The Future of Seed Amplification Assays and Clinical Trials

Abstract

Prion-like seeded misfolding of host proteins is the leading hypothesised cause of neurodegenerative diseases. The exploitation of the mechanism in the protein misfolding cyclic amplification (PMCA) and real-time quaking-induced conversion (RT-QuIC) assays have transformed prion disease research and diagnosis and have steadily become more widely used for research into other neurodegenerative disorders. Clinical trials in adult neurodegenerative diseases have been expensive, slow, and disappointing in terms of clinical benefits. There are various possible factors contributing to the failure to identify disease-modifying treatments for adult neurodegenerative diseases, some of which include: limited accuracy of antemortem clinical diagnosis resulting in the inclusion of patients with the “incorrect” pathology for the therapeutic; the role of co-pathologies in neurodegeneration rendering treatments targeting one pathology alone ineffective; treatment of the primary neurodegenerative process too late, after irreversible secondary processes of neurodegeneration have become established or neuronal loss is already extensive; and preclinical models used to develop treatments not accurately representing human disease. The use of seed amplification assays in clinical trials offers an opportunity to tackle these problems by sensitively detecting in vivo the proteopathic seeds thought to be central to the biology of neurodegenerative diseases, enabling improved diagnostic accuracy of the main pathology and co-pathologies, and very early intervention, particularly in patients at risk of monogenic forms of neurodegeneration. The possibility of quantifying proteopathic seed load, and its reduction by treatments, is an attractive pharmacodynamic biomarker in the preclinical and early clinical stages of drug development. Here we review some potential applications of seed amplification assays in clinical trials.

Nanoparticle-Enhanced RT-QuIC (Nano-QuIC) Diagnostic Assay for Misfolded Proteins

“…Biological samples, especially from clinical settings, are extremely complex, thus making diagnostics challenging. ,,,, Similar to inhibitors that negatively impact PCR performance, RT-QuIC is susceptible to inhibitors that hamper detection and/or the misfolding of protein substrates. ,,, Hypothesized RT-QuIC inhibitors include mucin family proteins and polar lipids , although a variety of inhibitory factors likely exist. Traditionally, RT-QuIC inhibitors are overcome by diluting tissue samples, and concordantly reaction-limiting compounds, until diagnostic sensitivity performs as expected for true-positive samples.…”

“…Together, these antibody-based assays are limited in the identification of early stage MPs and are primarily used on tissues collected post-mortem. Real-time quaking-induced conversion (RT-QuIC) has emerged as one of the most promising assays for early diagnosis of various MP-related neurodegenerative diseases. ,,,, Briefly, in RT-QuIC, small volumes of biological samples (e.g., lymph nodes, cerebral spinal fluid, skin, etc.) are seeded into a solution containing an excess of recombinant protein substrate (e.g., recombinant hamster prion protein [rHaPrP]) that, in the presence of a specific MP template, begins to misfold and form amyloids after a period of shaking and incubation.…”

“…For example, the assay has low kinetic efficiency, often taking up to 48 h to complete a standard test. Additionally, although it is more sensitive than conventional protein detection methods (i.e., ELISA and immunohistochemistry), natural inhibitors present within biological samples can interfere with reaction kinetics, producing false-negative results. ,,, To harness RT-QuIC’s potential for early neurodegenerative diseases diagnosis, it is imperative to develop novel methodologies that enhance assay performance, especially diagnostic sensitivity, and facilitate the transition of RT-QuIC from basic research to clinical application.…”

Automated microscopic measurement of fibrinaloid microclots and their degradation by nattokinase, the main natto protease

“…kinetically isolated from other macrostates) that they are even heritable [122,[145][146][147][148][149][150][151]. Homo-and hetero-polymerisation and their catalysis are then referred to, respectively, as (self-)'seeding' [140,[152][153][154][155][156][157][158][159][160][161][162][163][164][165][166] and 'cross-seeding' [153,[167][168][169][170][171][172][173][174].…”

Investigation of Inflammation in Lewy Body Dementia: A Systematic Scoping Review

“…Previous attempts at ELISA-based measurements of α-synuclein in CSF and blood have failed to produce reliable results [145]. Keen interest now surrounds the measurement of aggregated α-synuclein using real-time quaking-induced conversion (RTQuIC) seeding assays, which have been successfully demonstrated for the brain, CSF, skin, submandibular gland, gut, and mucosal tissue [146][147][148]. In clinical LBD studies, CSF α-synuclein measurement by RTQuIC detects cortical Lewy body disease [149] and predicts the conversion of prodromal idiopathic RBD to clinical Lewy body syndromes [150].…”