The development of an RT-qPCR Assay for early diagnosis of Alzheimer's Disease

Alzheimer's disease (AD) is the leading cause of dementia, affecting nearly 52 million people worldwide and 6.9 million people in US. The number of deaths from disease increased in the US by 141% between 2000 and 2021. Although one out of three seniors die from AD and other forms of dementia, currently, there is no cure for AD, and the disease poses a substantial economic burden. In 2024, AD and other dementias cost the US healthcare system $360 billion. By 2050, these costs could rise to as high as $1.1 trillion. Imaging of Aβ and Tau in the brain are proven to be early diagnostic biomarkers for AD, but high costs prevent their use as conventional markers. Peripheral blood (PB) has always been the source for biomarker discovery due to the ease of accessibility and low cost of repetitive sampling. Included in PB are several different immunological cells, such as T lymphocytes (T cells) and B lymphocytes (B cells). T cells are an essential part of the adaptive immune system and engage with antigens through their surface receptors (T cell receptors, or TCRs) to exert their function. TCR clonality or clonal expansion (loss the diversity) is one of the hallmarks of adaptive immune responses upon T cells exposure to antigens. Using previously collected peripheral blood mononuclear cell (PBMC) samples from age and gender- matched AD, MCI, and Normal Subjects at the Florida Alzheimer’s Research Center (FADRC), MegaNano Diagnostics’ preliminary research has demonstrated that the clonality of TCR variable β (Vβ) chain repertoires in CD8+ T cells increased in a disease-dependent manner compared to control samples (normal subject groups). Our preliminary results strongly demonstrate Vβ change in CD8+ T cells in AD and MCI samples. This change has been confirmed through study replication and resulted in the identification of disease-dependent Vβ clonality. Through gene scan, clonal sequencing, and RNA Next Generation Sequencing (NGS), strong evidence demonstrates that the Vβ clonality is Aβ-driven. The NGS data sets displayed several unique CDR3 sequences exclusively found in AD and MCI samples, thus confirming the existence of clonality-based biomarkers. With the STTR phase I grant, the final goal for the project phase is to have completed and validated an IVD RT-qPCR diagnostic kit prototype that uses a panel of unique Vβ sequences generated from long-term exposure to Aβ consistent with the pathology of AD. This will be accomplished through Aim 1: identifying and validating any additional Vβ sequences as biomarkers which enable differentiation of AD, MCI, and normal samples and generating a biomarker cluster-specific prototype RT-qPCR IVD kit. We will conduct NGS sequencing and TCR repertoire analysis from 90 samples consisting of 30 samples per clinical group received from the FADRC to isolate the cluster of sequence biomarkers. These biomarkers will be validated through immunoassays measuring cytokine expression and single-cell sequencing data generated from in vitro tissue culture T cells exposed to Aβ. After primers are designed, Aim 2 detection studies will be conducted with the prototype kit to predict disease categories from 180 randomly selected age and gender-matched human samples collected through FADRC, which, when compared to the clinical diagnosis, will determine the accuracy of the prototype. Once this is accomplished, we will be able to use immune modulation as an effective diagnostic and treatment tool against AD.