Long-Wu Zhou

Antisense RNA gene therapy for studying and modulating biological processes

Abstract. Agents that produce their effects through an antisense mechanism offer the possibility of developing highly specific alternatives to traditional pharmacological antagonists, thereby providing a novel class of therapeutic agents, ones which act at the level of gene expression. Among the antisense compounds, antisense RNA produced intracellularly by an expression vector has been used extensively in the past several years. This review considers the advantages of the antisense RNA approach over the use of antisense oligodeoxynucleotides, the different means by which one may deliver and produce antisense RNA inside cells, and the experimental criteria one should use to ascertain whether the antisense RNA is acting through a true antisense mechanism. Its major emphasis is on exploring the potential therapeutic use of antisense RNA in several areas of medicine. For example, in the field of oncology antisense RNA has been used to inhibit several different target proteins, such as growth factors, growth factor receptors, proteins responsible for the invasive potential of tumor cells and proteins directly involved in cell cycle progression. In particular, a detailed discussion is presented on the possibility of selectively inhibiting the growth of tumor cells by using antisense RNA expression vectors directed to the individual calmodulin transcripts. Detailed consideration is also provided on the development and potential therapeutic applications of antisense RNA vectors targeted to the D2 dopamine receptor subtype. Studies are also summarized in which antisense RNA has been used to develop more effective therapies for infections with certain viruses such as the human immunodeficiency virus and the virus of hepatitis B, and data are reviewed suggesting new approaches to reduce elevated blood pressure using antisense RNA directed to proteins and receptors from the renin-angiotensin system. Finally, we outline some of the problems which the studies so far have yielded and some outstanding questions which remain to be answered in order to develop further antisense RNA vectors as therapeutic agents.

Developmental and age-related changes in the D2 dopamine receptor mRNA subtypes in rat brain

The influence of ontogeny and aging on the D2 dopamine receptor mRNA in rat brain were examined using in situ hybridization histochemistry and Northern analysis utilizing oligonucleotide probes complementary to the different D2 mRNA subtypes. At birth, there was a high level of D2 dopamine receptor mRNA in corpus striatum relative to that found in the cerebral cortex and other brain areas. The hybridization signal of striatum (using a probe that hybridizes to both the D2A and D2B mRNA) increased during the first two postnatal weeks, reached a peak at day 16, then declined slightly. The D2A mRNA showed a similar distribution and developmental pattern. Intracisternal injection of 6-hydroxydopamine into neonates did not significantly alter the increase of the D2 dopamine receptor mRNAs, suggesting that neuronal input does not influence the ontogenetic development of this mRNA. In striatum, olfactory tubercule and inferior colliculus, the D2A mRNA declined between 3 and 24 months of age. By contrast, there was an age-related increase in the D2A mRNA in the anterior and intermediate lobes of the pituitary. The mRNA for the D2B dopamine receptor showed very low but nevertheless detectable levels in striatum, olfactory tubercule and pituitary. Like with the D2A mRNA, in 24-month-old rats the D2B mRNA declined in striatum and olfactory tubercule and increased in pituitary. These results show that there are differential tissue-related changes in the mRNAs for the D2 dopamine receptor during both development and aging.

