This invention introduces a novel inducible transthyretin monomer trap that selectively binds and measures amyloidogenic TTR monomers. This assay offers a potential clinical tool for diagnosing transthyretin amyloidosis in plasma from patients.
Transthyretin amyloidosis (ATTR) is a chronic condition characterized by the accumulation of amyloid TTR fibrils in various organs, leading to cardiac and nervous system dysfunctions. TTR, a liver-produced plasma protein, naturally exists as a tetramer. However, genetic mutations or environmental factors can destabilize this structure, leading to monomeric forms prone to amyloid fibril formation. Current diagnostic methods lack the specificity to measure these amyloidogenic TTR monomers due to the prevalence of normal tetrameric TTR in circulation. As new TTR stabilizing treatments emerge, there is an urgent need for diagnostic tools that can accurately monitor these monomers to assess treatment efficacy and ATTR progression.
The synthetic mono-TTR trap is a pioneering reagent designed to selectively bind unstable TTR monomers in plasma. The assay capitalizes on the unique binding properties of the trap to differentiate between monomeric and tetrameric TTR, enabling the measurement of mono-TTR self-aggregation potential. The assay can be adapted for used with different TTR mutations (i.e. TTR V30M) and different detection methodologies, including fluorescence, luminescence, enzymatic, or radiolabeling. The technology provides a means to monitor disease progression and evaluate the effectiveness of TTR stabilization therapies. The implementation of this tool in clinical settings could significantly enhance prognostic capabilities and therapeutic evaluations for ATTR patients, potentially filling a critical gap in ATTR diagnostics.
In figure to the right: (A) Crystal structure of tetrameric TTR (PDB 5H0V). The 2 sets of antiparallel β-strands that form the β-sheets of DAGH and CBEF were marked in 1 of the subunits (upper-right). Biotin moieties (represented by spheres) were added to the N-termini of all TTR subunits. (B) Schematics of site-specific biotinylation by BirA and subsequent multimeric induction by SA. Full-length TTR variants (asterisks) of WT, V30M, and V122I were each fused with an N-terminus AviTag, which was biotinylated. In the presence of SA (lower left: follow arrow), 4 biotin-TTR monomers formed SA-TTR tetramers. In the absence of SA (right panel: follow arrow), soluble TTR formed a mixture of monomer (M), dimer (D), and tetramer (T), in which the broken circle represents the natural tetrameric fold of TTR. Following SA induction of these mixed TTR forms (Bottom right: follow arrow), larger TTR complexes were formed, jointed by monomer, dimer, and tetrameric TTRs (in box).