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We previously reported the in vitro selection and characterization of a DNA aptamer capable of stimulating remyelination in a mouse model of multiple sclerosis. This aptamer was selected for its ability to bind to suspensions of crude murine myelin in vitro. Our initial studies in vitro and in vivo involved a 40-nucleotide derivative (LJM-3064) of the original 100-nucleotide aptamer. LJM-3064 retained robust myelin-binding properties. Structural characterization of LJM-3064 revealed that the guanosine-rich 5' half of the sequence forms different G-quadruplex-type structures that are variably stable in the presence of physiologically relevant ions. We hypothesized that this structured domain is sufficient for myelin binding. In this study, we confirm that a 20-nucleotide DNA, corresponding to the 5' half of LJM-3064, retains myelin-binding properties. We then optimize this minimal myelin-binding aptamer via systematic evolution of ligands by exponential enrichment after sparse rerandomization. We report a sequence variant (LJM-5708) of the 20-nucleotide myelin-binding aptamer with enhanced myelin-binding properties and the ability to bind cultured human oligodendroglioma cells in vitro, providing the first evidence of cross-species reactivity of this myelin-binding aptamer. As our formulation of DNA aptamers for in vivo remyelination therapy involves conjugation to streptavidin, we verified that the myelin-binding properties of LJM-5708 were retained in conjugates to avidin, streptavidin, and neutravidin. DNA aptamer LJM-5708 is a lead for further preclinical development of remyelinating aptamer technologies.
DNA aptamer oligonucleotides and their protein conjugates show promise as therapeutics in animal models of diseases such as multiple sclerosis. These molecules are large and highly charged, raising questions about their biodistribution and pharmacokinetics in mammals. Here we exploit the power of quantitative polymerase chain reaction to accurately quantitate the tissue distribution of 40-nucleotide DNA aptamers and their streptavidin conjugates after intraperitoneal injection in mice. We show remarkably rapid distribution to peripheral tissues including the central nervous system. Modeling of tissue distribution data reveals the importance of DNA aptamer sequence, 3' modification, and protein conjugation in enhancing tissue exposure. These data help to interpret the previously observed effectiveness of aptamer conjugates, as opposed to free aptamers, in stimulating central nervous system remyelination in a mouse model of multiple sclerosis.
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