ABSTRACT
Intrathecal (IT) delivery and pharmacology of antisense oligonucleotides (ASOs) for the CNS have been successfully developed to treat spinal muscular atrophy. However, ASO pharmacokinetic (PK) and pharmacodynamic (PD) properties remain poorly understood in the IT compartment. We applied multimodal imaging techniques to elucidate the IT PK and PD of unlabeled, radioactively labeled, or fluorescently labeled ASOs targeting ubiquitously expressed or neuron-specific RNAs. Following lumbar IT bolus injection in rats, all ASOs spread rostrally along the neuraxis, adhered to meninges, and were partially cleared to peripheral lymph nodes and kidneys. Rapid association with the pia and arterial walls preceded passage of ASOs across the glia limitans, along arterial intramural basement membranes, and along white-matter axonal bundles. Several neuronal and glial cell types accumulated ASOs over time, with evidence of probable glial accumulation preceding neuronal uptake. IT doses of anti-GluR1 and anti-Gabra1 ASOs markedly reduced the mRNA and protein levels of their respective neurotransmitter receptor protein targets by 2 weeks and anti-Gabra1 ASOs also reduced binding of the GABAA receptor PET ligand 18F-flumazenil in the brain over 4 weeks. Our multimodal imaging approaches elucidate multiple transport routes underlying the CNS distribution, clearance, and efficacy of IT-dosed ASOs.
Subject(s)
Brain/metabolism , GABA-A Receptor Antagonists/pharmacokinetics , Muscular Atrophy, Spinal/drug therapy , Oligonucleotides, Antisense/pharmacokinetics , Animals , Arteries/diagnostic imaging , Arteries/metabolism , Brain/blood supply , Brain/cytology , Brain/diagnostic imaging , Flumazenil/administration & dosage , Flumazenil/analogs & derivatives , GABA-A Receptor Antagonists/administration & dosage , Gene Knockdown Techniques , Humans , Injections, Spinal , Intravital Microscopy , Male , Molecular Targeted Therapy/methods , Neuroglia/metabolism , Neurons/metabolism , Oligonucleotides, Antisense/administration & dosage , Pia Mater/diagnostic imaging , Pia Mater/metabolism , RNA, Messenger/analysis , RNA, Messenger/genetics , Rats , Receptors, AMPA/analysis , Receptors, AMPA/antagonists & inhibitors , Receptors, AMPA/genetics , Receptors, GABA-A/analysis , Receptors, GABA-A/genetics , Single Photon Emission Computed Tomography Computed Tomography , Spatio-Temporal Analysis , Thionucleotides/administration & dosage , Thionucleotides/pharmacokinetics , Tissue DistributionABSTRACT
Cellular traction forces are important quantitative measures in cell biology as they have provided much insight into cell behavior in contexts such as cellular migration, differentiation, and disease progression. However, the complex environment in vivo permits application of cell traction forces through multiple types of cell adhesion molecules. Currently available approaches to differentiate traction forces among multiple cell adhesion molecules are limited to specialized approaches to decouple cell-cell from cell-extracellular matrix (ECM) tractions. Here, we present a technique which uses indirect micropatterning onto a polyacrylamide gel to pattern multiple, spatially distinct fluorescently labeled ECM proteins, specifically gelatin and fibronectin (Fn), and confine the area to which cells can adhere. We found that cells interacting with both gelatin and Fn altered their traction forces significantly in comparison to cells on Fn-only substrates. This crosstalk interaction resulted in a decrease in overall traction forces on dual-patterned substrates as compared to cells on Fn-only substrates. This illustrates the unique need to study such interactions and demonstrates great potential in future studies in multi-ligand environments. Current micropatterning techniques on glass can easily be adapted to present other protein classes, such as cadherins, while maintaining control of adhesion spacing, cell spread area, and stiffness, each of which are important regulators of cell mechanobiology.
