SBIR-STTR Award

In this Phase I program we will establish the feasibility of a novel filter-paper whole blood sample based targeted sequencing assay based on the TempO-Seq® platform to measure DNA and RNA profiles as the basis for the development of a minimally invasive prognostic/diagnostic assay of Alzheimer’s Disease (AD). We will use our already functional whole transcriptome targeted gene expression assay to evaluate a set of whole blood samples from AD and normal donors provided by our collaborators, Dr. Allan Levey (Director, Alzheimer’s Disease Res. Center at Emory Univ., Chair, Dept. of Neurology) and Dr. Aaron Ritter (Cleveland Clinic, Lou Ruvo Center for Brain Health). We will utilize the resulting data to define a smaller targeted AD-specific gene subset, then redesign the probes for those genes to be compatible with the TempO-Seq DNA assay. This will allow us to generate a combined DNA/RNA assay capable of detecting both critical DNA genotypes (e.g. APOE alleles) and the diagnostic/prognostic gene expression RNA profile from dried blood spot samples, all in a single-well, addition-only assay, with no extraction, reverse transcription, or wash steps. Finally, we will combine the assay with a fill-in element, allowing mapping of significant stretches of genes which can harbor multiple unpredictable mutations (such as PSEN1/2 or APP), to create a “one-shot” assay capable of detecting all relevant genetic and transcriptomic biomarkers in one single step, all from small (1-3 mm2) areas of blood spots on filter paper. The functionality of the completed assay will be confirmed on the same set of normal/AD samples, allowing us to proceed to Phase II, where we will commercialize the assay and prove its predictive and diagnostic value. This novel assay will address the unmet need for a cost-effective, simple way to broadly screen at-risk populations, especially in hard-to-reach or otherwise underserved areas. Ability to derive data of such unprecedented depth from samples that are as non-invasive and simple to collect and ship holds promise to reshape AD detection and care. The proposed research program is responsive to the NIA/DBSR Special Interest Topic D.1. to “Develop multiple and reliable assays for limited blood-spot specimens for large surveys”, and Division of Aging Biology Topic J “Development of biomarkers for prognosis, diagnosis, or treatment monitoring of aging or aging-related diseases”, and DN Topic A “…development of minimally-invasive biomarkers that can be used for screening in the general populations... biomarkers that could serve as surrogate measures for disease progression in MCI, AD and ADRD…”. The outcome will be an assay that can be taken into Phase II to begin the development and validation of a prognostic/diagnostic screening assay for AD.

Public Health Relevance Statement:
Narrative Blood spots collected on filter paper represent an easy-to-collect, easy-to-store, and easy-to-ship sample type, but one that also presents significant technical challenges for transcriptomics, as extraction from filter paper tends to produce low RNA yield and quality. Having already developed a gene expression assay capable of detecting RNA levels directly on small samples of blood on filter paper, we now propose development of a targeted sequencing assay which is performed directly on the filter paper sample itself, and which will produce a full readout of gene expression biomarkers correlated with Alzheimer’s Disease detection and progression. Furthermore, we propose combining this assay with a readout of relevant DNA genotypes (such ApoE4, or presenilin mutants), for a fully one-shot, low-cost assay amenable to high throughput processing.

Project Terms:
Address; Adoption; Aging; Alleles; Alzheimer disease detection; Alzheimer's Disease; Alzheimer's disease related dementia; Alzheimer’s disease biomarker; apolipoprotein E-4; Area; base; Biological Assay; Biological Markers; Biology of Aging; biomarker development; Blood; blood filter; Blood specimen; brain health; Caring; Clinic; cohort; cost; cost effective; Data; design; detector; Development; Diagnosis; Diagnostic; diagnostic assay; diagnostic screening; Dideoxy Chain Termination DNA Sequencing; Disease; Disease Progression; disorder subtype; DNA; DNA purification; DNA sequencing; Early Diagnosis; Elements; Exons; Expression Profiling; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Mutation; gene panel; General Population; Genes; Genetic; Genetic Transcription; Genotype; high risk; innovation; interest; Measurement; Measures; minimally invasive; Monitor; mutant; Mutation; Neurology; novel; Nucleic Acids; Oligonucleotides; Outcome; outcome forecast; Paper; Patients; Phase; Population; Populations at Risk; Presenile Alzheimer Dementia; presenilin; presenilin-1; prevent; prognostic; programs; Research; response; Reverse Transcription; RNA; RNA Splicing; Sampling; screening; Ships; specific biomarkers; Specimen; Spottings; Stretching; success; Surveys; Symptoms; targeted sequencing; Testing; transcriptome; transcriptomics; Validation; Variant; Whole Blood

