A Method to Characterize In Vivo Binding of Morpholinos for Drug Design

Antisense nucleotide oligomer analogues such as morpholinos are actively investigated as drugs to treat or manage human diseases. Their binding affinity to the complementary target sequence is a crucial parameter. The in vitro measures such as association constant and melting temperature are useful but do not provide the exact information about the in vivo binding. This paper introduces a mouse model to which a circulating vehicle molecule carrying a target morpholino (t-morpholino of interest) is first injected followed by the effecting morpholino (e-morpholino). The 3-h blood concentration level of the e-morpholino is used as an indicator for its binding to the t-morpholino. Particular to this report, an amine-derivatized t-morpholino is conjugated to an NHS-activated lipid. Mixing the conjugate with albumin as a natural vehicle molecule forms a vehicle complex and the t-morpholino can circulate on the vehicle after injection. This method can be utilized for modulation of the in vivo binding affinity of e-morpholino oligomers to their targets and may be applied to other nucleotide oligomers as well.