Gray-Edwards, H. | Search bioRxiv

bioRxiv2021

Quantifying and Mitigating Motor Phenotypes Induced by Antisense Oligonucleotides in the Central Nervous System

Antisense oligonucleotides (ASOs) are emerging as a promising class of therapeutics for neurological diseases. When injected directly into the cerebrospinal fluid, ASOs distribute broadly across brain regions and exert long-lasting therapeutic effects. However, many phosphorothioate (PS)-modified gapmer ASOs show transient motor phenotypes when injected into the cerebrospinal fluid, ranging from reduced motor activity to ataxia or acute seizure-like phenotypes. The effect of sugar and phosphate modifications on these phenotypes has not previously been systematically studied. Using a behavioral scoring assay customized to reflect the timing and nature of these effects, we show that both sugar and phosphate modifications influence acute motor phenotypes. Among sugar analogues, PS-DNA induces the strongest motor phenotype while 2-substituted RNA modifications improve the tolerability of PS-ASOs. This helps explain why gapmer ASOs have been more challenging to develop clinically relative to steric blocker ASOs, which have a reduced tendency to induce these effects. Reducing the PS content of gapmer ASOs, which contain a stretch of PS-DNA, improves their toxicity profile, but in some cases also reduces their efficacy or duration of effect. Reducing PS content improved the acute tolerability of ASOs in both mice and sheep. We show that this acute toxicity is not mediated by the major nucleic acid sensing innate immune pathways. Formulating ASOs with calcium ions before injecting into the CNS further improved their tolerability, but through a mechanism at least partially distinct from the reduction of PS content. Overall, our work identifies and quantifies an understudied aspect of oligonucleotide toxicology in the CNS, explores its mechanism, and presents platform-level medicinal chemistry approaches that improve tolerability of this class of compounds.

pharmacology and toxicology↗

bioRxiv2020

PEA15 loss of function and defective cerebral development in the domestic cat

Cerebral cortical size and organization are critical features of neurodevelopment and human evolution, for which genetic investigation in model organisms can provide insight into developmental mechanisms and the causes of cerebral malformations. However, some abnormalities in cerebral cortical proliferation and folding are challenging to study in laboratory mice due to the absence of gyri and sulci in rodents. We report an autosomal recessive allele in domestic cats associated with impaired cerebral cortical expansion and folding, giving rise to a smooth, lissencephalic brain, and that appears to be caused by homozygosity for a frameshift in PEA15 (phosphoprotein expressed in astrocytes-15). Notably, previous studies of a Pea15 targeted mutation in mice did not reveal structural brain abnormalities. Affected cats, however, present with a non-progressive hypermetric gait and tremors, develop dissociative behavioral defects and aggression with age, and exhibit profound malformation of the cerebrum, with a 45% average decrease in overall brain weight, and reduction or absence of the ectosylvian, sylvian and anterior cingulate gyrus. Histologically, the cerebral cortical layers are disorganized, there is substantial loss of white matter in tracts such as the corona radiata and internal capsule, but the cerebellum is relatively spared. RNA-seq and immunohistochemical analysis reveal astrocytosis. Fibroblasts cultured from affected cats exhibit increased TNF-mediated apoptosis, and increased FGFb-induced proliferation, consistent with previous studies implicating PEA15 as an intracellular adapter protein, and suggesting an underlying pathophysiology in which increased death of neurons accompanied by increased proliferation of astrocytes gives rise to abnormal organization of neuronal layers and loss of white matter. Taken together, our work points to a new role for PEA15 in development of a complex cerebral cortex that is only apparent in gyrencephalic species. SummaryGyrification is the neurodevelopmental process in certain mammalian species during which the cerebral cortex expands and folds resulting in the classic wrinkled appearance of the brain. Abnormalities in this process underlie many congenital malformations of the brain. However, unlike many other human malformations, genetic insight into gyrification is not possible in laboratory mice because rodents have a lissencephalic or smooth cerebral cortex. We identified a mutation in domestic cats that likely causes failure of the cerebral cortex to expand and fold properly, and discovered that the mutation impairs production of a protein, PEA15 (phosphoprotein expressed in astrocytes-15), involved in intracellular signaling. Affected cats have profound abnormalities in brain development, with minimal changes in their superficial behavior and neurologic function. Additional studies of tissue and cultured cells from affected animals suggest a pathophysiologic mechanism in which increased death of neurons accompanied by increased cell division of astrocytes gives rise to abnormal organization of neuronal layers and loss of white matter. These results provide new insight into a developmental process that is unique to animals with gyrencephalic brains.

genetics↗