Technology and Product Development

Exosome Diagnostics is developing biofluid-based molecular diagnostic tests for personalized medicine applications. We are initially focused on commercializing high sensitivity/specificity RNA in vitro diagnostic assays for use in disease recurrence monitoring and as companion diagnostics for use with targeted molecular therapies. Because exosomes provide a protected source of highly enriched nucleic acids that can be harvested under standard clinical laboratory conditions from blood, urine and cerebrospinal fluid, we believe our exosome technology has a wide range of molecular diagnostic applications. We have accumulated extensive expertise and intellectual property related to the isolation of exosomes and other microvesicles from biofluids, and the extraction and analysis of their RNA and DNA contents.

The existence of exosomes and other microvesicles has been known for many years. Until recently, literature described microvesicles as primarily containing lipids and proteins. Researchers at Massachusetts General Hospital, who now make up the core of Exosome Diagnostic's development team, were able to demonstrate that microvesicles contain an abundance of nucleic acids, which given the appropriate methods, can be harvested with high quality yields and used diagnostically. Original findings conducted in blood exosomes of glioblastoma patients were published in Nature Cell Biology (December 2008). This publication was the first to show that mutations in the tumor could be detected in RNA extracted from microvesicles isolated from patient blood and that this discovery could be used diagnostically.

"Researchers … were able to demonstrate that microvesicles contain an abundance of nucleic acids, which … can be harvested with high quality yields and used diagnostically."

Biofluid microvesicles are particularly useful for differential nucleic acid profiling due to their presence in metastatic, non-metastatic, inflammatory and immune-response conditions, and as result of their direct release from solid tumors. Expression analysis using microarrays and ultra-deep sequencing in cancer and other indications reveals a large number of both coding and non-coding RNA biomarkers available for analysis. Depending on the biofluid medium and the nucleic acid target of interest, successive levels of enrichment can be achieved by applying various isolation technologies to increase sensitivity.

In recent years, researchers at Exosome Diagnostics have been investigating the optimum methods and conditions under which biofluid microvesicles may be captured, purified and treated to produce the level of enrichment required for direct RNA or cDNA analysis of target biomarkers using different analytical techniques. Using our findings from these studies, we are conducting a series of clinical studies through academic and industrial collaborations on both known and novel rare transcripts and gene expression in various disease states including oncology, diabetes and Alzheimer's disease.

We have developed several quality controls which we believe are necessary to pass a threshold for high quality nucleic acid preparation. Without these quality controls, it may not be possible to achieve the level of enrichment for target transcripts and/or expressions required for diagnostically reproducible results.

A number of different techniques can be applied to isolate microvesicles from biofluids. Exosome Diagnostic's primary technology is based on the use of filtration concentrators, which remove solvent and small molecule analytes while retaining and concentrating microvesicles as well as affinity purification based on microvesicle surface markers. This is followed by proprietary RNA extraction protocols to achieve high quality RNA preparations.

Depending on the diagnostic indication, the application and the transcripts in question, we analyze microvesicle RNA using a range of molecular biology analytical techniques. Qualitative real-time PCR is often applied for rare transcript variant detection, whereas high content techniques such as microarray analysis and massively parallel/next generation sequencing are applied for biomarker discovery applications.

One of the important features of microvesicle composition is the stability of nucleic acid content achieved during long periods of biofluid archiving in biobanks. We have shown that under many conditions, assuming proper storage and procedures, that there is little degradation in nucleic acid integrity in fresh vs. multi-year biobanked specimens.

"…the use of molecular diagnostic testing for cancer diagnosis, prognosis and treatment selection has greatly expanded driven by the need for more cost and treatment efficient applications of expensive therapies."

Because microvesicles carry with them nucleic acids from their cell-of-origin, we can achieve real-time access to molecular genetic information about cells in the body without direct access to the actual cells. Direct cellular biopsy may be difficult or otherwise unattainable.

Cancer is one of the primary focus areas of Exosome Diagnostics due to the growing importance of molecular diagnostics in patient stratification and drug resistance monitoring. As research continues to uncover more details about the molecular mechanisms behind cancer, more is also known about how to optimally treat a patient's disease based on the molecular makeup of his or her particular cancer. Over the past few years, this has led to a significant increase in the development of therapies specifically targeting the gene mutations involved in disease progression. In parallel, the use of molecular diagnostic testing for cancer diagnosis, prognosis and treatment selection has greatly expanded driven by the need for more cost and treatment efficient applications of expensive therapies. Current molecular diagnostics almost exclusively rely on assaying cancer cells from tissue biopsy by needle aspiration or surgical resection.

The ability to detect and genotype nucleic acids of tumor origin from a blood draw or a urine sample without the need for a biopsy or surgically resected tissue has a number of advantages. Frequently, fresh tissue is difficult or impossible to access, and archival tissue samples are often less relevant to the current state of the patient's disease. The ability to monitor biomarkers in real-time presents several opportunities including continuous monitoring of disease progression and recurrence, and monitoring the development of patient resistance over the course of therapy.

Exosome-enabled biofluid diagnostics are also potentially applicable to other diseases or medical conditions where genetic testing is beneficial, but where access to tissue is difficult or impossible. Examples include neurological diseases, muscular disease, kidney disease, diabetes and other metabolic diseases.

"…microvesicles arising from the body's immunologic response to the presence of a tumor may be an important contributor to a biomarker fingerprint in combination with microvesicles of tumor origin."

Since the shedding of exosomes and other microvesicles take place from many different cells in the body, all contribute to the complex mixture detectable in biofluids. In assays detecting mutations or transcripts that exclusively arise from cancer cells, it is important to isolate as many exosomes as possible to avoid disregarding any unknown or as of yet unidentified subpopulation. This holistic view is also important if gene signatures or biomarkers are assayed that have contributions from several cell types. In many cancer types, microvesicles arising from the body's immunologic response to the presence of a tumor may be an important contributor to a biomarker fingerprint in combination with microvesicles of tumor origin. In other applications where a sub-population of microvesicles is well characterized and known to contain the relevant targets, it can be beneficial specifically to isolate that subpopulation by affinity capture by antibodies against surface markers present on these microvesicles.

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