Antisense strategies in dopamine receptor pharmacology

Recent advances in molecular biology have provided pharmacologists the opportunity of developing an entirely new type of agent for studying and treating a variety of biological disorders. These agents, termed antisense oligodeoxynucleotides, have as their target the messenger RNAs encoding specific proteins. They act by binding to selected portions of these mRNAs through complimentary interactions and thereby prevent the synthesis of these proteins. These novel pharmacological tools have the promise of being easier to design and being more selective and predictable in their actions. In addition, insofar as agents targeted to receptors for neurotransmitters are concerned, unlike the classical pharmacological agents, these new compounds may not lead to the upregulation of the very receptors the drugs are designed to inhibit. The present review summarizes briefly studies on the effect of oligodeoxynucleotides antisense to the mRNAs encoding the various subtypes of the dopamine receptor. The studies show that oligodeoxynucleotides antisense to the D2 dopamine receptor when intracerebroventricularly into brains of rodents are rapidly taken up into the brain tissue, distributed to brain cells, and produce effects characteristic of highly selective D2 dopamine antagonists. The compounds also produced specific reductions in the levels of D2 dopamine receptor mRNA and D2 dopamine receptors. Similarly, injecting an antisense oligodeoxynucleotide targeted to the D1 dopamine receptor mRNA produces effects characteristic of D1 dopamine receptor antagonists. Other studies using these agents has produced evidence that there is a small pool of receptors that turn over very rapidly and which constitute the functional pool of these receptors. The evidence suggests further that antisense oligodeoxynucleotides inhibit the synthesis of this small functional pool of dopamine receptors, thereby providing an explanation of why there is often a discordance between changes in dopaminergic function and changes in the levels of dopamine receptors. Studies of antisense oligodeoxynucleotides targeted to the other subtypes of dopamine receptor may help reveal the biological roles that these and other newly discovered subtypes of neurotransmitter receptors have. They may also provide an entirely new and potentially more selective therapeutic regimen for altering the functions of these receptors.

Uptake and distribution of fluorescein-labeled D2 dopamine receptor antisense oligodeoxynucleotide in mouse brain.

To determine the uptake and distribution of oligodeoxynucleotides in brain, a 20-mer phosphorothioated oligodeoxynucleotide complementary to a portion of the D2 dopamine receptor mRNA was fluorescently labeled with fluorescein isothiocyanate (FITC) and injected into the lateral cerebral ventricles of mice. At various survival times after the injection, the brains were removed, fixed, sectioned, and viewed under a fluorescent microscope. The results showed that the oligodeoxynucleotide was rapidly taken up into the brain. Initially the label was relatively diffusely spread throughout the interstitial spaces of the brain, then became redistributed to the cellular compartments. The signal extended from those forebrain nuclei located immediately in contact with the ventricles, such as the corpus striatum, septum, and hippocampus, to areas further removed from the ventricles, such as the cerebral cortex, nucleus accumbens, and substantia nigra. When the FITC-labeled D2 antisense oligodeoxynucleotide was given once daily for 4 d, the signal intensity seen 24 h after the last injection appeared to be of greater intensity overall compared to that seen after a single injection. At early time-points the oligodeoxynucleotide signals appeared to be punctuated and were found in cell bodies as well as in proximal dendritic processes. However, not all cells were equally labeled, suggesting an uneven uptake and accumulation of the D2 antisense into the various cell types. At later time-points the fluorescent signal appeared granular; at these times the injected material was largely degraded. These studies show that a D2 dopamine receptor antisense oligodeoxynucleotide is rapidly taken up from cerebral ventricles into brain, becomes widely distributed throughout the brain tissue to areas far removed from direct contact with the ventricles, and appears to accumulate to a different extent in the various brain areas and cell types.

Cholinergic lesions of mouse striatum induced by AF64A alter D2 dopaminergic behavior and reduce D2 dopamine receptors and D2 dopamine receptor mRNA

To determine whether dopamine receptors are expressed in acetylcholine-containing neurons intrinsic to the striatum, and to study further the interactions between the dopaminergic and cholinergic systems, the irreversibly acting cholinergic neurotoxin, ethylcholine mustard aziridinium ion (AF64A), was injected unilaterally into the mouse corpus striatum, and rotational behavior induced by dopamine agonists and certain molecular events associated with this lesion were determined 7 days after lesioning. Brains were analyzed for D2 dopamine receptors by autoradiography, using [3H](-)sulpiride as a ligand, and for D2 dopamine receptor mRNA and glutamic acid decarboxylase mRNA by Northern blot analysis, using selective radiolabelled oligonucleotide probes. Choline uptake sites were determined by binding assays using [3H]hemicholinium-3, a selective choline reuptake blocker, as a ligand. Mice with intrastriatal injections of AF64A showed ipsilateral rotational responses to the non-selective dopamine agonist apomorphine and to the D2 dopamine agonists, pergolide and quinpirole, but not to the D1 dopamine agonist SKF 38393. This was associated with a significant reduction in D2 dopamine receptors in the ipsilateral striatum and a significant decrease in the amount of D2 dopamine receptor mRNA. That AF64A produced a relatively selective cholinergic deficit was supported by the evidence showing that AF64A lesions significantly reduced [3H]hemicholinium-3 binding sites but did not alter glutamic acid decarboxylase (GAD) mRNA. Further, hemicholinium-3, prevented the AF64A-induced changes in rotational behavior. These results suggest that striatal cholinergic interneurons contain D2 dopamine receptors and express the D2 dopamine receptor gene, and that these interneurons are involved in dopamine-mediated rotational behavior.

