Tabard-Cossa, V. | bioRxiv Science

bioRxiv2021

Central and peripheral delivery of AAV9-SMN target different pathomechanisms in a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of the SMN1 gene. Although lower motor neurons are a primary target, there is evidence that peripheral organ defects contribute to SMA. Current SMA gene therapy uses a single, high titre intravenous bolus of AAV9-SMN resulting in impressive, yet limited amelioration of the clinical phenotype. However, risks of this treatment include liver toxicity. Intrathecal administration is under clinical trial but was interrupted due to safety concerns in a concomitant animal study. As there is no direct comparison between the different delivery strategies while avoiding high dose toxicity, we injected SMA mice with low dose scAAV9-cba-SMN either intravenously (IV) for peripheral SMN restoration or intracerebroventricularly (ICV) for CNS-focused SMN restoration. Here, IV injections restored SMN in peripheral tissues but not CNS, while ICV injections mildly increased SMN in the periphery and the CNS. Consequently, only ICV treatment rescued motor neuron degeneration. Surprisingly, both treatments resulted in an impressive rescue of survival, weight, motor function, and peripheral phenotypes including liver and pancreas pathology. Our work highlights independent contributions of peripheral organs to SMA pathology and suggests that treatments should not be restricted to the motor neuron. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/467795v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1e4e882org.highwire.dtl.DTLVardef@15ccf93org.highwire.dtl.DTLVardef@3fe393org.highwire.dtl.DTLVardef@75cc20_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation

Solid-state nanopores have been used extensively in biomolecular studies involving DNA and proteins. However, the interpretation of signals generated by the translocation of proteins or protein-DNA complexes remains challenging. Here, we investigate the behavior of monovalent streptavidin and the complex it forms with short biotinylated DNA over a range of nanopore sizes, salts and voltages. We describe a simple geometric model that is broadly applicable and employ it to explain observed variations in conductance blockage and dwell time with experimental conditions. The general approach developed here underscores the value of nanopore-based protein analysis and represents progress toward the interpretation of complex translocation signals. STATEMENT OF SIGNIFICANCENanopore sensing allows investigation of biomolecular structure in aqueous solution, including electricfield-induced changes in protein conformation. This nanopore-based study probes: (1) the tetramerdimer transition of streptavidin, observing the effects of increasing voltage with varying salt type and concentration; (2) the possible conformational states of DNA-streptavidin complexes when confined inside a pore. We describe a broadly applicable geometric approach that maps stepwise changes in the nanopore signal to real-time conformational transitions. These results represent progress toward accurate interpretation of nanopore signals generated by molecular complexes.

biophysics↗