should consider as potential avenues include partnering with large pharmaceutical companies on cognitive disorders like
44
Autism Spectrum Disorder, identifying immune-modulatory targets and developing small molecules. However, diagnosing and treating parasite-related pathologies are underexplored within the microbiome research space, as well as a target for industry-side technology development. Finally, Vivante Health is a new competitor in the microbiome space. While not a traditional therapeutic company, Vivante is exploring the space of digital therapeutics. This is a relatively new space and would require its own exploration beyond the scope of this thesis. Vivante is developing a comprehensive digital health platform to monitor food sensitivities and triggers in patients with GI disease like
Crohn’s, IBD, and ulcerative colitis.
II.IX Therapeutic Pipeline FDA Regulations
The drug development process is time intensive and requires significant capital to reach key milestones. There are multiple routes to market beginning with the pursuit of an Orphan Drug designation where you gain additional market exclusivity for treating a rare disease. You could also follow a traditional Investigational New Drug pathway, but, again, this would be very expensive and Biohm risks not having the data grounding in a novel indication to compete successfully.
II.X 1Intellectual Property Relevant to Biohm
Biohm does not control or license any of the necessary IP to generate the interest of external private equity or venture capital investment. Patents related to the research of
Dr. Ghannoum, and thus needed to develop any of the three potential verticals, is not even assigned to Dr. Ghannoum. Rather, patents related to his research name Afif
Ghannoum as the inventor, and, in some cases, are assigned to a different company called
Arms Pharmaceutical. Table 4 lists granted patents and applications that list Afif or
45
Mahmoud Ghannoum as inventors, as well as the assigned company or institution.
Finally, the sequencing methods used by Biohm are not proprietary nor is the NGS tech, data analysis exists as know-how, or trade secrets, and there isn’t a novel compound or biomarker identified to center a therapeutic or medical food program.
Patent Number Title Inventor Recent Description Actions Method for US20190358149A1 “Method of Brian Vincent Application applying inhibiting harmful Sokol, Afif published on therapeutically microorganisms Mahmoud 11/28/2019. Filed active amount and barrier-forming Ghannoum by ARMS of composition composition Pharmaceutical, to prevent therefor” LLC. Status is microorganisms pending. from building pathogenic biofilm Mahmoud US10426761 “Method for Ghannoum, Afif Published to Method for treatment of Ghannoum ARMS treating biofilm disease caused or Pharmaceutical associated aggravated by inflammation in microorganisms or the gut relieving symptoms thereof”
Table 4: Most relevant patents assigned to Dr. Ghannoum
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III. Strategic Conclusions for Biohm’s Business Development
The aim of this section is to synthesize the research and analysis performed previously and provide concluding thoughts to the developmental strategy for Biohm.
This will include attention to risk mitigation factors, necessary team composition, and the viability of growing a sustainable business in the three proposed verticals.59 First, the scientific and technical capabilities of Biohm are assessed as a foundation for a new business vertical. Second, an analysis of the markets and industry show that three verticals can be viable, but only if specific business and strategic decisions align with the company’s technological position.
Biohm uses sequencing techniques and analytical tools that are non-proprietary and would be considered the gold standard for research and any commercial development. The at-home testing kits are sequenced using 16S rRNA techniques to identify well-known phyla of bacteria and 18S rDNA for fungal communities. No indication of metagenomic techniques are present in the literature available. For research, these are sufficient for the identification and composition of an individual’s microbiome, and this depth and coverage is sufficient for the different projects studying longitudinal, case
In The Innovators Solution, Clayton Christensen and Michael Raynor describe the process by which innovation can serve as the basis of disruption for an industry. Rather than see innovation as a spurious, random, or black-box process, Christensen and Raynor
59 This section ultimately serves, in part, as the praxis of my master’s education and experience as an analyst at BioEnterprise. I realize this is unconventional, and possibly impermissible, for a master’s thesis in a biology program. However, my program is unconventional and has taught me to weigh the risks of an action against possible outcomes, and that often unconventional and potentially impermissible ideas become commonplace. If this premise is true, and as I was taught on the first day of my first class with Dr. Jolly, that everything is negotiable, then I propose some elements of unconventionality be allowed here. 47 believe that innovation can be understood as an iterative process that can be observed, understood, and harnessed to develop strategies for new business strategies. They are specifically speaking about companies seeking new-growth opportunities through a strategic and innovative process. The second section of this thesis provides the analysis and understanding to provide strategic insight into which direction Biohm should innovate and move. This does not mean reinventing the wheel or doing something completely unseen before. Rather, the process of determining the strategic direction of
Biohm should necessarily be informed by the analysis performed in that thesis section.