BioSpyder proposes to develop and validate a classifier test for Alzheimer's Disease (AD) that may alsoclassify other dementias. Biospyder will market this as an RUO, then pursue development as a definitive invitro diagnostic (IVD) test, addressing a major unmet need. The program is based on a very successful PhaseI. It was found that a classifier using whole blood from a finger-stick spotted on filter paper and assayed usingthe whole transcriptome TempO-Seq® gene expression assay (the WBFP TempO-Seq assay) producedsignatures for both AD and Parkinsons's Disease that permitted a classifier to be built that identified eachinitially with 87% accuracy. After retraining with additional AD samples, the WBFP TempO-Seq AD classifieridentified 100% of an independent testing cohort of AD samples correctly, and identified immune cell functionalpathways underlying the signature. AD is the most common form of dementia, with the risk of developing AD 1in 5 for women, 1 in 10 for men. Mild cognitive impairment (MCI) can be noticed years before those patientscan be diagnosed with AD or another dementia, years of uncertainty, no treatment, lost time to prepare for afuture with AD. That diagnosis requires cognitive testing administered by a neurologist, with in some casesconfirmation by a -amyloid PET scan. Biomarker immunoassays recently cleared under the BreakthroughDevice designation are not definitive and require cognitive testing or a -amyloid PET scan for diagnosis, andrequire drawing blood or collecting cerebral spinal fluid. Thus, the still unmet need for a definitive IVD. In thisPhase II we will validate the performance of the WBFP TempO-Seq assay to classify AD patients, ability toclassify patients with other dementias, establish how early after presenting with MCI patients can be identifiedwith AD, correlate the WBFP TempO-Seq assay classification to -amyloid PET scans and immunoassay,implement/validate performance of a focused WBFP TempO-Seq AD or pan-dementia test, and prepare thematerials for, and meet with, the FDA in a pre-IDE meeting to discuss the studies required for an application forclearance as an IVD. The WBFP TempO-Seq whole transcriptome, focused, AD and focused pan-dementiatests will be marketed for RUO use to identify novel therapeutic targets, characterize the progression from MCIto AD, facilitate drug discovery efforts and selection/classification of patients, and potentially for developmentas companion diagnostic tests. If the WBFP TempO-Seq AD or pan-dementia tests are FDA cleared, they willbe transformative, not just by providing a definitive diagnosis of AD or other dementias, but potentially byproviding that diagnosis before a diagnosis by cognitive testing can be made, and as importantly, by enablingself-collection of samples to address health disparities in families and populations that do not have affordableor easy access to a neurologist. If providing early, definitive diagnosis, any clinic or physician (or familymembers) can evaluate persons for AD and potentially other dementias, broadening the network able toprovide diagnosis and focusing the role of the neurologist onto therapy and care.

Public Health Relevance Statement:
NARRATIVE Phase I data demonstrates the feasibility of producing a gene expression-based assay for the highly accurate classification of patients with Alzheimer's Disease and other dementias using a sample of blood spotted on filter paper which is easy to collect in the doctor's office, clinic, or even by self-collection. The validation of this assay in Phase II with multiple cohorts of samples, correlation to FDA-approved tests, and use to elucidate potential novel therapeutic targets and the launch of RUO whole transcriptome, Alzheimer's, and pan- dementias tests will be transformative for the field of neurodegenerative diseases at the levels of basic research, translational research, and drug discovery and development, as will the subsequent development into a definitive Alzheimer's or pan-dementia diagnostic assay. By enabling self-collection, this test may also reduce health disparities and transform the diagnosis of Alzheimer's Disease and other dementias to permit neurologists to focus on confirmation and treatment rather than diagnosis.

Project Terms: