Antisense inhibition of brain angiotensin in rat model of genetic hypertension

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Antisense inhibition of brain angiotensin in rat model of genetic hypertension
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Gyurko, Robert
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Gene Expression Regulation   ( mesh )
Oligonucleotides, Antisense -- genetics   ( mesh )
Oligonucleotides, Antisense -- pharmacology   ( mesh )
Angiotensin II   ( mesh )
Receptors, Angiotensin   ( mesh )
Hypertension   ( mesh )
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Thesis:
Thesis (Ph. D.)--University of Florida, 1995.
Bibliography:
Includes bibliographical references (leaves 113-130).
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Typescript.
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Vita.
Statement of Responsibility:
by Robert Gyurko.

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Full Text











ANTISENSE INHIBITION OF BRAIN ANGIOTENSIN IN RAT MODEL
OF GENETIC HYPERTENSION













by
ROBERT GYURKO














A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1995







I dedicate this work
to my wife and my parents













ACKNOWLEDGMENTS


To M.Ian Phillips I owe a tremendous debt of gratitude for his continuous support

throughout my training. He is not only an excellent scientist, but also a philosopher and

mentor, whose friendship I value greatly. Of the Phillips Laboratory, I thank Sara Galli,

Birgitta, Gayle, Leping, Tibor, Phillip, and my fellow students, Bing, Hongbin and Dan, for

their friendship and support. A friend and scientific fellow, Jon Bui, is also recognized for the

endless discussions on scientific and other matters. I am grateful to the faculty of the

Department of Physiology for giving me the opportunity to train in this highly competitive

atmosphere. I am particularly grateful to the members of my committee: Drs. Mohan Raizada,

Colin Sumners, Nick Muzyczka and Fulton Crews.













TABLE OF CONTENTS




ACKNOWLEDGMENTS ....................................... iii

AB STRA CT ................................................ v

CHAPTERS
1 INTRODUCTION .................................... 1
2 HYPOTHESIS AND SPECIFIC AIMS ...................... 4
H ypothesis ...................................... 4
Specific Aims ..................................... 4
3 REVIEW OF THE LITERATURE ......................... 6
Blood Pressure Regulation ........................... 6
The Renin-Angiotensin System ....................... 9
The Antisense Paradigm ............................. 27
Gene Therapy with Antisense Expression Vector .......... 41
Summary of the Literature ........................... 47
4 MATERIALS AND METHODS ........................... 49
Oligonucleotide Experiments ......................... 49
Expression Vector Experiments ...................... 54
5 RESULTS ............................................ 58
The Plan of the Study .............................. 58
Antisense Oligonucleotide Experiments in Vivo ........... 59
Antisense Oligonucleotide Experiments in Vitro ........... 73
Antisense Expression Vector Experiments ............... 79
Summary of the Results ............................. 90
6 CONCLUSIONS ....................................... 91
Antisense Oligonucleotide Design ..................... 91
Lowering Blood Pressure with Antisense Oligonucleotides... 92
Intracellular Mechanism of Action ...................... 101
Inhibition with Antisense Expression Vector ............. 104
7 IMPLICATIONS OF THE STUDY ......................... 109

REFERENCES ............................................... 113

BIOGRAPHICAL SKETCH ................................... 131








Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor
of Philosophy

ANTISENSE INHIBITION OF BRAIN ANGIOTENSIN IN RAT MODEL
OF GENETIC HYPERTENSION

By

Robert Gyurko

December 1995


Chairman: M. Ian Phillips
Major Department: Physiology


A novel method of inhibition of the synthesis of specific proteins, antisense inhibition,

was used to decrease the activity of the brain renin-angiotensin system (RAS). Since

overactivity of the brain RAS has been implicated in the development of essential hypertension,

the hypothesis was tested that attenuation of angiotensin type-1 receptor protein synthesis with

antisense DNA in the brain of spontaneously hypertensive rats can decrease high blood

pressure. Antisense oligonucleotides and antisense expression vectors were developed. The

ability of antisense oligonucleotides to lower blood pressure was tested in vivo, when antisense

oligonucleotides were injected intracerebroventricularly in spontaneously hypertensive rats. A

long-lasting, significant drop in blood pressure showed that antisense inhibition is applicable to

physiological processes. The functional downregulation of brain angiotensin receptors was

demonstrated by an attenuated drinking response to angiotensin A, whereas the decrease in

receptor number was shown by receptor binding and autoradiography. The cellular uptake was

followed using fluorescent labeled oligonucleotides, and the mechanism of action was







investigated with northern blots. In vitro experiments established that the antisense expression

vectors are capable of decreasing the synthesis of their target proteins. More experiments are

needed to develop an effective method for antisense delivery to the brain.












CHAPTER 1

INTRODUCTION

Angiotensin II (Angll) is one of the major physiological regulators of blood pressure.

Circulating AngI is generated in the blood from angiotensinogen through a cascade of

proteolytic events by the renin-angiotensin system (RAS). The RAS was initially described

only in the peripheral circulation, but neurochemical and functional data points to the existence

of an independent RAS in the brain as well as in other tissues (1,2). All components of the

RAS have been shown to be produced in the brain, and distinct functions have been linked to

brain AngIl: stimulation of vasopressin release, stimulation of the sympathetic nervous system

and induction of drinking behavior are the best recognized among them (3).

The brain RAS is implicated in the development of essential hypertension (4,5).

Increased levels of AnglI and AnglI receptors in the brain of spontaneously hypertensive rats

(SHRs) suggest an overactive brain RAS in the SHR (6,7,8,9). Inhibition of AngII in the brain

by a peptide antagonist saralasin decreases blood pressure in SHR (4). The angiotensin-

converting enzyme inhibitor captopril prevents the development of hypertension in SHR (5).

The choice ofantisense inhibition for further dissection of the brain RAS was appealing

primarily because of the specificity and the versatility of the approach and also because of its

potential clinical applicability. The unique specificity of antisense inhibition is due to base to

base matching of the oligonucleotide (ODN) to the target mRNA, allowing for differentiation

between e.g. subtypes of receptors with known nucleotide sequence. The versatility is intrinsic

1






2
to antisense technology: antisense oligonucleotides can be constructed to any part of any

mRNA with known nucleotide sequence. As with any new approach of course, there are yet

unknown factors that might influence the applicability: these include cellular uptake and

mechanism of action (10). The application of antisense ODNs as clinical drugs is particularly

appealing not only because it practically takes out the "guess-work" from drug design, but it

also uses natural components, nucleotides, as building blocks, therefore the chance of a toxic

reaction is decreased. In addition, antisense sequences can be cloned into expression vectors

which carry the potential for long-term depression of specific genes.

Blood pressure regulation is one of the fundamental physiological tasks in man. It is a

fine balance between providing the necessary blood perfusion to particular organs and

preventing the heart and blood vessels from undue strain. It has to ensure not only a

continuous delivery of blood but it has to accommodate to changing needs of all the organs

under different conditions.

Its importance is well reflected by epidemiological data: even slight elevations in blood

pressure shorten life expectancy, and there is an inverse linear relationship between systolic as

well as diastolic blood pressure and lifespan (11).

According to its importance, blood pressure is regulated by multiple organs including

the heart, blood vessels, kidney and the nervous system. While classic physiology describes the

hemodynamic, hormonal and neural regulation of blood pressure on the whole animal and

single organ level, our understanding of the molecular and cellular aspects of hemodynamic

control is still at its infancy.







3
The deficiency of classic techniques in answering some fundamental questions is

demonstrated by the fact that while some 100 years of hypertension research enriched us with a

formidable knowledge, the origin of 95% of the hypertensive cases is still unaccounted for, and

there is an ongoing debate whether researchers should look at the kidneys, the blood vessels or

the nervous system for the culprit.

The advent of genetics and molecular biology permits us to investigate a basic aspect of

this complex disease: these new techniques investigate the roles of individual genes and their

protein products in biological processes, thus narrowing the gap between physiology and its

underpinning, biochemistry. While there is no guarantee that molecular biological

investigations will answer all the questions about hypertension, its use will undoubtedly bring

us closer to understanding this and possibly other complex diseases. In this project, we

attempted to apply one of the new molecular biological tools, antisense molecules to

investigate the role of the brain renin-angiotensin system in hypertension.












CHAPTER 2

HYPOTHESIS AND SPECIFIC AIMS

Hypothesis

We hypothesize that antisense oligonucleotides injected into the brains of

spontaneously hypertensive rats can decrease the synthesis of angiotensin type-1 (AT-1)

receptor protein and decrease blood pressure. We also hypothesize that an expression vector

expressing antisense RNA delivered to the brain can decrease AT-1 synthesis and consequently

decrease high blood pressure.

Specific Aims

Specific Aim 1

Antisense oligonucleotides will be designed based on available information on the base

sequence of cloned AT-1 receptor cDNA. The ability of AT-1 antisense oligonucleotides to

modulate physiological responses, such as blood pressure and drinking in spontaneously

hypertensive rats will be tested.

Specific Aim 2

The effect of antisense oligonucleotides on AT-1 receptor levels in rat brain following

intracerebroventricular injection will be tested using radioligand binding on brain slices or brain

membrane homogenates.








Specific Aim 3

The intracellular mechanism of action of antisense oligonucleotides will be investigated.

The oligonucleotides will be tested along with their controls in cultured cells for their

efficiency in inhibiting AT-1 receptor protein synthesis. The cellular uptake of fluorescent

labeled oligonucleotides will be followed with confocal microscopy, and the fate of the target

mRNA will be investigated with northern blot.

Specific Aim 4

The possibility of long-term delivery of antisense molecules will be explored with and

adeno-associated virus (AAV)-derived antisense expression vector. A portion of the AT-1

receptor cDNA will be cloned into the expression vector in the antisense direction. This

recombinant molecule will be tested in vitro for its ability to inhibit AT-1 receptor synthesis,

and in vivo whether it can influence hypertension when injected in the brains of SHRs.












CHAPTER 3

REVIEW OF THE LITERATURE

Blood Pressure Regulation

Our current understanding of blood pressure regulation states that two main factors

determine blood pressure: cardiac output and vascular resistance (12). Cardiac output in a

healthy heart is determined by the venous return, which, in the long run, is a function of the

circulating blood volume. Thus, the more blood is circulating, or the higher is the vascular

resistance, the higher is the blood pressure.

Regulation of Blood Volume

Blood volume is mainly regulated by the kidneys as well as by the brain. The

juxtaglomerular cells of the kidney release renin when the blood pressure decreases in the

afferent arteriole or the sodium concentration is reduced in the tubular fluid. Renin cleaves the

decapeptide angiotensin I from angiotensinogen in the blood, which is converted to angiotensin

II (AngDI) by angiotensin converting enzyme (ACE) in the lungs.

Circulating AnglI regulates blood volume by stimulating aldosterone secretion in the

zona glomerulosa of the adrenal cortex, which stimulates sodium reabsorption in the distal

tubule. Sodium retention by the kidney results in more efficient water reabsorption. Thus a

decrease in blood volume elicits a cascade of events which improves water conservation.

The brain, on the other hand, has its own effector to restore fluid homeostasis.

Vasopressin, also known as antidiuretic hormone, is synthesized by neurons in the supraoptic

6







7
and the paraventricular nuclei of the hypothalamus, and released from the posterior pituitary in

response to increased plasma osmolarity as well as decreased blood pressure. Slight increases

in plasma vasopressin vastly improves water permeability of the collecting ducts of the kidney,

thus promoting water conservation.

Although it does not directly influence the total blood volume, the heart determines the

blood volume on the arterial side of the circulation, thus playing an important role in

determining the blood pressure. Its role can be demonstrated upon syncope or heart stop,

when the blood pressure decreases to the mean arterial filling pressure (about 7 mmHg), which

is the sum of the effects of blood volume and vascular tension only, without heart function.

Regulation of Peripheral Resistance

Regulation of the vascular resistance involves some of the hormones responsible for

fluid homeostasis, and also other humoral and neural actions. AngI and vasopressin, discussed

in the earlier paragraph as water-conserving hormones, are also the two most potent

vasoconstrictors of the body. AngI binds to its type-1 receptors on the vascular smooth

muscle cells and increases intracellular calcium, which is a signal for contraction. Vasopressin,

upon binding to its V-1 receptors on the vascular smooth muscle cell, elicits vasoconstriction

with a similar mechanism.

Many other hormones can elicit vasoconstriction including catecholamines, endothelin

and neuropeptide Y, whereas some are potent vasodilators such as nitric oxide or ANP.

Neural Control

The brain exerts control over vascular tone as well as cardiac output via the

sympathetic and parasympathetic nervous system. The sympathetic postganglionic neurons







8
innervate the blood vessels, heart and adrenal medulla. They release norepinephrine and elicit

vasoconstriction in different vascular beds, increase venous return by contracting large veins,

increase heart rate and the release of epinephrine from the adrenal medulla.

The sympathetic preganglionic neurons are found in the intermediolateral column of the

thoracic and upper lumbar spinal chord, and provide tonic stimulation of the peripheral

sympathetic system. Loss of this tonic activity results in collapse of blood pressure. The origin

of tonic excitatory activity are the adrenaline-synthesizing cells in the Cl area of the rostral

ventrolateral reticular nucleus of the medulla oblongata (RVM), the integrative center of

regulation of circulation. Cl cells fire in phase with cardiac systole because baroreflex inhibits

them (13).

Baroreflex

Baroreflex originates from stretch-receptors in the carotid artery and the aorta, and

travels through the IX. and the X. cranial nerves to the nucleus tractus solitarii (NTS).

Increased blood pressure activates the stretch-receptors which results in inhibition of the RVM

vasomotor cells by GABA-erg neurons from the NTS. Inhibition of RVM in turn causes

vasodilation by decreasing sympathetic activity. Baroreflex activation also causes a decrease in

heart rate by activating cardiovagal efferents.

Diencephalic and Telencephalic Control of Blood Pressure

There are supramedullary pathways that are shown to exert profound effects on blood

pressure regulation. The specific nuclei involved in these networks are found in all levels of the

brain: the parabrachial nuclei of the pons, the periaqueductal gray of the midbrain,

hypothalamic nuclei including the paraventricular, supraoptic, lateral and anterior nucleus, the







9
central and medial amygdaloid nucleus and areas of the limbic cortex including the cingulate,

parahippocampal, insular and orbitofrontal cortices (14). The functions of these areas are only

partially understood, but they are thought to play a role in integrating cardiovascular functions

with other physiological functions including stress reaction, emotion and behavior, and

malfunction of some of these brain areas are implicated in the development of essential

hypertension.

It is apparent from this brief overview of blood pressure regulation that blood pressure,

a vital body function, is secured with several feed-back loops. These regulatory mechanisms

are apparently built onto each other, and on the top of this regulatory hierarchy is the brain.

The Renin-Angiotensin System

The best recognized function of the renin-angiotensin system is to directly or indirectly

preserve fluid homeostasis and normal blood pressure. The renin-angiotensin system (RAS)

was originally described in the peripheral circulation (15). According to the classical

description, the only endogenous ligand for angiotensin receptors was the octapeptide

angiotensin II (AngIl), which is derived from its prohormone angiotensinogen through

generation of angiotensin I by renin and consequent conversion of angiotensin I to angiotensin

II by angiotensin converting enzyme (ACE). This of course still holds true with the addition

that some other derivatives of the octapeptide such as AngII (Ang 2-8) and AnglI(3-8) have

since been proposed to be endogenous ligands, and alternative routes for synthesis of AngIl

from angiotensinogen have been postulated (16,17). In the classical view, the prohormone

angiotensinogen is synthesized and secreted constitutively by the liver into the circulation,

where some of it is transformed to AngI by renin in the kidney and further cleaved by ACE







10
mainly in the lungs to form AngII. Now we know that there are several other RASs located in

tissues rather than in the circulating blood and they act independently of each other (18). The

primary sites of action for the circulating AngIl are the blood vessels, kidney and the adrenal

cortex, where AT-1 receptors mediate the well known effects of AngI: vasoconstriction in the

blood vessels, decreased glomerular filtration in the kidney and aldosteron secretion of the

glomerulosa cells of the adrenal gland (19). All three actions are aimed at conserving body

fluids and elevating blood pressure. Accordingly, the rate limiting step in AngII synthesis is the

conversion of angiotensinogen to AngI by renin, which is released by juxtaglomerular cells of

the kidney in response of decreased fluid and/or Na+ load.

The function of the local tissue RASs are considerably less well understood. What

northern blots and in situ hybridizations tell us is that components of RAS are not only found

but also synthesized locally in tissues (18). It is also suggestive for the existence of the

independent tissue RAS's that these tissues, the brain, kidney, heart, blood vessel wall, and

testis are capable of maintaining local AngII concentrations significantly different from that of

the plasma. Evidence accumulates that the locally produced AnglI in these tissues acts in a

paracrine and/or autocrine fashion serving physiological and perhaps developmental regulatory

functions.

Angiotensin Receptors

Classification of angiotensin 1 receptors

Angiotensin II binding sites were characterized first in 1974 (20) in adrenal cortex and

soon after in blood vessels and brain (21). Although isolation and chemical characterization of

the receptor protein proved to be difficult due to the instability of the receptor protein,







11
pharmacological data suggested that there is probably more than one type of AnglI receptors.

For example, the peptidic AngII analogue des-Asp-AngII proved to be equipotential with

AngII in increasing serum aldosterone, but increased blood pressure 10 times less efficiently

(22). Receptor binding assays with radiolabeled AngII suggested the presence of a high affinity

and a low affinity AngII binding site in rat hepatic membranes, and the GTP analogue GppNHp

was found to influence receptor binding only in the high-affinity site (23,24). Dithiothreitol, a

sulfhydryl-reducing agent cleaving covalent bonds between cystein amino acids, reduced AnglI

binding only on the high-affinity site suggesting structural differences between the two AngII

binding sites (25). Different second messenger systems seemed to be activated by AnglI

depending on tissue localization: in the kidney AngII was found to activate phospholipase C in

glomerular mesangial cells, whereas in tubular epithelial cells it appeared to be coupled to

adenylate cyclase (26).

Defining the two major AnglI receptor subtype was greatly facilitated by the

development of the new, nonpeptidic AngIl antagonists. The first of these, losartan (formerly

DuP753), displaced AngI completely in rabbit adrenal cortex, liver and vascular smooth

muscle and displaced AngII partially in rat adrenal cortex, adrenal medulla and uterus. AngII

binding in human uterus and PC12W cells was not influenced by losartan. The losartan-

sensitive binding sites were sensitive to dithiothreitol and also to nonhydrolyzable GTP

analogues such as GppNHp and GTP-gamma-S. The losartan-insensitive AngII binding site in

rabbit uterus was shown to be sensitive to another type of nonpeptidic AngII antagonist,

PD123319. The PD123319-sensitive site was not sensitive to the nonhydrolyzable GTP

analogue 5'-guanylyl-imidodiphosphate, and was dissimilar to the losartan-sensitive site in that







12
it did not influence inositol phosphate metabolism, vascular contractile activity or blood

pressure regulation (27).

Together, these two drugs serve as basis for classification of AngI receptors: the

accepted nomenclature calls AT-1 the losartan-sensitive binding site, whereas PD123319-

sensitivity defines the AT-2 subtype (28).

Recent advances in molecular cloning of the cDNAs and consequently the whole genes

for angiotensin receptors not only validated the classification of AngI receptors based on their

pharmacological properties, but also expanded the number of the different receptor subtypes.

Clones of the AT-1 receptor cDNA from rat vascular smooth muscle (29) and adrenal gland

(30) was found to be different, and screening rat genomic library showed that there is two

similar but distinct genes for AT-1 receptor in rat, which were later termed AT-la for the

vascular type and AT-lb for the adrenal type. The presence of two AT-1 genes has been also

shown in mice but not in humans (31).

Pharmacological data suggested AT-2 receptor heterogeneity in the brain and in

neuroblastoma cell line NIE-115. AT-2 receptors located in the ventral and mediodorsal

thalamic nuclei, medial geniculate nuclei and in the locus ceruleus of the rat brain are sensitive

to guanine nucleotides, pertussis toxin and dithiothreitol and named AT-2a, whereas AT-2

receptors in the inferior olive and in the hypoglossal nucleus are not sensitive to any of these

reagents and suggested to be classified as AT-2b (32). In N1E-1 15 cells two population of

AT-2 receptors were distinguished based on their sensitivity to PD123319 and dithiothreitol

(33).








Molecular structure of the AT-1 receptor

Regardless of species or subtype, the open reading frame of all AT-1 receptor genes

code for a protein of 359 amino acids constituting a 41 kDa protein, which is in good

agreement with the molecular weight of the deglycosylated form of AT-1 receptor protein

purified from adrenal gland (34). Hydropathy analysis of the amino acid sequence showing

seven groups of non-hydrophilic amino acids suggest that the AT-1 receptor protein belongs to

the superfamily of G-protein coupled receptors possessing seven membrane-spanning regions.

All four extracellular loops connecting the transmembrane regions have a cysteine

residue capable of forming disulfide bridges. It is suggested that the cysteines on the second

and the third loop form one disulfide bridge, whereas the first and the fourth loop form the

other creating a well-like structure transversing the cell membrane (35). Disruption of these

disulfide bridges with sulfhydryl reagents such as dithiothreitol would destroy this tertiary

structure and this is the probable explanation for the effect of these reagents on decreasing

binding affinity to AnglI.

Cloning AT-1 cDNA into expression vectors and expressing it in COS-7 cells which

normally do not possess AnglI receptors has shown that upon Angnl stimulation these

recombinant receptors are capable of activating inositol trisphosphate (IP3) and causing

transient elevation of intracellular Ca', as well as stimulating phospholipase C activation (29).

Transfection of CHO cells showed that the cloned AT-1 is capable of inhibiting cAMP

production and that the IP3 stimulation is mediated by Gq protein, whereas the cAMP inhibition

is mediated by ,,. These observations confirmed that the cloned AT-1 cDNA codes for a

functional receptor and it is similar in its actions to the naturally occurring one.







14
Further dissection of the functional domains of the AT-1 receptor required molecular

techniques such as site-directed mutagenesis. Mutating individual amino acids revealed that

Lys-199 of the fourth transmembrane domain is critical for ligand binding: it was proposed that

the negatively charged charboxyl-terminus of AngI lands on the positively charged amino

group of this lysine. Mutating of any of the cysteine residues reduced AngI binding

drastically, whereas eliminating the three Asn residuals which are potential sites for N-

glycosylation had no effect on receptor binding (36). It appears that intracellular domains

responsible for G-protein binding are found on the second intracellular loop and on the

intracellular carboxyl terminal of the protein: mutation of any of these areas prevented the

characteristic shift from high-affinity to low affinity state upon exposure to non-hydrolizable

GTP analogues such as GTP-y-S. This is in apparent contrast with the adrenergic receptors

where a large third cytoplasmic loop is thought to interact with G-proteins.

Regulation of AT-1 gene expression

The gene encoding the AT-1 receptor is comprised of at least five exons and four

introns in rodents as well as in humans. The whole gene spans a region over 55 kilobases. The

first four exons are short sequences and are subject to alternative splicing: whereas the fifth

exon is always present in the mRNA, the first, second, third and/or the fourth might be missing.

The entire coding region (1077 bp) is found on the fifth exon. Whereas there is two AT-1

gene in rats and mice, called AT-la and AT-lb, coding for two proteins about 95% similar but

differing significantly in their untranslated regions, there is only one AT-1 gene is found in

southern blots of human DNA. The 5' flanking region of the human gene contains two putative







15
TATA boxes, two CAAT boxes, two overlapping SPI sequences, a GC box and a cAMP-

responsive element (CRE) (37).

The rat AT-la gene promoter region located upstream of the first exon also includes

several consensus elements: two TATA boxes (a third can be found 5' to the fourth exon),

several transcription factor binding sites including two AP-1 site capable of binding early gene

products, an SP-1 site which can interact with RNA polymerase I, several glucocorticoid-

responsive elements (GRE) and a CRE site (35). The presence of the GREs gives explanation

for the stimulation of AT-1 mRNA in response to aldosterone and dexamethasone, and the

CRE is thought to be responsible for AT-1 downregulation by AngII (38).

AngII downregulates AT-1 mRNA rapidly: AngIl treatment causes 80%/ loss of AT-1

mRNA in 3 hours in adrenocortical carcinoma cells. A similar effect can be achieved by the

combination of calcium ionophore and phorbol ester, suggesting the involvement of protein

kinase C in the downregulation of AT-1 mRNA (39). Protein kinase C in turn, is thought to

act on the AP-1 and AP-2 sites of the promoter region of the AT-1 gene (40).

Downregulation of AT-1 mRNA by AngII was also observed in vascular smooth muscle cells

and mesangial cells (41,42). In vivo studies, however, suggest that AngII also can upregulate

its receptor expression: AngII infusion for two weeks increased AT-1 mRNA in the adrenal

gland, whereas bilateral nephrectomy decreased it (43).

The life-cycle of the AT-1 receptor

The AT-1 receptor, like many other receptors, undergo cellular internalization upon

ligand binding. Electronmicroscopic studies utilizing colloidal gold-labeled Angl showed that

the ligand bound first diffusely to the cell surface. In the following minutes, however, receptor-







16
ligand complexes aggregated into groups and internalized forming small membrane vesicles

called coated pits (44). The receptor-ligand complexes are thought to dissociate in lysosomes,

where AngI is degraded. The fate of the receptor molecule is uncertain: a fraction of the

receptors is recycled to the cell surface, whereas most of it degrades in lysosomes (45). In

vascular smooth muscle, there is significant perinuclear accumulation of radiolabeled AngII

following intravenous injection suggesting direct nuclear action of AngII-receptor complex

(44).

Binding of an antagonist to the AT-1 receptor generally does not result in

internalization. Whether internalization is actually necessary for activating the appropriate

second messenger systems remains to be seen.

Intracellular messenger systems coupled to AT-1 receptors

The AT-1 receptors, based on their predicted molecular structure, belong to the

superfamily of the G-protein coupled receptors. Experimental evidence also shows the active

involvement of at least two types of G proteins- Gq and G,- in intracellular signaling. Gq has

been shown to mediate AT-1 stimulation on phospholipase C, whereas i is thought to mediate

AT-1 inhibition of adenyl cyclase. Stimulation of phospholipase C by AngII in several tissues

results in hydrolysis of phosphatidyl-inositol-bisphosphate, a minor membrane protein into

inositol-(1,4,5)trisphosphate (IP3) and diacylglycerol. IP3 then binds to its intracellular receptor

and releases Ca' into the cytosol. These events are observed within 1-2 minutes after AT-1

receptor stimulation (46,47,48). A slower component of the stimulation can be observed when

diacylglycerol, along with cytoplasmic Ca+, activates protein kinase C. Increased Ca++ also

contributes to the activation of calcium-calmodulin dependent protein kinase. Protein kinase C







17
activation leads to the cell-specific response by phosphorylation of MAP kinase, tyrosine

kinases and myosin light chain (49). Vasoconstriction by blood vessel smooth muscle cells or

aldosterone secretion by glomerulosa cells in the adrenal are examples of cellular responses

mediated by these intracellular mechanisms.

Coupling AT-1 to the inhibitory G, protein results in inhibition of the action of

adenylate cyclase in hepatocytes, bovine adrenal cells and heart myocytes (50-52). Inhibition

of cAMP production antagonizes actions of protein kinase A such as sodium transport

inhibition in proximal tubule epithelium. In rat brain homogenates, however, stimulation of

adenylate cyclase was detected (53).

Angi is also known for inducing Ca' influx from the extracellular fluid. AngU can

operate ligand-gated channels or modify the action of voltage-gated T or L type Ca++-channels

either through direct intramembrane action or by activating intracellular protein kinase C

(54,55).

Besides phospholipase C, AngII can activate other phospholipases through the AT-1

receptor such as phospholipase D and phospholipase A2. Phospholipase D hydrolizes

phosphatidylcholine to phosphatidic acid and choline upon stimulation with AngII in vascular

smooth muscle cells. The proposed second messenger is the phoshatidic acid, whereas the

choline is released into the extracellular medium. Activation of phospholipase A2 in proximal

tubule cells and vascular smooth muscle cells by AngII results in the release of arachidonic

acids from phospholipids. Arachidonic acid is the precursor for prostaglandin and leukotriene

synthesis. Indeed, AngI has been shown to induce prostaglandin synthesis in vascular smooth

muscle cells (50).







Genetics of the Renin-Angiotensin System and Hypertension

Studies of monozygotic and heterozygotic twins indicate that the development of

hypertension is due to inherited as well as environmental factors (56). The success of animal

breeding experiments in producing inbred hypertensive rats (SHR) is also an evidence for a

strong genetic component in the etiology of hypertension.

Blood pressure is a quantitative trait which shows continuous variations from low to

high values in human populations. Quantitative traits are generally thought to be polygenic,

that is, a product of the effect of several genes.

Genetic linkage studies searching for chromosomal loci cosegregating with high blood

pressure in animal models and in humans indicated several candidate genes that might cause

hypertension. Most persistently, molecular variants of angiotensinogen are shown to be linked

to the hypertensive phenotype. M235T, a variant of the angiotensinogen gene coding for

threonine instead of methionine at position 235 (which is outside the renin cleavage site) has

been shown to be present significantly more often in hypertensive subjects than in

normotensives, and plasma angiotensinogen levels were also significantly higher in female

M235T-positive subjects. An other molecular variant, T174M was also associated with

essential hypertension (57).

The linkage of T235M variant to hypertension was confirmed in a different population

in Japan (58), but not in the United Kingdom (59), although these latter investigators also

found significant linkage between angiotensinogen locus and hypertension.

The same molecular variant of angiotensinogen gene was also found to be associated

with the development of preeclampsia, a severe form of pregnancy-induced hypertension (60).







19
Transgenic experiments also indicate the possible role of angiotensinogen in the

development of hypertension. Transgenic mice TGM(rAOGEN)123 carrying a copy of the rat

angiotensinogen gene expressed rat angiotensinogen in the liver and the brain and developed

hypertension (61). Duplication of one allele of the angiotensinogen gene in mice resulted in

elevated blood pressure, while disruption of one allele caused hypotension (62).

Other components of the renin-angiotensin system were also investigated as candidate

genes for hypertension. Investigations of the potential involvement of the renin gene were

inconclusive (63). Linkage analysis of the rat genome strongly suggested the pathogenic role

of ACE (64,65), but cloning expression and functional analysis showed only few differences in

base sequence and no apparent difference in enzyme function between SPSHR and WKY

ACEs (66).

A deletion-insertion polymorphism in the ACE gene, however, appeared to be

associated with heart disease. The homozygous deletion (DD) genotype was found to be in

excess in middle-aged men with left ventricular hypertrophy (67).

The Brain Renin-Angiotensin System

It was suggested as early as in 1961 that AngIl might have physiological effects on the

brain (68). The possibility that AngII is synthesized in the brain was suggested in 1971, and

conclusive evidence for the presence of AngII in neurons was provided by Ganten, Fuxe and

Phillips showing renin- and AngI-immunoreactivity in neurons in the brain (69,70). Further

immunocytochemical studies revealed that AngII is found in the brain in neurosecretory

vesicles, which is suggestive for synthesis of AngII by these neurons (71). Physiological data

showed that acute or chronic changes in circulating AngII levels failed to change brain AngI







20
levels, which points to the existence of an independently regulated brain RAS (72). In vitro

experiments showed that radiolabeled amino acids are incorporated into AngIl by cultured

primary rat brain cells which is a direct evidence for the ability of brain tissue to synthesize

AngII (73).

Components of the brain RAS: angiotensinogen

Even though there is little doubt that AngII is synthesized in the brain, it is not filly

understood where and how the synthesis occur. The prohormone angiotensinogen is a

relatively abundant protein of the brain extracellular fluid. Angiotensinogen secretion has been

shown from brain slices to the fluid media (74). Angiotensinogen mRNA is widely distributed

in the brain; highest levels were measured in the hypothalamus (75,76). Angiotensinogen

mRNA is found to be colocalized with glial fibrillary acid protein (GFAP)-staining cells,

suggesting that glial cells are responsible for the production of brain angiotensinogen (77). In

contrast, immunocytochemical data shows angiotensinogen-like immunoreactivity in neurons

as well as in glia (78). Angiotensinogen secretion is thought to be constitutive rather than

regulated, based on observations showing that cells themselves contain little angiotensinogen,

and depolarizing agents such as K' do not evoke increase in cerebrospinal angiotensinogen

levels (79). It seems, therefore, that most of the angiotensinogen in the brain is synthesized by

glial cells of the hypothalamus, and it is constitutively secreted into the extracellular fluid.

Renin

The only substrate for the aspartyl protease renin is angiotensinogen. Other enzymes

such as cathepsin D can also cleave angiotensinogen, but whether this actually happens under

physiological circumstances is not known (80). The presence of renin in brain tissue has been







21
shown with functional assays showing renin-like activity as well as immunohistochemically.

Renin-like activity has been found ubiquitously but in a low level in the brain, with the highest

levels in the brainstem and in circumventricular organs such as the pituitary and the pineal gland

(79). Immunocytochemical assays using anti-salivary and anti-kidney renin antibodies showed

highest staining in the supraoptic and paraventricular nucleus (81,82). Others found more

widespread immunoreactivity covering most parts of the brain (83). Renin mRNA has been

found in the cortex and in low amounts in the brainstem, but not in the hypothalamus (84).

Brain renin appears to be regulated independently from plasma-renin: infusion of renin or renin

antibody into the peripheral circulation failed to produce significant elevation or decrease in

brain renin-like activity (85). Sodium depletion, which increases plasma renin activity,

decreased brain renin activity (86).

Cultured primary brain cells also produce immunochemically detectable renin, showing

the capacity of brain cells for endogenous renin synthesis. Brain renin appears to be in

synaptosomal preparations, and depolarization of cortical cells results in renin release (87).

These data suggest that renin-like enzyme activity and immunoreactivity is present in the

mammalian brain, even though the two do not necessarily overlap. The overall renin levels are

low: most of it is found on the cortex and it appears to be intracellular.

Angiotensin converting enzyme (ACE)

The enzymatic properties of brain ACE closely resembles to that of the lung and

kidney ACE: all of them are peptidylpeptide carboxyhydrolases anchored to the outside of the

cell membrane. Besides AngI, ACE also cleaves bradykinin, luteinizing hormone releasing

hormone, enkephalins and substance P (88). ACE is found mostly in the choroid plexus,







22
subfornical organ, area postrema, and, in lesser amounts, in the substantial nigra, locus ceruleus

and the hypothalamus (89). There is also soluble ACE in the cerebrospinal fluid.

Angiotensin I[

AngII immunoreactivity in the brain has been demonstrated in the hypothalamus,

medulla oblongata and limbic system. AngIl has been found in synaptic nerve terminals in the

median eminence, paraventricular nucleus, supraoptic nucleus and subfomrnical organ. The

caudate-putamen and the sympathetic lateral column also contains immunoreactive AnglI (69).

Biochemical identification of brain AngLI using HPLC showed that brain AnglI is identical to

its circulating counterpart (90).

Several pathways have been identified having AngII-containing neurons. AngI-

containing fibers from the subfomical organ and organum vasculosum lamina terminalis

(OVLT) converge at the median preoptic nucleus and send fibers to either to the brainstem

through the medial periventricular hypothalamus or to the paraventricular and supraoptic

nucleus from where angiotensinerg fibers go to the neurohypophysis (91). A separate

pathway responsible for the drinking response (see below) has been identified starting from the

OVLT through the median preoptic area projecting to the midbrain.

In summary, while acknowledging that our knowledge is far from complete about how

AnglI is generated in the brain, the current theory states that angiotensinogen, synthesized and

secreted constitutively into the extracellular space by glial cells is cleaved by renin, which is

secreted from nerve endings upon depolarization and appears to be the rate limiting factor.

The generated AngI is then converted to AnglI by either membrane bound or soluble ACE.






23
AngII then can be taken up by neurons and utilized as a peptide neurotransmitter or it can act

extracellularly as neuromodulator.

Angiotensin Receptors of the Brain

Angiotensin receptors are fairly widespread in the mammalian brain (92). They are

found in circumventricular organs where they are accessible to circulating AnglI as well as in

areas protected by the blood-brain barrier. The method of choice for identifying angiotensin

receptors in the brain is autoradiography, where slide-mounted brain slices are incubated with

radiolabeled AngI analogues, and the slices then are exposed to x-ray film. Such experiments

yielded precise maps of angiotensin receptor localizations in the brain.

The two major receptor subtypes--AT-1 and AT-2--which can be differentiated in

autoradiographic studies by using subtype-specific receptor antagonists show different spatial

and temporal (developmental) distribution (93). There are areas which contain exclusively AT-

1 receptors: the piriform cortex, organum vasculosum lamina terminalis, median preoptic

nucleus, supraoptic and suprachiasmatic nucleus, subfomrnical organ, basolateral amygdaloid

nucleus, paraventricular nucleus, lateral olfactory tract, dentate gyms, subiculum, area

postrema, nucleus of the solitary tract and choroid plexus. Other areas contain only AT-2

receptors: the lateral septal nucleus, ventral and mediodorsal thalamic nucleus, parasolitary,

medial amygdaloid and medial geniculate nucleus, locus coeruleus and inferior olive. Some

areas contain both AT-1 and AT-2 receptors within the same nucleus: in the superior

colliculus, cingulate and cerebellar cortex (94).

During fetal development, angiotensin receptors first appear in the last third of the

pregnancy in the rat brain. The angiotensin receptor number progressively increases up until







24
the first two weeks after birth, when the receptor density reaches an approximately ten times of

the adult level. The majority of these receptors are type-2, such as the ones in the inferior

olive, paratrigeminal nucleus, hypoglossal nucleus and in the meninges. There are some areas

in the fetal brain with AT-1 receptors, such as the nucleus of the solitary tract and the choroid

plexus. As the rats reach young adulthood, there is no major change in the pattern of AT-1

receptor expression, whereas a drastic decrease can be detected in the AT-2 receptors (95). In

the lateral septal nucleus, ventral and mediodorsal thalamic nuclei, locus ceruleus, sensory

trigeminal nucleus, parasolitary nucleus, inferior olive, medial amygdaloid nucleus and medial

geniculate nucleus the AT-2 binding significantly decreases, whereas in other areas such as the

cerebellar cortex, anterior pretectal nucleus, nucleus of the optic tract, ventral tegmental area,

posterodorsal tegmental, hypoglossal nucleus, central medial and paracentral thalamic nuclei,

laterodorsal thalamic nucleus and oculomotor nucleus the AnglI binding disappears.

Effects of Central Angiotensin on Brain Receptors

Three major effects of Angi on brain angiotensin receptors can be observed most

consistently: pressor response, drinking behavior and vasopressin release from the posterior

pituitary (1). Even though these are distinct reactions, they are related to each other and all of

them result in restoration of body fluid homeostasis. A fourth, less obvious effect serving the

same goal is the increase in salt appetite (96). AnglI injected into the brain also can elicit

oxytocin release form the posterior and luteinizing hormone and ACTH release from the

anterior pituitary. AngI may also influence higher brain functions such as memory (97,98).

The most readily observable reaction to i.c.v. AngII is the drinking response (99). Fifty

ng AngI injected into the lateral brain ventricle induces drinking behavior in 1-2 minutes, and







25
the animals will drink 5-15 mnil water in 30 minutes (100). The main brain areas involved in

mediating the drinking response are localized in the anterior hypothalamus and include the

SFO, the AV3V region including the organum vasculosum lamina terminalis (OVLT) and the

median preoptic nucleus (MnPO). It is generally thought that the SFO, as a circumventricular

organ, is responsible for drinking reaction induced by circulating AngII, whereas the AV3V

region transmits central AngII signals (101). Preventing AngIl to reach the AV3V region

blocked central AngI effects (102). Applying AngII to the OVLT resulted in neuronal

excitation which can be blocked by specific antagonists (103). On the other hand, lesioning

the SFO attenuated the central effects of intravenously injected AngII (104).

It is believed that there are two drinking pathways, both of them originating in the

AV3V region: one passes through the periventricular zone of the hypothalamus and projects to

the brainstem, while the other sends fibers to the PVN and SON and to the posterior pituitary

(16). As to how these neurons bring about the motor and cognitive functions required for the

act of drinking is less well understood.

The other well known reaction to i.c.v. AngII is increased blood pressure. Low doses

of AngII will result in pronounced and prolonged increase in blood pressure when given i.c.v.

(68). The main coordinating center in the brain for mediating this pressor response is thought

to be the PVN. The PVN receives efferents from the SFO, which is shown to mediate the

effects of circulating AngII of central blood pressure regulation, and the OVLT, which is a

major site of action for brain-borne AngII (105,101). The PVN has large amounts of AngII

immunoreactivity as well as one of the highest concentrations of AngII receptors. The PVN,

among other efferents, projects to the posterior pituitary, NTS and the intermediolateral horn







26
of the spinal cord. It is thought that brain AngII elevates blood pressure by increasing

vasopressin release from the posterior pituitary, stimulating the sympathetic nervous system

and decreasing vagal input, including baroreflex input (1). The involvement of the sympathetic

nervous system is suggested by results showing that sympathetic blockade can diminish pressor

response to AnglI (106,107). Norepinephrine release was found to be increased in the

hypothalamus in response to i.c.v. AngI administration (108,109). The possible involvement

of the baroreflex modulation is supported by the notion that some of the AT-1 receptors in the

NTS are located on nerve endings of vagal afferents (110,111).

The third major effect of central AngI involves the posterior pituitary. AngI

receptors in the supraoptic nucleus (SO) and in the PVN can stimulate vasopessin as well as

oxytocin secretion from the posterior pituitary (112,113). AVP in turn, has immediate

(vasoconstriction) and prolonged antidiureticc action) effect on blood pressure. Inhibition of

AVP's action by V1 AVP antagonists or hypophysectomy decreases, but does not eliminate the

pressor effect of i.c.v. AngII (114,115). Pressor response to i.c.v. AngHI is also attenuated in a

genetic model of diabetes insipidus, the Brattleboro rat (116).

Besides these better known actions, AngHI has been proposed to influence other

neurosecretory cells. Lesioning dopaminerg nigrostriatal pathways inhibited AngI induced

drinking (117). The opiate-antagonist naloxone was also shown to inhibit both pressor and

drinking response to AngII (118,119). The involvement of central inhibitory GABA-erg

neurons was proposed when it was shown that GABA injections into brain ventricles inhibited

the pressor response and the release of vasopressin (120). Whether these neurons are

interneurons, modulatory neurons or parallel pathways to the AngI-neurons, is not clear. The







27
final efferents of the brain RAS on blood pressure are thought to be sympathetic motor fibers

and vasopressin secretion. Indeed, hypophysectomy and pharmacological blockade of alpha-1

adrenoceptor abolished the pressor response to centrally injected AngI (121).

The Antisense Paradigm

Antisense inhibition of gene expression was first proposed by Zamecnik et al. in 1978,

who inhibited Rous sarcoma virus replication with a 13mer antisense oligonucleotide (122).

Consequently it was discovered that prokaryotes use endogenous antisense RNA molecules

(123). These natural antisense molecules are produced most frequently by transcribing parts of

the gene to be regulated, but from the non-template strand under a promoter directed opposite

to the "sense" promoter. The endogenous antisense RNAs influence gene expression, plasmid

replication and transposition by binding to mRNA coding for essential replication proteins, or

by binding to a primer RNA required for DNA replication (124). It has been shown that

eukaryotic cells also synthesize antisense RNAs (125). It was, however, not until the advent of

the automatic DNA synthesizers that custom-made oligonucleotides became widely available,

and functional testing of antisense oligonucleotides targeted to different proteins began.

Antisense inhibition is an extremely attractive investigative and pharmacological

approach, since it offers base-to-base specificity to the target protein and versatility comparable

to the complexity of the genetic code itself It has become clear, however, that there are a

number of issues to be considered before using antisense in any experimental or clinical setting.

These include (1) selection of target sequence; (2) stability of antisense oligos in body fluids;

(3) the mechanism of cellular uptake; (4) possible intracellular sites of action and (5)

effectiveness, specificity and toxicity.








Selection of the Target Sequence

An antisense oligodeoxynucleotide is a short (15-30 nucleotides long), synthetic, single

stranded DNA molecule, which is complementary in sequence to the messenger RNA (mRNA)

of the target protein. When the antisense DNA hybridizes with the mRNA, it can inhibit the

synthesis of the target protein. Thus in principle, the concept is very simple. Designing an

effective antisense oligonucleotide, however, is not simple. The ambiguity in antisense

oligonucleotide design stems from incomplete knowledge about the mechanism of action of

such molecules, as well as from conflicting experimental results.

The original concept of antisense inhibition assumes that antisense molecules bind to

the mRNA of the target protein in the cytoplasm, and prevent either ribosomal assembly or

read-through (124). Most oligonucleotides therefore are targeted to the initiation codon AUG,

or part of the coding region downstream from it. When designing such an antisense

oligonucleotide, one has to consider two antagonistic factors: the affinity of oligonucleotide to

its target sequence, which is dependent on the number and composition of complementary

bases, and the availability of the target sequence, which is dependent on the folding of the

mRNA molecule (126).

There have been attempts to correlate oligonucleotide efficiency and these two factors:

affinity and availability. There is a general agreement that increasing affinity of antisense

oligonucleotides positively influences effectiveness, but results are conflicting on the

importance of the secondary structure of the target molecule. Several reports suggested that

antisense oligonucleotides targeted to different regions of the RNA have unequal efficiencies

(127,128). These differences may be related to the predicted secondary structure of the target







29
mRNA (129-131), but other experimenters did not find significant differences in antisense

efficiency between oligonucleotides targeting different segments of the RNA (126,132,133). It

would seem logical that the folding of the mRNA influences target sequence availability, but

one also has to consider that the stretches of the RNA double helices which are responsible for

the secondary structure of the mRNA incorporate a weaker G-U base pairing besides A-U and

G-C and are generally short and rarely perfect. Therefore an ODN that has only strong

(Watson-Crick) base pairing with 100% complementarity will form the more

thermodynamically favorable structure with its target RNA (134). If this is the case, the

antisense DNA could resolve the RNA secondary structure, and hybridize with the targeted

RNA region.

Besides inhibition of translation, other possible antisense mechanisms of action have

been proposed. Based on studies of cellular uptake of labeled oligonucleotides, the picture

emerged that most antisense oligonucleotides quickly migrate to the cell nucleus, suggesting an

intranuclear site of action (135). This suggests a more direct action on gene expression:

antisense molecules might inhibit pre-RNA splicing, transport of mRNA from the nucleus to

the cytoplasm, or bind to the DNA inhibiting transcription. Accordingly, effective antisense

oligonucleotides were designed targeting exon-intron splicing sites (136) or the major groove

of the DNA (137).

The experimental evidence accumulated in the past few years of antisense research

suggest that a large number of antisense oligonucleotides targeted to different regions of a

variety of mRNAs were successful in downregulating their target proteins, although the

number of failed attempts can not be gauged. It is, however, hard to recognize a general







30
pattern by which one can design an effective antisense oligonucleotide to a particular target.

The three regions that are repeatedly suggested to be better targets are the 5' cap region, the

AUG translation initiation codon and the 3' untranslated region of the mRNA (126,138,139).

Since most mRNAs have an AUG initiator codon site, targeting 12-15 of the neighboring bases

should produce inhibitory oligos. Unfortunately, many mRNAs in vertebrates have the Kozak

consensus sequence around the AUG start codon, and there is a danger of creating nonspecific

oligos by taking this approach. In any case, all designed oligos must be checked with the

GeneBank for existing sequences to avoid homology with other mRNAs.

A possible reason why there is no general rule of thumb for antisense oligonucleotide

design is that different mechanisms of action are present in different experimental situations

(131). If this is the case, the safest way for finding the optimal target sequence is through

testing a series of oligonucleotides. Common rules for antisense action may exist, but factors

like differential cellular uptake and discharge of different oligonucleotides or intracellular

feedback mechanisms on mRNA or protein production make them difficult to identify. Further

systematic investigation is needed to elucidate the molecular mechanisms of antisense inhibition

in order to be able to design antisense molecules with predictable results.

Stability of Oligonucleotides

Oligonucleotides in their natural form as phosphodiesters are subject to rapid

degradation in the blood, intracellular fluid or cerebrospinal fluid by exo- and endonucleases.

The half-life of phosphodiester oligonucleotides is in the range of minutes in blood and tissue

culture media. The half-life of oligonucleotides is .somewhat longer in cerebrospinal fluid, and

there are reports of detecting intact oligonucleotides 24 hours after injection into the cerebral







31
ventricles (140). Several chemical modifications have been proposed to prolong the half-life of

oligonucleotides in biological fluids while retaining their activity and specificity. The majority

of these modifications are backbone-modifications, which effect the phosphodiester bond

between nucleosides.

Phosphorothioates

Probably the most widely used modified oligonucleotides are phosphorothioates, where

one of the oxygen atoms in the phosphodiester bond between nucleosides is replaced with a

sulfur atom. These phosphorothioate oligonucleotides have greater stability in biological fluids

than normal oligos. The half-life of a 15-mer phosphorothioate oligo is 9 hours in human

serum, 14 hours in tissue culture media with 10% fetal bovine serum and 19 hours in

cerebrospinal fluid (141). Phosphorothioate oligos can be synthesized with automated DNA

synthesizers, but because these machines replace either of the oxygen atoms in a given

phosphodiester bond, the resulting product is a 2-diastereoisomer in the case of an n-mer

instead of a uniform compound, which may complicate not only experimental data

interpretation but also pharmacological production of a given phosphorothioate antisense

oligonucleotide (142). By replacing both oxygen in the phosphodiester bond to sulfur one

would expect even greater resistance to nucleases, and also the problem of diastereoisomerism

could be eliminated. However, this type of oligonucleotides, the phosphorodithioates is hard to

synthesize, and even though they show high resistance to endonucleases, they have been

studied much less than monothioates (143).

Phosphorothioate oligonucleotides retain the negative charge of each nucleotide which

is characteristic to phosphodiester oligonucleotides, which might explain the similar behavior of







32
phosphorothioate oligos and phosphodiester oligos in cellular uptake and possibly intracellular

action.

Methylphosphonates

Replacing one of the oxygens in the phosphodiester bond with a methyl group results

in two degrees of magnitude of increase of nuclease resistance. Single base modification can

even protect the neighboring phosphodiester bonds. In serum containing tissue culture media

where 3'-exonuclease activity is the most prominent, methylphosphate modification of the 3'

nucleotide can increase the half-life of an oligonucleotide from 2 hours to 10 hours (144).

Other modifications

One or more of the oxygen atoms can be replaced with a variety of other compounds

such as alkyl phosphotriester, phosphoramidate or boranophosphate which all expand the half-

life of oligonucleotides in ex-vivo experiments, but only limited data is available on their

biological activity. Other modifications aiming at protecting oligonucleotides from enzymatic

digestion include 2'-sugar modifications (145), Y'-cholesterol modifications (146) and peptide

nucleic adcids (147).

Cellular Uptake of Oligonucleotides

In order to hybridize with the target mRNA, antisense oligonucleotides have to cross

the cell membrane. Investigators used two major methods for detecting intracellular

oligonucleotides: radiolabeling and fluorescent labeling. Fluorescent labeling offers convenient

detecting methods with fluorescent or confocal microscopes, whereas radioactive tags offer

labeling without changing the chemical configuration of the molecule. One of the first

comprehensive studies dissecting oligonucleotide uptake by cultured cells utilized fluorescent







33
(acridine)-labeled oligonucleotides (148). This group detected saturable uptake of

oligonucleotides plateauing within 50 hours with 11% of the administered oligonucleotides

taken up by the cells. The uptake was faster for shorter oligonucleotides than for longer ones.

Decreasing the temperature prevented the oligonucleotide uptake almost completely, indicating

an active uptake mechanism. Any sequence or size of unlabeled oligonucleotide competed

with the labeled oligonucleotide for uptake, even DNA as small as a single nucleotide or as big

as yeast DNA. Free nucleosides however, were not competitive, showing that the phosphate

group is essential for this uptake mechanism. An 80 kDa oligonucleotide binding protein was

isolated using oligo(dT) cellulose beads and this protein has given rise the theory of the

receptor mediated uptake of oligonucleotides.

Others used 35S labeled oligonucleotides to study their uptake and intracellular

localization without altering the chemical structure (149). They described oligonucleotide

uptake which was concentration-dependent, reached a plateau in 16 hours and resulted in an

intracellular oligonucleotide concentration 100 times of that of the tissue culture medium

indicating an active uptake mechanism. An efflux mechanism has also been described

indicating temperature-dependent secretion of the oligonucleotides from the cells to the

extracellular space.

Separation of the nuclei from the rest of the cells indicated that only about 20% of the

cell-associated oligonucleotides was found in the nucleus, which was indicative for a

predominantly cytoplasmic uptake. Other groups, however, describe fast translocation of the

oligonucleotides from the cytoplasm to the nucleus which was found to be independent of the

ATP pool or temperature, suggesting diffusion through nuclear pores (150,151).







34
While some of the data might seem to be controversial, most of the differences are

probably due to different cell types and different oligonucleotides used. Indeed, quite dramatic

differences in oligonucleotide uptake rate has been shown between different cell lines (149). It

is generally believed that most types of oligonucleotides enter the cell by adsorptive or fluid

phase endocytosis, but the existence of the oligonucleotide receptor has to be proven by

purification of the protein.

Mechanism of Action

Antisense oligonucleotides have several potential sites of action, and different types of

oligonucleotides might not utilize the same one. According to the most obvious theory

antisense oligonucleotides inhibit translation by hybridizing to their respective mRNA thus

preventing either ribosomal assembly or ribosomal sliding along the mRNA. This kind of

action would assume that antisense oligonucleotides are acting in the cytosol and they do not

affect measurable mRNA levels. Indeed, there are several articles reporting antisense effects

without detectable change in target mRNA levels (152).

In cases where decreased mRNA levels are detected, the most likely explanation is the

digestion of the RNA portion of the mRNA-antisense DNA hybrid by RNase H. RNase H is

found in the cytoplasm as well as in the nucleus, and it is normally involved in DNA

duplication. In cell-free systems the role of RNase H is clearly demonstrated where the cleaved

RNA products were detected as smaller fragments, and in the case of chicken lysozyme, even

truncated polypeptides were detected (153). In cells, however, detection of the RNA

fragments appears to be difficult probably because the accelerated degradation of the truncated

mRNA fragments. Indeed, mRNA stripped from its cap sequence or poly-A tail is subject to







35
fast degradation. RNase H is thought to be activated by not only phosphodiester but also

phosphorothioated oligonucleotides, whereas oligonucleotides with modifications that do not

maintain the negative charge of the natural form will not activate RNase (153). Activation of

RNase H also seems to be advantageous because the enzyme leaves the antisense

oligonucleotide intact, so it is free to hybridize with another mRNA, making the reaction

catalytic rather than stochiometric.

In several cases however, the antisense oligonucleotides seem to be taken up by the

nucleus, suggesting other possible mechanisms of action. Antisense DNA can hybridize to its

target mRNA or pre-mRNA in the nucleus, forming a partially double-stranded structure which

would inhibit its transport from the nucleus to the cytoplasm, thus preventing transcription.

Antisense oligonucleotides targeted to intron-exon junction sites can prevent proper splicing,

and consequently the maturation of the transcript.

There is also a theoretical possibility of the antisense oligonucleotides' binding to the

DNA by triple helix formation or by binding to the locally opened loop created by RNA

polymerase (154). These appear to be possible only in special cases, e.g. triple helix formation

by antisense oligonucleotide binding in the major groove of the DNA can only happen if the

target sequence contains only purines on one strand and pirimidines on the other. Initial hopes

that triple helix formation by oligonucleotides or peptide nucleic acids can provide long-term

inhibition of transcription have been faded when it become clear that even covalent

modifications or DNA cleavage is corrected in the cell by various repair mechanisms. It is also

not clear how antisense oligonucleotides can have access to DNA in the chromatin.








Pharmacology ofAntisense Oligonucleotides



Antisense inhibition can be considered pharmacologically a drug-receptor interaction

where the oligonucleotide is the drug and the target sequence is the receptor. In order to

binding occur between the two, a minimum level of affinity is required which is provided by

hydrogen bonding between the Watson-Crick base pairs and base stacking in the double helix

which is formed. In order to achieve pharmacological activity, a minimum number of 12-14

bases can provide the minimum level of affinity (155).

Specificity

The specificity of the antisense oligonucleotide-target sequence interaction is provided

by the Watson-Crick base pairing. Considering that roughly 1% of the human genome

contains coding regions, an oligonucleotide of 11-15 nucleotides long is specific enough

statistically to be complementary to a single sequence (156). Increasing the length of the

antisense should result in higher level of specificity, but there is an upper limit of about 30

nucleotides above which oligonucleotides are not taken up effectively by cells. Instead of

increasing length, testing the antisense sequence against genetic databases for possible cross-

reaction with other than the target sequence is advisable.

Sequence-specificity can be ascertained experimentally by using control

oligonucleotides such as sense, which is identical to the target sequence, scrambled, which has

the same base composition as the antisense but in a scrambled order and mismatch, which is

similar to the antisense but contains 3-4 mismatches while maintaining the same base







37
composition. Naturally, the more the control sequence resembles the antisense, the more

stringent control it will provide.

Effectiveness

Antisense oligonucleotides can inhibit protein synthesis in cultured cells in pM doses.

This inhibition, however, never results in a total knockout of the target protein, but rather in a

partial decrease. Increasing the dose generally. does not result in complete inhibition, instead,

nonspecific effects might take place. Indeed, the therapeutical window for antisense

oligonucleotides is rather narrow: when testing for the optimal dose, small increments in the

low pM range should be used (140). This limited ability for protein synthesis inhibition is

certainly disadvantageous when targeting disease genes that need to be shut down completely,

but might be advantageous in e.g. neuropharmacology where modulation rather than

eradication of gene function is needed.

Toxicity

Phosphodiester oligonucleotides are degraded to their naturally occurring nucleotide

building blocks relatively quickly, therefore no toxic reaction is expected from even high doses.

Studies on phosphorothioated oligonucleotides in rats show that following intravenous

injection phosphorothioated oligonucleotides are taken up from the plasma mainly by the liver,

fat and muscle tissues. Phosphorothioate oligonucleotides are excreted virtually completely

through the urine in 3 days mainly in their original form. An apparent mild increase in plasma

LDH, and to a lesser extent in AST and ALT indicated a possible transient liver-toxicity (157).

The exceptionally long presence of the phosphorothioated oligonucleotide in the

organism is promising from a pharmacological point of view because sufficient tissue levels







38
could be maintained with infrequent administration. The fact that there is hardly any

metabolism of the compound is important because there is a theoretical possibility that these

unnatural nucleotides can be built into DNA of dividing cells possibly causing mutations.

In Vivo Applications of Antisense Oligonucleotides

Neuroendocrin functions such as vasopressin secretion has been successfully

suppressed with antisense oligonucleotides: injection of vasopressin antisense into the lateral

brain ventricle of the rat resulted in reduced vasopressin immunoreactivity in the hypothalamic

nuclei and in temporary diabetes insipidus which lasted up to 9 hours (158). In oxytocin

antisense treated rats where oligonucleotides were infused into the supraoptic nucleus of the

lactating rat there was a decrease in the number of milk ejection reflexes, and consequently,

there was significantly less weight gain observed in the litter (159).

Opioid receptors are also susceptible to antisense treatment. When injected

intrathecally in mice, the delta opioid receptor antisense has been shown to significantly inhibit

the antinociceptive action of deltorphin, a selective delta opioid agonist, whereas it was

ineffective in altering the pain response to g or K opioid agonists (160).

Antisense oligonucleotides have proven to be valuable research tools in identifying

functions of proteins. With no classical receptor antagonist available, Wahlestedt et al. used

antisense oligos in establishing the role of neuropeptide Y (NPY) in anxiety.

Intracerebroventricular injections of an NPY-1 antisense resulted in decreased density of NPY-

1 receptors in the cortex, with consequent change in behavior (161).

The role of kinesins in axonal transport was demonstrated in the optic nerve: when

antisense oligonucleotides to kinesin heavy chain were injected into the vitreous of rabbits,







39
retinal kinesin synthesis decreased, and to a similar extent, rapid axonal transport of proteins

was inhibited (162).

Animal behavior can be changed by interfering the synthesis of specific proteins in the

brain. Suppression of the synthesis of adrenocorticotropin (ACTH) and beta-endorphin in the

hypothalamic arcuate nucleus of the rat substantially decreased the grooming behavior normally

observed in a novel environment (163).

Reproductive behavior such as lordosis in female rats can be changed by inhibiting

progesterone receptor synthesis in the hypothalamus with antisense injections into the third

brain ventricle, or directly into the ventromedial hypothalamic nucleus (164-166). Inhibition of

two different forms of GABA synthetic enzyme GAD65 and GAD67 both led to a decrease in

female rat receptivity when injected into the medial hypothalamus and the midbrain central

gray, but with different time courses: GAD67 took effect within 24 hours and lasted for 4 days,

whereas GAD65 acted with a 48 hours delay and lasted 5 days (167). These differences might

be explained with differing turnover rates of the targeted proteins and the oligonucleotides.

Inhibition of catecholamine synthesis in the ventral tegmental area of the brain by

antisense oligonucleotides targeted to tyrosine hydroxylase mRNA led to reduction in operant

behavior, and this effect was observable for several days after the injection (168).

Dopamine receptors, which are known to induce rotational behavior in rats lesioned in

the corpus striatum, can be inhibited subtype-specifically with antisense. D2 dopamine receptor

antisense inhibited rotational behavior induced by intracerebroventricular injection of the D2

specific agonist quinpirole, whereas D, antisense was not effective (169). Conversely, D,







40
antisense inhibited behavioral responses elicited by the D, agonist SKF38393, and the D2

antisense proved to be ineffective (170).

Inhibition of the immediate-early genes with antisense oligonucleotides has become a

popular tool for investigating specific cellular responses requiring gene expression.

Intrahippocampal injection of c-jun antisense oligonucleotides impaired learning and memory

formation in rats (171). Superfusion of c-fos antisense onto rat spinal cord was shown to

suppress heat-induced c-Fos protein expression (172). Inhibition of c-fos expression in the rat

nucleus accumbens blocked cocaine-induced locomotor stimulation (173). On the other hand,

inhibition of c-fos expression unilaterally in the neostriatum lead to induction of ipsilateral

rotational behavior after d-amphetamine administration (174).

Important pathophysiological events such as arterial restenosis after balloon

cathetherization can be effectively influenced with antisense oligonucleotides. Inhibition of two

cell cycle regulatory enzymes, cyclin-dependent kinase 2 kinase and cell division cycle 2 kinase

with antisense oligonucleotides resulted in a nearly complete abolition of neointima formation

which is thought to be responsible for arterial restenosis (175). Application of c-myc antisense

oligonucleotides in apluronic gel to the outside of balloon-cathether injured blood vessel

resulted in 75% decrease in c-myc expression and significantly reduced neointima formation

(176). This result also suggests the remarkable penetrating ability of the oligonucleotides by

which they reached the neointima of the rat carotid from the outside of the vessel.

The outcome of cerebral infarction can be influenced with antisense oligonucleotides.

Synthesis of NMDA receptors, which are thought to be responsible for neuronal death

following cerebral vascular occlusion, can be inhibited by antisense treatment. Such an







41
inhibition reduces the volume of the infarction produced experimentally by the occlusion of the

middle cerebral artery (152).

Inhibition of tumor growth is one of the major fields of research in antisense

development. Indeed, the first in vivo application of antisense inhibition was designed to

downregulate N-myc and consequently inhibit neuroectodermal tumor growth. Antisense

oligonucleotides were delivered in an osmotic pump to the vicinity if the xenographic tumor,

and loss ofN-myc protein along with 50% loss in tumor mass was observed (177).
Gene Therapy with Antisense Expression Vector

Why do we Need Gene Therapy?

The answer in short is, that because contemporary research shows that a score of

diseases are caused by malfunctioning genes. Some of these faulty genes are already identified:

the mutated chloride-channel gene CFTR for cystic fibrosis, or BRCA1 for hereditary breast

cancer, and there are certainly a lot more to come. Curing these diseases will require the

correction or replacement of the disease-genes with the correct ones.

Not only inherited diseases are caused by genetic disorders, however, most cancers

now are viewed as acquired genetic diseases where the irregular expression of a cell regulatory

protein causes uncontrolled cell division.

Viral diseases are clearly caused by genes: viruses can be viewed as genetic

misinformation packaged into a tiny protein capsid. Some of the foreign viral genes can

integrate into the host cell's chromosome and cause disease in various ways, including cell

death or malignant transformation.







42
There are other conditions where there is no mutation, foreign DNA invasion or

expression of normally silent genes, but there is malfunction in the degree of the expression of

normal genes. It would be difficult to estimate the number of diseases which can be included

into this group, but experimental evidence suggests that hypertension is one of them.

What also links these disorders together is that currently there is no cure for them, only

treatment of the symptoms or in some instances, not even that. Gene therapy aims at

eliminating the cause of these diseases by correcting the genetic malfunctions.

The ideal solution for genetic disorders would be the replacement of the faulty gene

segment with a normal one by homologous recombination in all or at least in a clinically

significant fraction of the affected cells. Unfortunately, the frequency of homologous

recombination upon introduction of the correct gene is very low. It seems, however, that it

might be not all that critical introducing the correct gene into the exact same location where the

faulty gene is; and suppression of an overactive gene can be achieved form another locus on the

genome without excising it, with e.g. an antisense gene.

Viral Vectors

As it turns out, some of the best agents to date in the fight against genetic malfunctions

are the same viruses that cause some of these disorders; only the correct genes have to be put

into them. Viruses evolved for the specific task of delivering DNA (or RNA) into cells, and

researchers take advantage of this ability by using viruses for delivering recombinant genes into

diseased cells.







43
There are several viruses which have been tested for gene delivery, and each has its

advantages, but none of them fits perfectly to the description of the "ideal viral vector". To be

the perfect vector, a virus should fulfill all of the following criteria:

-it should be safe. This means that it cannot be a virus known to cause disease, or it

has to be re-engineered to be harmless. It cannot elicit immune response, and it cannot

integrate into the genome randomly carrying the risk of disrupting other cellular genes. It also

has to be replication-deficient for the prevention of the spread of it to unwanted tissues or

subjects.

-it should be efficient. The virus has to infect the target cells with high frequency in

order to achieve biological effect.

-it should be easy to manipulate. The virus should to be able to accommodate the gene

of interest, along with its regulatory sequences (promoter), and the recombinant DNA has to

be packaged with high efficiency into the viral capsid proteins.

Viruses Adopted for Gene Therapy

The most popular viral vectors in the past have been retroviruses. These have been

used primarily because of their high efficiency in delivering genes to cells (178). They proved

to be effective in cell culture systems, but they are notorious for their random integration into

the genome which raises concerns about their safety when used in vivo. Retroviruses also

require cell division for chromosomal integration, which makes them limited mostly to tumor

therapy.

Adenovirus vectors have been tested successfully in their natural host cells, the

respiratory endothelium, as well as other tissues such as muscle and brain (179-181). Most







44
adenovirus vectors in their current form are episomal, that is they do not integrate into the host

DNA. They provide high levels of expression for a significant amount of time, but the

episomal DNA will invariably turn inactive. Repeated infections, on the other hand, result in

inflammatory response with consequent tissue damage.

Herpesvirus vectors hold the promise for delivering genes into the nervous system.

They have the ability to spread from one neuron to the other, raising the possibility of infecting

cells of the central nervous system from peripheral injections. Controlling of this spread,

however, proved to be difficult, and continuous expression of the inserted gene is hindered by

latency periods intrinsic to herpesviruses.

Adeno-associated virus (AAV), has been gaining attention because its safety and

efficiency (182). It has been successfully used for delivering antisense RNA against alpha-

globin (183) and HIV LTR (long terminal repeat) (184), and it is our vector of choice for

delivering antisense RNA to the AT-I receptor.

Adeno-Associated Virus

AAV is a parvovirus, discovered as contamination of adenoviral stocks. It is a

ubiquitous virus (antibodies present in 85% of US human population), which has not been

linked to any disease. It is also classified as dependovirus, because its replication is absolutely

dependent on the presence of a helper-virus, which can be adenovirus or herpes virus. Five

serotypes have been isolated, of which AAV-2 is the best characterized. AAV has a single-

stranded linear DNA which is encapsidated into capsid proteins VP1, VP2 and VP3 to form an

icosohedral virion of 20-24 nm in diameter.







45
The AAV DNA is approximately 4.7 kilobases long. It contains two open reading

frames and it is flanked by two terminal repeats. Each terminal repeat can form a T-shaped

hairpin structure. These terminal repeats are the only essential components of the AAV for

chromosomal integration. They also serve as origins of replication. There are two major genes

in the AAV genome: rep and cap. rep codes for proteins responsible for viral replication,

whereas cap codes for capsid proteins VP1-3. Three promoters have been identified and

named p5, p19 and p40, according to their map position. Transcription from p5 and p19 result

in production of rep proteins, and transcription from p40 produces the capsid proteins (185).

The life cycle of adeno-associated virus

Upon infection of a human cell, AAV integrates to the q arm of chromosome 19 (186).

For chromosomal integration the only essential component are the terminal repeats. The viral

components responsible for site-specific integration are yet to be identified. With no helper

virus present, AAV infection remains latent indefinitely. Upon superinfection of the cell with

helper virus the AAV genome is excised, replicated, packaged into virions and released to the

extracellular fluid.

Adeno-associated virus as expression vector

There are several factors that prompted researchers to study the possibility of using

AAV as an expression vector. One is that the requirements for delivering a gene to the host

chromosome is surprisingly little: the presence of the terminal repeats- which are each only 3%

of the AAV genome- is sufficient for chromosomal integration. No viral proteins required,

and the cellular proteins that are needed for integration are most likely ubiquitous cellular

components.







46
The second characteristics that makes AAV a good vector candidate is its relative

complicated rescue mechanism. As opposed to integration, productive infection requires

several factors including superinfection with a second virus as well as transcription of viral

proteins, which gives several opportunities for preventing the spread of recombinant AAV

vectors to non-target areas.

Another reason why AAV is a premier candidate for gene therapy is its exceptionally

broad host range. It infects all mammalian tissues tested apparently with the only exception of

vascular endothel cells (187).

Preparation of recombinant AAVs is relatively easy: 94% of the genome can be

replaced for the gene(s) of interest. There are, however, disadvantages to such a small virus.

Recombinant DNA larger than the wild-type AAV is not encapsidated effectively, so the 4.4

kilobases available space has to accommodate the promoter, coding sequence, polyA tail and

possibly a marker protein gene. Other difficulty is the preparation of the viral stocks: addition

ofadenovirus as helpervirus to packaging cells contaminates AAV stocks. It is also a matter of

concern that the site-specific integration of AAV into the ql3.4-ter of chromosome 19 appears

to be lost when cells are infected with recombinant AAV.

In summary, AAV appears to be one of the best candidates for gene delivery. Most of

the aforementioned problems appear to be technical and should be solved satisfactorily in the

near future.

Expression of Antisense Sequences with Vectors

Long-term suppression of synthesis of individual proteins is desirable in conditions

where overexpression of endogenous genes or expression of foreign genes is thought to be the







47
cause of the disease. Inhibition of synthesis of certain proteins for extended periods of time is

also useful when studying protein function, e.g. in development.

Antisense expression vectors produce antisense RNA, which can hybridize with the

target mRNA and prevent its translation by either promoting its degradation, preventing its

transport from the nucleus to the cytoplasm or interfering with ribosomal function.

AAV-derived expression vectors have been successfully used to inhibit alpha-globin

expression in an erythroleukemia cell line where a 1.0 kb fragment of the alpha-globin gene was

inserted in the antisense direction under either thymidine kinase, SV40 or the alpha-globin

promoter. 91% inhibition in alpha-globin mRNA production was achieved with the alpha-

globin's own promoter, whereas the other two promoters proved to be less efficient (183).

A much shorter antisense sequence (64 base pairs long) targeting the human

immunodeficiency virus (HIV) long terminal repeat was expressed with an AAV vector using

the SV40 promoter in 293 cells. Greater than 99% reduction in infectious HIV-1 production

and the absence of cellular toxicity was observed for over a period of 20 days (184).

Recombinant AAV in the Brain

An AAV expression vector has been successfully tested in the central nervous system.

A recombinant AAV virus expressing tyrosine hydroxylase significantly raised dopamine levels

in the striatum of monkey brains. The hormonal change was sufficient to produce long-lasting

behavioral changes (188).

Summary of the Literature

Despite the immense knowledge accumulated over decades in circulation research, the

cause of essential hypertension is still unknown. The prevailing view today is that blood







48
pressure is determined by both genetic and environmental factors. Moreover, the inheritable

component of hypertension is thought to be the sum of the effects of several genes. Most of

the candidate genes for hypertension are members of the renin-angiotensin system.

The role of the brain RAS in the development of the hypertension has been indicated by

large volume of experimental evidence. The recent development of subtype-specific AngII

receptor antagonists, however, failed to support unequivocally the pivotal role of brain AngII

in hypertension.

Emerging new molecular techniques such as antisense allow us to approach these

questions from a molecular genetic point of view. Besides using antisense as a research tool,

evaluating it as a potential clinical agent is equally important.












CHAPTER 4

MATERIALS AND METHODS

Oligonucleotide Experiments

Animals

Adult male spontaneously hypertensive rats (SHR) and Sprague-Dawley (SD) rats

(250-300g) were acquired from Harlan. The animals were kept in individual cages in a room

with 12 hours light-12 hours dark cycle. They were given tap water to drink and standard rat

chow to eat ad libitum. For intracerebroventricular cannulation, each animal was anesthetized

with sodium pentobarbital (65 mg/kg body weight) intraperitoneally, and a stainless steel (23-

gauge) cannula was placed in the right lateral brain ventricle, using a Kopf stereotaxic

instrument (coordinates: 1.0 mm lateral, 1.0 nmmn caudal to the bregma; 5.0 numm below the skull

surface). The animals were allowed to recover for 5 days before further surgical procedures or

blood pressure readings were carried out.

Blood Pressure Measurements

Blood pressure and heart rate were monitored by a Statham pressure transducer via a

heparinized (100 U/ml) catheter placed in the left common carotid artery. Animals were

allowed to recover from carotid catheterization for 24 hours before experimentation. All

experimental protocols were approved by the Animal Care Committee of the University of

Florida, Gainesville, FL.








Oligonucleotides

Antisense (AS), sense (S) and scrambled (SC) oligonucleotides were synthesized as

15-mer phosphorothioated oligonucleotides on an Applied Biosystems 394 DNA/RNA

Synthesizer at the DNA Synthesis Core Facility at the University of Florida. AS

oligonucleotides were complementary to bases +63 to +77 of the AT-la receptor mRNA (29).

S oligonucleotides were identical to this target sequence, whereas SCR oligonucleotides had

the same base composition as AS but in a scrambled order. Prior to synthesis, oligonucleotide

sequences were tested for sequence-homology against all available DNA sequences in the

GeneBank. The purity of oligonucleotides was routinely tested with capillary electrophoresis.

Oligonucleotides were dissolved in 0.9% saline solution, aliquotted and stored on -20 C.

Drinking Response

SHRs were treated with i.c.v. injections of 50 pg/4gl antisense, scrambled

oligonucleotide or saline 24 hours before the drinking tests. Through the same i.c.v. cannula,

50 ng AngII (acetate salt, Sigma) dissolved in 2 pgl artificial cerebrospinal fluid was injected in

conscious, freely moving rats. The rats were presented with scaled drinking bottles, and the

amount of consumed water was recorded over a 30 minutes period.

Autoradiography

24 hours after oligonucleotide treatment the rats were sacrificed, the brains were

removed and frozen on dry ice. Cryostat-sections were cut at 20 gm thickness on -20 C, and

thaw-mounted onto gelatin-coated slides. The slides were preincubated in assay buffer (150

mM NaCl, 50 mM sodium phosphate, 1 mM EDTA, 0.1 mM bacitradcin, pH 7.4) for 30

minutes and then incubated in the same buffer with the addition of 500 pM 12I-Sar-Ile-Angll







51
for 2 hours. Sections were incubated with the radioligand in the presence or absence of 1 pM

AngII to determine nonspecific and total receptor binding. The sections were washed in assay

buffer and in distilled water, and dried under cool air. Autoradiograms were generated by

apposition of the slide-mounted tissue sections with X-ray film (Hyperfilm-3H, Amersham) for

3 days. Densitometric analysis of the autoradiograms was carried out with a video-based

computerized image analysis system (MCID, Imaging Research, Ontario, Canada).

Receptor Binding Assay on Hypothalamic Cell Membranes

Under metophane anesthesia, rats were decapitated and the brains were dissected. The

hypothalamic block, including the hypothalamus, septum and thalamus was homogenized in 4

ml ice cold TE buffer (50 mM Tris, ImM EDTA, 150 mM NaCI, pH 7.2) for 10 s using a

Tekmar SDT 1810 homogenizer and centrifuged at 2,000 g for 10 minutes. The pellet was

discarded and the supernatant was re-centrifuged at 50,000 g on 4 C for 30 minutes. The

resulting pellet was resuspended in 1.5 ml TE buffer, homogenized using a dounce glass

homogenizer and assayed for protein content with the Lowry method. 100 jg of membrane

proteins were incubated with 200 pM of the radiolabeled AngII analog 12I-Sar-lle-AngI in the

presence of absence of 1 pM cold Angi, Losartan or PD 123319 to determine the nonspecific,

total, AT-1 and AT-2 binding, respectively. The membrane proteins were incubated in

triplicates with the receptor ligands in 500 gl of incubation medium containing TE buffer, ImM

bacitracin, 10 pM 0-phenantrolene and 0.1% bovine serum albumin for 90 minutes on room

temperature. At the end of the incubation procedure, membrane-bound ligand was collected







52
on Whatman filter paper using a Brandel cell harvester, and radioactivity was determined with

a Beckman gamma-counter.

Receptor Binding Assay on Cultured NG108-15 Cells

NG108-15 cells were grown in Dulbecco's modified Eagle's medium (DMEM)

supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin and 1

gig/ml Fungizone in 100 mm plates in a humidified atmosphere of 5% CO2 and 95% air at

37C. Oligonucleotides were administered to 1-day-old cell cultures (60-70% confluent) in

serum-free DMEM at final concentrations of 2.5 or 5 gM. After 24 hours ofoligonucleotide

treatment, differentiation was induced by replacing the DMEM with medium containing 1.5%

dymethylsulfoxyde (DMSO) and 0.5% FBS. 48 hours after the initiation of oligonucleotide

treatment the differentiating medium was removed, the cells were washed with cold phosphate

buffered saline (PBS) and collected in TE buffer (4 ml/plate) by mechanical dissociation. Cells

were homogenized with a dounce glass homogenizer and centrifuged on 50,000 g for 30

minutes on 4 C. The resulting pellet was resuspended in 1.5 ml TE buffer, rehomogenized and

assayed for protein content. Receptor binding assay was performed as described above.

Northern Blotting

NG108-15 cells were treated with oligonucleotides and differentiated as described

above. 48 hours after the initiation of oligonucleotide treatment, cells were washed with PBS,

and total RNA was extracted and purified with TRI-Reagent. 12 gig RNA/lane was run on a

1.2% agarose-formaldehyde gel on 30 V for 16 hours. RNA was transferred to a nylon

membrane and stabilized by UV-crosslinking. A 3'P-labeled riboprobe was generated by in

vitro transcription of the linearized AT-la cDNA using the Promega Gemini System II. The







53
AT-la clone pKSCalSb was a gift from T.G. Murphy (Emory University, Atlanta, GA).

Membranes were prehybridized in 50% formamide, 5X SSPE, 5X Demhardt's solution, 0,8%

SDS and 10 mg salmon sperm DNA for 4 hours at 56 C, then hybridized in the same solution

with approximately 106 cpm/ml AT-la riboprobe for 15-18 hours. The following day the

membranes were washed with 1 x SSPE, 0.5% SDS for 3X15 minutes at 65 C and in 0.1 x

SSPE, 0.5% SDS for 30 minutes at 65 C. The membranes then were wrapped in plastic foil

and exposed to X-ray film, which was developed 3-7 days later.

Detection of Fluorescent Labeled Oligonucleotides

AS and S phosphorothioated oligonucleotides were labeled on both 5' and 3' ends with

fluorescein isothiocyanate (FITC) at the DNA Synthesis Laboratory of the University of

Florida. Rats equipped with intracerebroventricular guide cannulas were injected with 50 p g

FITC labeled oligonucleotides dissolved in 4 p.1 saline. 1 hour later the rats were sacrificed, the

brains were removed and cryostat-sectioned into 20 pm-thick slices. The slide-mounted brain

slices were viewed with a BioRad 600 MRC laser-scanning confocal microscope. For cellular

uptake experiments, NG108-15 cells were cultured on 1.2 cm diameter glass cover slips, and

1-day-old cell cultures were treated with 5 or 25 pM FITC-labeled oligonucleotides for various

time periods. At the end of the incubation, the cells were washed with PBS, the cover slip

carrying the cells was removed from the culture dish and mounted upside down onto a glass

microscope slide using paraphenyline diamine mounting medium. The slides were stored in the

dark until viewed with laser-scanning confocal microscope.








Expresion Vector Experiments

Expression Vector Construction

pJDT95, a pBR322-derived plasmid containing the AAV-2 genome with truncated cap

gene was provided by E.M. Meyer (University of Florida, Gainesville). pTR-UF, a plasmid

carrying the terminal repeats but lacking the rep and cap genes of AAV was provided by N.

Muzyczka (University of Florida, Gainesville). pKSCal8b was carried in Esherichia Coli

DH5a, whereas pJDT95 was carried inKColi HB101. Both strains of bacteria were grown in

ampiciUin (50 g.g/ml) containing Terrific Broth. The bacterial transformation, growth and

alkaline lysis of bacteria was performed according to Sambrook et al.(189). Plasmids were

purified using Magic Maxipreps (Promega). The strategy for constructing the AT-1 antisense

expression vector paATI is summarized in Fig. 15. pJDT95 was linearized by restriction

enzyme digestion with HIindUl and KpnI. A 855 bp fragment of the AT-1 cDNA including the

first 761 bp of the coding region and 94 bp upstream of the transcription initiation codon

(ATG) was copied with polymerase chain reaction (PCR) using a 3' primer which incorporated

a HindiH recognition sequence on its 5' flanking region. The resulting PCR product was

digested with KpnI and HIindlH to generate compatible ends with pJDT95. This AT-1 cDNA

fragment was ligated into the linearized pJDT95 using T-4 ligase. The different recognition

sequences of Hindin and KpnI assured the proper antisense orientation of the AT-1 fragment.

The resulting recombinant products were analyzed by restriction enzyme digestion with NdeI

and subsequent 1% agarose gel electrophoresis. DNA fragments were visualized by ethidium

bromide staining and were photographed under ultraviolet illumination with Polaroid camera.

Restriction and modifying enzymes were purchased from Promega (Madison, WI).








Transfection ofNG108-15 Cells with Expression Vector

The antisense vector paATI was delivered to the cells using the cationic lipid

Transfectam (Promega). The positively charged Transfectam binds the vector DNA and it also

shows affinity to the cell membrane lipids. The exact mechanism of cellular uptake of plasmid

DNA is, however, unclear. Transfectam has been shown to deliver plasmid DNA effectively

into a variety of cell types. We mixed 15 pg of antisense vector DNA with 30 pl of

Transfectam in serum-free medium, and overlayed it onto 65-75% confluent NG108-15 cells

grown on 100 mm poly-L-lysine coated plates. The cells were incubated on 37C overnight,

then the medium was replaced with differentiating medium containing 1.5% DMSO and 0.5%

FBS. Differentiation ofNG108-15 cells continued for 48 hours, after which cells were washed

and subjected to receptor binding assay or immunocytochemistry. Control cells received

treatment with either expression vector without antisense sequence completed with

Transfectam, or Transfectam alone, or no treatment.

Helper Virus Superinfection of AAV-transfected Cells

A heat-sensitive mutant adenovirus ts149 was used as helper-virus to enhance antisense

RNA production, ts149 was grown in 293 cells, a human transformed kidney cell line.

Approximately 90 % confluent 293 cells were infected with 10 m.o.i. (multiplicity of infection)

ts149. Infected 293 cells were grown in DMEM supplemented with 10% calf serum (CS) and

100 U/ml penicillin/streptomycin on 32 C, which is the permissive temperature for ts149.

Four days later 293 cells were harvested, the cells were disrupted by brief sonication and the

cell debris was separated by centrifugation. The ts149 viruses were isolated by density gradient

centrifugation in CsCI2 gradient. Virus stocks were stored in 50% glycerol on -20 C.







56
Upon transfection of NG108-15 cells with the antisense vector paATI, 5-10 m.o.i. (5-10

helper virus/cell) ts149 was added to the transfection medium along with the expression vector.