Therefore, with an understanding of the dynamics and forces in the market, how each vertical is competitively positioned, and leveraging the assets of Biohm, this final section will provide the conclusions and recommendations necessary for Biohm to successfully and sustainably grow as a company.
The analysis of each vertical shows that it could survive as a successful business model for Biohm, but there are strong and non-negligible considerations that must be weighed heavily before any strategic decisions are made. The medical food market is expected to grow and is relatively void of direct competition. However, the competition that does exist is large and well-engrained with large pipelines and strong distribution networks that would be difficult to compete with as a new entrant. The types of indications that would necessitate the need for medical foods are not exclusive to microbiome-related insights, but they are often related, in part, to a microbiome connection. The most common indications needing specific nutritional diets are metabolic in nature, but the treatments designed for them are only indirectly related to the work done by Biohm.
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When considering competing in the medical foods market, the type of innovation necessary should be considered. In this case, Biohm would be an entrant in a market engrained with large players who have established manufacturing and distribution networks. Biohm could not compete with Cambrooke or Nestlé directly by developing a sustaining innovation. A sustaining innovation in this market would be providing a technology (product or service) to consumers that is significantly better, in multiple ways, than what the large players provide. Biohm could develop a medical food for diabetes or phenylketonuria, but even if their solution is significantly better than Nestlé’s, Nestlé will leverage their resource and capital-intensive infrastructure and ultimately recapture any consumer that Biohm attracted.
Rather, if Biohm were to succeed in the medical foods market, they would either need to develop a solution that is so low-cost that margins are unattractive or address current non-consuming customers. The preference in this dilemma is for Biohm to develop medical foods for populations of consumers with indications that are currently unaddressed by the other large market players. If this were the case, then Biohm would need to develop a research and development pipeline leveraging their rich database from their testing kits. However, if such a process were to be developed at Biohm, then it would be more advantageous to operate as a therapeutic/pharmaceutical company performing drug discovery and development. The alternative pathway, while containing its own risks, provides a more lucrative opportunity as a business model.
On the other hand, the separate but related industry of consumer probiotics and supplements is just as large as medical foods. Biohm already generates revenue as their core business is selling probiotic supplements to consumers. While this opportunity may
49 not seem like the immense opportunity that a medical foods or pharmaceutical company would provide, this model is already established within the culture of Biohm. The market for probiotic supplements is fragmented with multiple, boutique providers, and a sustainable growth plan could be developed to scale what Biohm is currently providing.
The groundwork has already been established for Biohm to continue growing as a probiotic and consumer lifestyle company.
Turning to the at-home testing kit and data platform, Biohm has a wealth of data from consumer test kits that can provide the basis for a novel discovery platform. The market is fresh for innovation in this industry as we have seen uBiome close due to fraud investigations by the FBI and SEC. In fact, most other competitors in this market have moved away from pursuing insurance reimbursement due to expensive pilot studies and the heavy regulatory load of reimbursement.
Rather, data platforms show the most promise in this area for successful development. Bolstered by the data from consumer testing, preclinical assessment of new modulatory targets as in silico experimentation is the model that many companies like
CosmosID or In Sitro are now following. These companies develop extensive machine learning algorithms to interrogate those deep gene interactions and molecular interaction pathways to discover new metabolic compounds. These platforms are then licensed as therapeutics to be developed by large pharmaceutical companies who have the motivation and capital to develop novel drugs.
While it’s not “the hot topic” or “sexy” to be an in silico CRO, this pathway could provide that better segmentation and cohort study selection that remains a major issue in the microbiome industry. However, I worry that in order to capitalize on this kind of
50 radical disruption Biohm may not have the necessary staff. Two full-time computer scientists or bioinformaticians would be necessary to begin forming a machine learning platform around Biohm’s vast mycobiome dataset. I see that this could be similar to some competitor’s business models such as CosmosID, Human Longevity, Finch Therapeutics, and Freenome. These companies provide a data platform that form the foundations of partnerships with larger pharma companies. The company would need a post-doc or PhD- level technical coordinator to translate discovery into commercial opportunities.
My final thoughts surround new potential high value areas that are underrepresented or neglected by industry. Two examples are: (1) diagnosing and treating parasite-related pathologies that are underexplored within the microbiome research space, as well as a target for industry-side technology development; and (2) digital health therapeutics developed from a data platform. The connection between the gut microbiome and parasite infection is well-documented. However, innovation and the development of truly radically innovative technologies have not followed this research.