INTRASTRIATAL ADMINISTRATION OF AN OLIGODEOXYNUCLEOTIDE ANTISENSE TO THE D2 DOPAMINE RECEPTOR mRNA INHIBITS D2 DOPAMINE RECEPTOR-MEDIATED BEHAVIOR AND D2 DOPAMINE RECEPTORS IN NORMAL MICE AND IN MICE LESIONED WITH 6-HYDROXYDOPAMINE

Previous studies have shown that the intracerebroventricular injection of antisense oligodeoxynucleotides targeted to the mRNAs encoding the different subtypes of dopamine receptors inhibited behaviors mediated by these receptors. The present studies were designed to determine whether such antisense oligodeoxynucleotides could produce similar effects when injected into a discrete brain area. A D2 dopamine receptor antisense oligodeoxynucleotide (D2 antisense) was repeatedly injected into one corpus striatum of either normal mice or mice with unilateral lesions of the striatum induced by 6-hydroxydopamine. In the latter, intrastriatal injection of D2 antisense blocked the contralateral rotational behavior induced by the parenteral administration of the D2 dopamine receptor agonist quinpirole. The inhibitory effect of D2 antisense was dose- and time-related and was reversed upon cessation of D2 antisense treatment. This inhibitory effect was also selective in that D2 antisense treatment inhibited the rotational behavior induced by quinpirole but not that induced by the D1 dopamine receptor agonist SKF 38393 or by the muscarinic cholinergic agonist oxotremorine. Following repeated intrastriatal injections of D2 antisense into normal mice, parenteral administration of quinpirole caused rotational behavior ipsilateral to the side in which the D2 antisense was injected. No such rotational behavior was seen when similarly treated mice were challenged with SKF 38393 or oxotremorine. The quinpirole-induced rotational behavior in mice given intrastriatal injections of D2 antisense disappeared upon cessation of D2 antisense treatment. Repeated intrastriatal administration of D2 antisense also caused a significant reduction in the levels of D2, but not D1, dopamine receptors in striatum, as determined by receptor autoradiography. The levels of D2 dopamine receptors returned to normal upon cessation of D2 antisense treatment. Intrastriatal administration of an oligodeoxynucleotide with randomly placed nucleotides failed to alter the rotational response to quinpirole in either 6-hydroxydopamine-lesioned or normal mice and failed to alter the levels of D2 dopamine receptors in striatum. These results show that selective inhibition of behavioral responses mediated by D2 dopamine receptors can be achieved by the direct injection of a D2 antisense oligodeoxynucleotide into a discrete brain area.

D2 dopamine receptor antisense increases the activity and mRNA of tyrosine hydroxylase and aromatic l-amino acid decarboxylase in mouse brain

Abstract A D 2 dopamine receptor antisense oligodeoxynucleotide was administered intracerebrovetricularly to mice twice on the first day and then once daily for 2 days. The animals were killed 2 h after the last injection, and tyrosine hydroxylase and aromatic l -amino acid decarboxylase activities assayed in the corpus striatum, olfactory tubercle and frontal cortex. Tyrosine hydroxylase activity increased in corpus striatum but not in the olfactory tubercle or in the frontal cortex, while the activity of aromatic l -amino acid decarboxylase increased in all three brain regions. The treatment with the antisense oligomer also elevated the mRNA levels for the two enzymes in the midbrain. In contrast, repeated injection of a vehicle or a random oligomer was without effect on enzyme activity or mRNA D 2 antisense oligodeoxynucleotides appear to be selective tools to investigate the role of D 2 dopamine receptors in brain.

Continuous infusion of clozapine increases mu and delta opioid receptors and proenkephalin mRNA in mouse brain.

The biochemical mechanisms involved in the actions of the atypical antipsychotic clozapine are still unclear. Because elevated levels of enkephalin in certain areas of the central nervous system may be necessary for antipsychotic activity, we have examined the effect of clozapine on certain receptors and mRNA transcripts involved in the opioid peptidergic system. Clozapine was infused continuously into mice for 21 days and the density of mu and delta opioid receptors was measured in the brains by quantitative receptor autoradiography, and the level of proenkephalin mRNA and dopamine D1 and D2 receptor mRNA were measured by in situ hybridization histochemistry. The results showed that continuous infusion of clozapine increased the density of D1 but not D2 receptors. However, it failed to alter the levels of either D1 or D2 dopamine receptor mRNA. By contrast, clozapine increased the density of mu and delta opioid receptors and increased the levels of proenkephalin mRNA. These results indicate that continuous treatment with clozapine increases opioid peptidergic activity in mouse brain and suggest that alteration of peptidergic activity as well as alteration of dopaminergic activity may be involved in its antipsychotic action.

Continuously infusing quinpirole decreases Ca2+/Calmodulin-dependent phosphorylation in mouse striatum

Continuously administering the D2 dopamine agonist quinpirole to mice for 6 days produces an initial stereotypy that is reduced by 2 h and is absent from 3 h to the 6 day duration of the infusion. In an attempt to determine the biochemical correlates for this down-regulation of stereotypic behavior, the effects of continuously administering quinpirole on a number of biochemical parameters were measured in mouse corpus striatum. After 6 days of infusion with quinpirole, the striata were analyzed for D1 and D2 dopamine receptors, for the activities of several protein phosphorylation reactions which are thought to be involved in receptor activity and for the levels of calmodulin-binding proteins. Quinpirole decreased the D2 receptors in striatum and produced a small but statistically non-significant increase in D1 receptors, resulting in a significant increase in the ratio of D1 to D2 dopamine receptors. An examination of the effects of quinpirole on protein phosphorylation systems showed that the agonist failed to alter the activity of cyclic AMP-dependent protein kinase or protein kinase C but significantly decreased the Ca2+/calmodulin-dependent phosphorylation of striatal membranes. However, this decrease in Ca2+/calmodulin-dependent phosphorylation was not associated with changes in the levels of calmodulin-binding proteins. The results suggest that behavioral down-regulation following the continuous administration of a D2 dopamine agonist is associated with at least two biochemical events: a down-regulation of D2 dopamine receptors and a decrease of Ca2+/calmodulin-dependent phosphorylation of striatal membranes.

Triazolam blocks the initial rotational effects of quinpirole but permits the later developing reduction of dopamine D2-mediated rotational behavior and dopamine D2 receptors

Continuous infusion of the dopamine D2 receptor agonist quinpirole into mice with unilateral striatal 6-hydroxydopamine lesions initially produces a supersensitive rotational behavior. This is followed by reductions of dopamine D2-mediated behavior and dopamine D2 receptors. In this study we attempted to determine if it is possible to inhibit the acute increase in D2-mediated behavior while still allowing the reduction of D2-mediated behavioral responses and dopamine D2 receptors to occur. Mice were implanted with Alzet minipumps containing either quinpirole alone or quinpirole combined with the GABA receptor modulator triazolam or the dopamine D2 receptor antagonist sulpiride, and rotational behavior was monitored for the 6 days of infusion. The pumps were then removed, and D2 receptors in striatal membranes were determined. Triazolam completely blocked the initial rotational behavior normally induced by implanting quinpirole. However, the quinpirole-induced reduction of D2-mediated behavioral responses and D2 receptors still occurred. Continuous infusion of sulpiride also inhibited the rotational behavior produced by quinpirole, but it prevented the reduction of dopamine D2 receptors. We conclude that up-regulated dopamine receptors and dopaminergic behaviors can be reversed by the continuous administration of a dopamine receptor agonist and that this reversal can occur without producing an initial exacerbation of dopaminergic responses. These results suggest that this type of treatment regimen might be useful for treating clinical conditions associated with dopaminergic supersensitivity.