Distribution of phosphodiester and phosphorothioate oligonucleotides in rat brain after intraventricular and intrahippocampal administration determined by in situ hybridization.

The distribution and stability of exogenously administered oligonucleotides (oligos) are important variables determining the potential utility of antisense oligos as agents for modifying gene expression within a given brain region in vivo. In the present study, phosphodiester (PO) and phosphorothioate (PS) oligos antisense with respect to a recently cloned rat hsp70 sequence were localized in rat brain following intraventricular and intrahippocampal administration using an in situ hybridization detection method. Unlabeled PO and PS oligos were dissolved in artificial cerebrospinal fluid and infused under stereotaxic control using a syringe pump. At various intervals after administration frozen brain sections were collected on gelatin-coated slides and hybridized with 35S-labeled probe consisting of the corresponding phosphodiester sense sequence. After intraventricular administration the unmodified PO oligo exhibited a limited and strictly periventricular distribution. In contrast the PS oligo showed significant penetration into and accumulation within brain, with extensive uptake in ipsilateral striatum and dorsal hippocampus, as well as in midline periventricular structures. Both oligos remained detectable for at least two days after administration. Following intrahippocampal injection the PO oligo was rapidly lost from the injection site, with detectable signal persisting only along the hippocampal fissure at 24 h. The PS oligo exhibited a more diffuse initial distribution as well as greater stability. While there was no indication of specific accumulation in the major hippocampal neuron layers through 24 h, there was some indication of selective localization in neuronal soma by 48 h. These results confirm that the relative instability of unmodified oligos may severely limit their utility as antisense reagents in brain in vivo. While PS oligos show more widespread distribution than PO oligos after intraventricular infusion, even these do not detectably accumulate in cortex and other structures without immediate access to the ventricular space under the dosing conditions employed here. The hybridization approach used in these studies should prove to be of general use in verifying the targeting of specific brain structures with antisense oligos by various routes of administration.