Parasite and viral infection affect nearly every person on Earth yet developing accessible technology and healthcare around these area (based in microbiome study) have not met the demand.
Digital health therapeutics are rapidly become accepted and are capital non- intensive to develop. These are solutions that are potentially accessible to all people and all markets, yet only one company has explored this space in connection to the microbiome. Vivante Health is a new competitor in the microbiome space. While not a traditional therapeutic company, Vivante is exploring the space of digital therapeutics.
This is a relatively new space and would require its own exploration beyond the scope of
51 this thesis. Vivante is developing a comprehensive digital health platform to monitor food sensitivities and triggers in patients with GI disease like Crohn’s, IBD, and ulcerative colitis.
My final thoughts are regarding the team and management of Biohm. Dr.
Ghannoum is not directly involved either as a CSO or executive, and I worry that the direction of his intellectual property and wealth of expertise may go unleveraged in pursuing any new vertical. His IP is held by another pharmaceutical company, and the potential to work as a CRO seems to be a non-starter. This could be because he has another LLC called Next Trillion Ventures that provides CRO work in the mycobiome and fungicide space. I do not know the full relationship here, but Biohm’s current executive management seems either unaware of this competing interest or unwilling to see how it conflicts with their interest in one of the explored verticals.
There is a lot of potential with Biohm, and with the right team and strategic capital, it could potentially be a highly innovative company that solves real problems for people. I will end with one quote from The Innovators Solution that I think is especially relevant here:
“A dearth of good ideas is rarely the core problem in a company that struggles to launch exciting new-growth businesses. The problem is in the shaping process.
Potentially innovative new ideas seem inexorably to be recast into attempts to make existing customers still happier…Managers who understand these forces and learn to harness them in making key decisions will develop successful new-growth businesses much more consistently than historically has seemed possible.” 60
60 Christensen, Clayton M. The Innovator's Solution: Creating and Sustaining Successful Growth. Boston, Massachusetts: Harvard Business Review Press, 2013. Pg. 11. 52
Works Cited
1. Silicon Valley Bank. 2017 Annual Report. San Francisco, CA: SVB, 2017. Accessed February 15, 2020. 2. Cox, Michael J, Cookson, William OCM, Moffatt, Miriam F. “Sequencing the human microbiome in health and disease.” Human Molecular Genetics 2013; 22: R88-R94. 3. The Human Microbiome Jumpstart Reference Strains Consortium. “A catalog of reference genomes from the human microbiome.” Science 2010; 328: 994-999. 4. Human Microbiome Project / Program Initiatives. The NIH Common Fund. Retrieved March 12, 2020. 5. NIH Human Microbiome Portfolio Analysis Team. “A review of 10 years of human microbiome research activities at the US National Institutes of Health, Fiscal Years 2007-2016. Microbiome 2019; 7:31 6. Goodrich, Julia K., Di Rienzi, Sara C, et al. “Conducting a Microbiome Study.” Cell 2014; 158(2): 250-262. 7. Sinha, Rashmi, Abnet, Christian, et al. “The microbiome quality control project: baseline study design and future directions.” Genome Biology 2015; 16:276. 8. Ling, Yiwei, Watanabe, Yu, and Okuda, Shujiro. “The human gut microbiome is structured to optimize molecular interaction networks. Computational and Structural Biology Biotechnology Journal. 2019; 17:1040-1046. 9. Kurokawa K, Itoh T, Kuwahara T, Oshima K, Toh H, Toyoda A, et al. “Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes.” DNA Res 2007;14:169–81. 10. Levy R, Borenstein E. “Metabolic modeling of species interaction in the human microbiome elucidates community-level assembly rules.” Proceedings of the National Academy Sciences USA. 2013; 110:12804–9. 11. Browne, Hillary P, Forster, Samuel C, et al. “Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation.” Nature 2016; 533:543- 546. 12. Hugenholtz, Philip, Goebel, Brett M., Pace, Norman R. “Impact of culture- independent studies on the emerging phylogenetic view of bacterial diversity. Journal of Bacteriology 1998; 180(18): 4765-4774. 13. Cox, Michael J, Cookson, William OCM, Moffatt, Miriam F. “Sequencing the human microbiome in health and disease.” Human Molecular Genetics 2013; 22: R88-R94. 14. Thomas, Torsten, Gilbert, Jack, and Folker, Meyer. “Metagenomics – a guide from sampling to data analysis.” Microbial Informatics and Experimentation 2012; 2:3-12. 15. Blaxter, Mark, Mann, Jenna, Chapman Tom, et al. “Defining operational taxonomic units using DNA barcode data. Philosophical Transactions of the Royal Society 2005; 360: 1935-1943. 16. Meyer, Achim, Todt, Christaine, Mikkelsen, Nina T, and Bernhard, Lieb. “Fast evolving 18S rRNA sequences from Solenogastres (Mollusca) resist standard PCR amplification and give new insights into mollusk substitution rate heterogeneity.” BMC Evolutionary Biology 2010; 10:70.
