Unmodified Antisense and the BBB Are Modifications Necessary 41 Antisense Therapy in Pathophysiology Involving Increased Protein Production

Although many studies have investigated various modification strategies designed to enhance penetration of antisense across the BBB, few studies have considered if unmodified antisense can cross the BBB. It is possible that enzy-matically resistant analogs of unmodified AS-ODNs enter the CNS by binding to transporters/receptors present in the BBB for the import of endogenous substances. By analogy, viruses often use this method to enter cells. For example, human immunodeficiency virus-1 binds to the CD4 receptor to promote fusion and entry of the virus into T-cells, dendritic cells, and macrophages (44). Currently, studies examining the ability of unmodified antisense to cross the BBB have shown that two AS-ODN analogs are capable of permeation into the CNS: PNAs and phosphorothioate analogs (1,2). To illustrate the points above, we now consider studies that have focused on treatment of pathophysiology through the use of unmodified AS-ODN analogs to target the proteins or receptors associated with disease states such as schizophrenia, pain perception, Alzheimer's disease, and alcoholism.

4.1.1. Schizophrenia and Pain Perception

Neurotensin (NT) is an endogenous peptide that exerts potent effects on the CNS. Because NT is rapidly degraded in the blood, direct injection into the brain is necessary for NT to exert its CNS effects of hypothermia, antino-ciception (decreased perception of pain), modulation of dopamine (DA) neurotransmission, and increased locomotor activity. Because the pharmacological activity of NT is similar to that of known neuroleptic agents, NT agonists have implications in the treatment of schizophrenia.

Schizophrenia is a disorder that is often associated with increased levels of the neurotransmitter DA. Many of the drugs designed to treat schizophrenia are DA receptor agonists. Because administration of NT is associated with increased DA turnover, this peptide could provide a useful tool in the treatment of schizophrenia. Peripheral administration of NT will not produce the effects seen on direct administration of NT into the CNS (45). Peripheral administration of NT analogs has been shown to induce the CNS responses associated with the direct administration of NT into the brain (46,47). NT69L, an NT analog developed by Tyler et al. (47), has been shown to cross the BBB at levels sufficient to cause CNS activity, as assessed by hypothermic and antinociceptive effects. Like NT, the NT69L analog increases DA turnover rates, as demonstrated by increased DA metabolite (DOPAC) (48). The effects of NT are mediated through binding to its receptors, NTR-1 and NTR-2 (49,50). Both are G-pro-tein-coupled receptors, but they are distributed differentially throughout the brain (51). NT knockout animals would be useful in the elucidation of the specific roles of these receptors in the maintenance of critical DA concentrations within the brain. However, these animals may produce misleading results. Some knockout animals are not compatible with life, and others may develop mechanisms to compensate for the lack of the deleted protein. Antisense technology would provide a useful tool to better understand the interaction between the NT and DA systems in the CNS.

A study by Tyler et al. (47) investigated an unmodified 12mer PNA antisense directed at the NT receptor NTR-1. The normal physiological response to an increase in NT is hypothermia and antinociception. Using a gel-shift assay, Tyler et al. (2) demonstrated that the unmodified PNA could cross the BBB and affect CNS function after a single ip injection in rats. The effects of NT microinjected into the brain were inhibited within 24 h of ip administration of the PNA directed at NTR-1. The effects of the antisense PNA were completely reversible. The normal physiological response to NT returned 48 h postinjection. Radioligand binding assays showed that administration of antisense PNA was accompanied by a reduction in receptor sites; however, there was no change in the levels of mRNA, possibly because of the nature of inhibition caused by antisense PNAs. Unlike AS-ODN-RNA complexes, PNA-RNA complexes do not activate RNase H, so protein levels are reduced by sterically blocking translation machinery (35,52).

4.1.2. Alzheimer's Disease

Alzheimer's disease is characterized by the presence of amyloid plaques in the extracellular space of the CNS. The amyloid plaques consist mainly of a 40- to 42-amino-acid peptide known as amyloid p protein (AP). These aggregates are toxic and could accumulate around blood vessels, leading to apoptosis of the vascular smooth muscle cells (53). Ap is a cleavage product of the amyloid precursor protein (APP). Increased brain levels of Ap in Alzheimer's patients could result from many factors including increased production of APP. Another possibility is altered cleavage of APP, resulting in the formation of a mutant form of Ap that is more likely to aggregate. Regardless of the cause, levels of Ap could be reduced with antisense therapy.

Kumar and colleagues (54,55) tested a series of P-ODNs directed against Ap with the above in mind. They tested the P-ODNs in a strain of mice that spontaneously overexpresses Ap peptide as it ages, the senescence accelerated mouse (SAMP8). By 12 mo of age, this strain demonstrates a twofold increase in the brain levels of Ap and severe learning and memory deficits (54,55). This increase is similar to that which occurs in patients with Alzheimer's disease. Kumar and colleagues (54,55) found that either antibody or a phosphorothioate antisense directed against Ap reversed the cognitive impairments present in mature SAMP8 mice after injection directly into the brain.

To determine whether the phosphorothioate antisense directed at Ap could cross the BBB, it was radioactively labeled with 32P and administered intravenously (1). Purification techniques using gel electrophoresis and autoradiography revealed that the AS-ODN was transported intact across the BBB. Because of its large molecular weight and poor lipid solubility (partition coefficient = log[-3.52]), passive transmembrane diffusion was an unlikely means of transport for the antisense. Penetration of this antisense into the CNS was inhibited by an excess of unlabeled antisense directed against Ap, thus demonstrating saturable transport. This system was named OTS-1 (1).

Transport of AS-ODN across the BBB was also verified behaviorally. Intravenous administration of the antisense against Ap reversed the learning and memory deficits normally seen in the mature SAMP8 mouse.

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