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New Blood-based Alzheimer's Biomarker Panel Uses Exosomal miRNA

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Exosomes

NEW YORK (GenomeWeb) – Researchers at the University of Melbourne have used unbiased next-generation deep sequencing of exosome-derived microRNAs to identify a signature of 16 blood-based biomarkers predictive of Alzheimer's disease.

In a report published last month in Molecular Psychiatry, sequencing was validated by reverse-transcription quantitative PCR, and the panel was shown to have sensitivity and specificity for predicting the disease of 87 percent and 77 percent, respectively. The research team also demonstrated that the miRNA signature correlated with amyloid neuroimaging, suggesting it could ultimately be used to screen patients with cognitive impairment prior to referral for more involved testing. 

Complicated and invasive tests, like PET and cerebrospinal fluid analyses, are expensive and difficult to access in some regions, Lesley Cheng, lead author on the study, noted in an interview. Blood testing could enable a patient with memory complaints or a family history of Alzheimer's to "not have to worry or spend the money to go through more invasive testing, when it could just be normal aging and nothing to worry about," Cheng said.

With this laudable aspiration of developing non-invasive assays, biodiscovery of blood-based biomarkers is a hot research topic. Critics, however, say that sequencing and PCR-based methods are prone to bias, particularly when evaluating signature nucleic acid levels in blood that may have no causal relationship with disease.

For example, issues of reproducibility in blood-based biomarker studies were noted in a recent white paper in Alzheimer's & Dementia, which emphasized standardization of pre-analytical steps as one way to foster cross-validation in research. A recent review in The Journal of Cellular and Molecular Medicine highlighted methodological challenges for analyzing miRNA biomarkers. And preparation of a cDNA library from miRNA remains a problematic step, as reverse transcription has recently been characterized as highly variable

Unlike many previous studies of miRNA biomarkers for Alzheimer's disease, the Melbourne group used purified circulating exosomes from blood serum. A type of microvesicle, miRNA-containing exosomes can be secreted by brain cells, are known to cross the blood-brain barrier, and are protected from RNases in the bloodstream, Cheng said. Thus, it is possible that they may prove to be a more reliable indicator of neurodegenerative disease than other circulating nucleic acids.

The miRNA-containing exosomes for the initial phase of the Melbourne study were derived from blood serum of 49 Alzheimer's patients and controls from the Australian Imaging, Biomarkers, and Lifestyle Flagship Study. There were 1,419 microRNAs detected in this training set, but only 17 showed a significant +/-1.2-fold change between the groups after bioinformatics and statistical analyses.

Standard exosome purification protocols require ultracentrifugation and gradient purification. For the Molecular Psychiatry study, the group evaluated a number of exosome purification kits, Cheng said, and found one from Norgen Biotek gave "the most similar profile to the ultracentrifuge." Using such a kit could ultimately shave about five hours from the protocol and allow labs without an ultracentrifuge to do the testing.

After the discovery phase, the signature was validated on a blinded reference cohort of 60 blood samples. Interestingly, the researchers also discovered eight individuals among the control samples whose miRNA signature and imaging results indicated they may progress to Alzheimer's in the future.

"We took a look at the PET imaging ... and the ones that had been [grouped] as Alzheimer's disease [using the miRNA panel] but who are currently cognitively healthy, with no memory complaints, also showed progression towards Alzheimer's disease according to their brain imaging," Cheng said. This group will now be followed to see if they begin to develop other symptoms of the disease.

Commercialization of miRNA biomarkers

In an interview earlier this year, miRNA researcher Muneesh Tewari said that even he has been "surprised at the pace, the number of different applications, the amount of research going on, and number of papers being published with respect to circulating or other extracellular miRNAs in diverse areas." 

Although Tewari's most recent study has shown most exosomes in blood may not carry substantial amounts of miRNA — an average of one copy of miRNA was detected per 121 exosomes across a range of sample types — exosome subpopulations may contain larger numbers and could potentially be purified based on markers signifying tissue of origin.

A number of commercial entities are currently developing miRNA-based Alzheimer's diagnostics. For instance, Rosetta Genomics recently inked a deal with an undisclosed global pharmaceutical firm to develop its test, while Diamir is pushing to make an miRNA biomarker panel of mild cognitive impairment available through a CLIA lab partner in 2015.  

Eugenia Wang, founder of Advanced Genomic Technology, also hopes to commercialize a plasma-based miRNA marker in the next three years. Experimental validation with large cohorts will be key to bringing this technology to patients, Wang said in an interview this week.

Advanced Genomic Technology recently received funding to establish the reproducibility of its miRNA assays, as well as to validate candidate biomarkers. A future diagnostic would ultimately be a PCR-based test, Wang said, and the company is now pursuing commercial partnerships and licensing for further development. They also recently published a new study of their circulating miRNA marker, mi-34c, which is obtained from blood plasma and, interestingly, was not among the panel of 16 markers in the Melbourne study.  

Commercialization is the ultimate goal for the Melbourne biomarker panel as well, which is now protected by a patent. "We have gotten ... inquiries and have a couple of interested parties from all sorts of areas and industries," Cheng said.

"We still have to do further validation [of the assay] across a larger set of people," she noted. "It would be great if it becomes a diagnostic, but at the moment I believe that it will be complimentary to other diagnostic tools."

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