53
17. Schoch, C.L., Seifert, K.A., Huhndorf, S., Robert, V., Spouge, J.L., Levesque, C.A., Chen, W., Bolchacova, E., Voigt, K., Crous, P.W., et al. "Nuclear Ribosomal Internal Transcribed Spacer (ITS) Region as a Universal DNA Barcode Marker for Fungi". PNAS. 109;16: 6241–6246. 18. Iliev ID, Funari VA, Taylor KD, Nguyen Q, Reyes CN, Strom SP, et al. “Interactions between commensal fungi and the C-type lectin receptor dectin-1 influencecolitis.” Science 2012;336:1314–7. 19. Hoffmann C, Dollive S, Grunberg S, Chen J, Li H, Wu GD, et al. “Archaea and fungi of the human gut microbiome: correlation with diet and bacterial residents.” PLoS ONE 2013; 8:e66019. 20. Hager, C. and Ghannoum, M. “The mycobiome: Role in health and disease, and as a potential probiotic target in gastrointestinal disease.” Digestive and Liver Disease 2017; 49:1171-1176. 21. Hager, Christopher L, Isham, Nancy, Schrom, Kory P. Chandra, Jyotsna, McCormick, Thomas, Miyagi, Masaru, Ghannoum, Mahmoud. “Effects of a novel probiotic combination on pathogenic bacterial-fungal polymicrobial biofilms.” American Society for Microbiology 2019 (2); e0038-19. 22. Baumgart, Daniel, Sandborn, William. “Inflammatory bowel disease: clinical aspects and established and evolving therapies. The Lancet 2007; 9573: 1641- 1657. 23. Rodriguez-Palacios, A, Harding, A, Menghini P, et al. “The artificial sweetener Splenda promotes gut protebacteria, dysbiosis, and myeloperoxidase reactivity in Crohn’s disease-like ileitis.” Inflammatory Bowel Disease 2018; 24(5): 1005- 1020. 24. Baumgart, Daniel, Sandborn, William. “Inflammatory bowel disease: clinical aspects and established and evolving therapies. The Lancet 2007; 9573: 1641- 1657. 25. FDA Medical Food Definition – Orphan Drug Act of 1988. Accessed February 25, 2020. 26. “Frequently Asked Questions About Medical Foods; Second Edition – A Guidance for Industry.” U.S. Department of Health and Human Services Food and Drug Administration Center for Food Safety and Applied Nutrition May 2016. Accessed Feb 10, 2020. 27. Kumar, Aneesh. “Probiotic Market.” BCC Market Research 2018. Report Code: FOD035F. 28. Sator RB. Efficacu of robiotics for the management of inflammatory bowel disease. Gastroenterol Heaptology. 2011; 7:606-8. 29. Phillips, Kathryn A, Deverka, Patricia A., et. al. Genetic Test Availability and Spending: Where Are We Now? Where Are We Going? Health Affairs 37, No. 5 (2018):710-716. 30. “Next-generation Sequencing: Emerging Clinical Applications and Markets. https://www-bccresearch-com.eu1.proxy.openathens.net/market- research/biotechnology/next-generation-sequencing-markets-report.html. Accessed March 3, 2020. 31. https://www.genome.gov/about-genomics/policy-issues/Genetic-Discrimination. Accessed February 19, 2020.
54
32. Proctor, Lita. “What’s next for the human microbiome?” Nature 569, 623-625 (2019). 33. Zaheer, Rahat, Noyes, Noelle, Ortega Polo, Rodrigo, et al. “Impact of sequencing depth on the characterization of the microbiome and resistome.” Scientific Reports 2018; 8:5890. 34. Meyer M, Kircher M. "Illumina sequencing library preparation for highly multiplexed target capture and sequencing". Cold Spring Harbor Protocols 2010: 5448.
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