Transfection of Brain Cells in Vivo

SHRs were equipped with guide cannulas aiming at the anteroventral third ventricle

(AV3V) area of the rat brain. The coordinates were: 1 mm lateral, 0.3 mm rostral from the

bregma, 8.1 mm deep in 7 degrees angle in a 250-275 g rat. The nose bar was set at -3.3 mm.

Following recovery, a mixture of 0.5 gg paATI, 1 Wl Transfectam and 0.5 pIl (5x105) helper

virus (ts149) was slowly (0.5 pl/minute) infused into the AV3V region of the brain of SHRs.

Systolic blood pressure was recorded before and 3 days after the infusion using the tail cuff

method. After the last blood pressure recordings, the rats were sacrificed and the brains were

removed for immunohistochemical detection of expressed viral rep proteins.

Immunohistochemistry

Rats were anesthetized and the heart was exposed. The brain was perfused after

clamping the descending aorta and the inferior vena cava, opening the right auricle and

inserting a needle into the left ventricle. 50 ml saline containing 100 U/ml heparin was infused

through the heart followed by 100 ml Zamboni's fixative. The brain was removed and stored in

Zamboni's fixative overnight. The anterior hypothalamic region was sectioned into 40 pm

thick slices and washed in PBS. The slices were then pre-treated with 3% hydrogen peroxide,

10% ovalbumin, 3% goat serum and 0.2% Triton 100X in PBS. The primary rep antibody

IF811 was diluted 1:1000 in PBS containing 0.2% Triton 100X and 1% goat serum, and

incubated with brain slices on a shaking table on 4 C overnight. The next day the brain slices

were washed in PBS, and incubated with rabbit anti-mouse IgG for 45 minutes on 4 C. The







57
slices were washed again, and incubated with peroxidase-antiperoxidase for 45 minutes on 4

C. The complex was stained with diaminobenzidine (DAB) in hydrogen peroxide, the brain

slices were mounted on microscope slides, coverslipped and viewed under light microscope.

Immunocytochemnistry

For detecting rep expression in cultured cells, NG108-15 cells were grown on poly-L-

lysine coated 35 numm culture dishes to 50-60% confluence, and treated with the expression

vector paATI and helper virus ts149 as described above. Cells were incubated on 37 C for 48

hours, then were washed three times with PBS. Cells were fixed with 3.5% formaldehyde in

PBS on room temperature for 30 minutes, then rinsed with PBS three times. Cells were

permeabilized by incubating them with 0.2% Triton 100X and rinsed with PBS. The primary

antibody IF811 was diluted 1:25,000 in PBS, and incubated with the cells overnight on 4 C.

Following three rinses with PBS the cells were incubated with the secondary antibody, and

stained with anti-peroxidase and DAB as described above. The primary antibody IF811 (a

generous gift of Dr. Nick Muzyczka, University of Florida, Gene Therapy Center) was raised

in mouse and it recognizes all four rep proteins.

Statistical Procedures

All numerical values are expressed as mean + SEM. Statistical analysis was performed

using Sigmastat (Jandel Scientific) computer software. One-way ANOVA was used to

determine treatment effect in the receptor binding studies, whereas two-way ANOVA was

used to analyze blood pressure data. Duncan multiple range test was used for individual

comparisons. A probability (p) value of<0.05 was considered statistically significant.












CHAPTER 5

RESULTS

The Plan of the Study
Antisense Oligonucleotide Experiments In Vivo

1. First we designed antisense olgonucleotides targeting the AT-1 receptor mRNA.

Two oligonucleotides were designed: the first was a 18-mer phosphodiester targeting bases 1-

18, while the second was a 15-mer phosphorothioate targeting bases 63-77. Corresponding

sense and scrambled oligonucleotides had also been synthesized.

2. These oligonucleotides were then tested for their ability to lower blood pressure

upon their injection into the lateral brain ventricle of SHRs.

3. Autoradiography or membrane receptor binding was performed on oligonucleotide-

treated rats to detect any changes in AngI receptor number.

4. Functional activity of brain AT-1 receptors following antisense oligonucleotide

treatment was tested by measuring drinking response to i.c.v. AngI injection.

5. Oligonucleotide uptake was studied using fluorescein-labeled oligonucleotides.

Antisense Oligonucleotide Experiments In Vitro

6. A glioma-neuroblastoma hybrid cell line NG108-15 was used to study the cellular

mechanism of antisense inhibition. Membrane receptor binding experiments established the

effectiveness of antisense oligonucleotides in inhibiting AT-1 receptor synthesis.

7. Northern blotting was performed to detect changes in the AT-1 mRNA.

58







59
8. The cellular uptake of fluorescein labeled oligonucleotides was studied with laser-

scanning confocal microscope.

Antisense Expression Vector Experiments

1. Antisense expression vectors were constructed, and their ability to downregulate

AT-1 receptor or angiotensinogen synthesis was tested by receptor binding or RIA,

respectively.

2. Expression of transcripts from the AT-1 antisense vector was detected by

immunocytochemistry.

3. The AT-1 antisense expression vector was injected into SHRs, followed by blood

pressure recording and immunohistochemical detection of a viral protein.

Antisense Oligonucleotide Experiments In Vivo

Blood pressure of antisense oligonucleotide treated SHRs

First an 18-base long ("18-mer") antisense oligonucleotide was tested for its ability to

lower blood pressure in the SHR. The 18-mer oligonucleotide targeted bases -6 to +12 of the

AT-la mRNA, thus covering the AUG translation initiation codon (Fig. 1). This

oligonucleotide contained natural phosphodiester bonds with the exception of the 17th, which

was thioated to resist 3' exonucleases. Spontaneously hypertensive rats were equipped with

guide cannulas to the right lateral brain ventricle. The 18-mer oligonucleotides were

administered every 12 hours for 3 days in a dose of 50 tg/ 4 gl i.c.v. Systolic blood pressure

decreased on the 2nd day after the beginning of the injections and remained low on the third

day as determined by tail cuff blood pressure recordings (Fig. 2).








(A)

Antisense: 5'-GTT AAG GGC CAT TTT GTT-3'

Sense: 5'-AAC AAA ATG GCC CTT AAC-3'



(B)

Antisense: 5'-TAA CTG TGC CTG CCA-3'


Sense:


5'-TGG CAG GCA CAG TTA-3'


Scrambled: 5'-CTT ACT AGC CTA GGC-3'




Figure 1. Base sequence of oligonucleotides. (A) The 18 bases long (18-mer) antisense
oligonucleotide was complementary in sequence to bases -6 to +12 of the AT-la mRNA
(target sequence). A sense oligonucleotide served as control, the base sequence of which was
similar to the target sequence. The 18-mers were phoshodiester oligonucleotides, except the
17th base, which was modified by phosphorothioation. (B) The second set of oligonucleotides
were 15-mers and were targeted to bases +63 to +77. Besides the antisense oligonucleotide, a
sense oligonucleotide, which has similar base composition as the target sequence on the AT-1
mRNA, and scrambled oligo having the same base composition as the antisense but in a
random order, served as control.











220-
I-- I Control
210- Sense
Antisense
0)
X 200-
E
190-
h.
180-
40
0*
1 170*

160
0
m 150-
0
2*, 140-

0- 130-

120
baseline 1 2 3
Days treatment


Figure 2. Blood pressure lowering effect of a 18-mer phosphodiester antisense oligonucleotide
targeted to the translation initiation site of the AT-1 receptor mRNA. 50 gig oligonucleotide
was injected every 12 hours for 3 days into the lateral brain ventricle of spontaneously
hypertensive rats. Systolic blood pressure was monitored daily with a tail cuff blood pressure
recorder (*: p<0.05, two-way ANOVA followed by Duncan multiple range test; n=3-4).







62
Then fully phosphorothioated antisense, sense and scrambled oligonucleotides were

synthesized corresponding to nucleotides +63 to +77 of the AT-la receptor mRNA. SHRs

were fitted with carotid catheters for direct blood pressure recording, and were injected with

50 p.g of sense or antisense oligonucleotide dissolved in 4 ,Ll saline through an i.c.v. cannula.

Baseline blood pressure data was recorded 1 hour before oligonucleotide injection. Three

measurements were taken 10 minutes apart and averaged. The mean arterial pressure (MAP)

was calculated using the equation MAP=0.43(S-D)+D, where S and D represent systolic and

diastolic pressure, respectively. Antisense oligonucleotide injection into the lateral brain

ventricle resulted in a significant fall of 27 + 8 mmHg in the mean arterial pressure of SHR

(Fig. 3). For the antisense oligonucleotide treated rats, the pre-treatment mean blood pressure

was 178 + 5 mmHg, and the 24 h post-treatment mean blood pressure was 150 + 6 mmHg

(n=7). Saline, scrambled or sense oligonucleotide injections did not alter blood pressure in

SHR.

Drinking response to AngHi stimulation

SHRs treated with antisense or scrambled oligonucleotides or saline i.c.v. 24 hours

earlier were stimulated to drink by i.c.v. injections of 50 ng/ 2jgl AngI. The amount of water

consumed over a 30 minutes period immediately after the AngII injection decreased from the

control levels (11.7+0.5 ml/30 minutes) to 5.0 + 0.8 ml/30 minutes in the antisense

oligonucleotide treated rats (p<0.01) (Fig. 4). The water intake in the scrambled

oligonucleotide treated rats showed no significant difference from that of the animals without

oligonucleotide treatment.











200-

190 -

180 -

170 -

160 -

150-

140 -

130 -

120-

110 -


pre-treatment
24 h
T


TT


100 MIT= -II
Control Scrambled Sense Antisense

Oligonucleotide treatment


Figure 3. Decrease in mean arterial pressure in SHR 24 hours after a single i.c.v. injection of
50 gg of AS-ODN specific for AT, receptor mRNA. Baseline blood pressure data was
recorded prior to the injection. All blood pressure data were obtained by direct measurement
through a carotid catheter. The mean arterial pressure was calculated using the equation:
[0.43(S-D)]+D where S and D represent systolic and diastolic pressure respectively (*: p<0.05,
two-way ANOVA followed by Duncan multiple range test; n=6-7).







64




16-

S14-

o 12-
Elo

o0
8_ -- -

V= 6-----
4-*



CO

0T -------


Control Scrambled Antisense
Oligonucleotide treatment


Figure 4. Effect of AT-1 antisense oligonucleotides on drinking in response to i.c.v. AngII.
Spontaneously hypertensive rats were administered 50 ng AngIl 24 hour after saline (0.90/%),
scrambled or antisense oligonucleotide treatment. The water intake of each rat was measured
for 30 minutes after AngII injection. (**:p<0.01, ANOVA followed by Duncan multiple range
test; n=5).








Autoradiography of AT-1 receptors after antisense oligonucleotide treatment

After the 24 h blood pressure measurements, rat were sacrificed, the brains were

removed and cryostat-sectioned for autoradiography. 12I-Sar-Ile-AngII binding was decreased

by 16% in the paraventricular nucleus (PVN), 9 % in the suprachiasmatic nucleus (SCN) and

17 % in the median preoptic nucleus (MnPO) in the antisense oligonucleotide (50 Rg/4 jgl)

injected rat brains compared to saline-injected controls. Binding in the sense and scrambled

controls in the MnPO, however, was also decreased to some extent (about 10%). Similar

tendency was observed in the SON, which showed a decrease of 14 % after antisense

treatment and 8-9 % decrease after sense or scrambled oligonucleotide treatment. There were

no changes in the PVN in the sense or scrambled oligo treated rats (Fig. 5). Receptor numbers

in the mediodorsal thalamic nucleus and in the lateral septum, areas that are known to have

type-2 Angl (AT-2) receptors, did not decrease after antisense injection.

In an effort to improve the delivery of antisense oligonucleotides to their proposed site

of action, the hypothalamus, we injected 50 pgg/ 2 gl oligonucleotides into the third brain

ventricle once a day for three days (coordinates: 1mm lateral, 1mm caudal to the bregma, 8.5

mm ventral in 8 degree angle). We observed 14% decrease in the PVN, 14% in the OVLT and

10% in the MnPO 3 hours after the third antisense oligonucleotide injection. Similar treatment

with sense oligonucleotides resulted in no change in the PVN, 14% decrease in the OVLT and

29 % increase in the MnPO. Direct injections of 25 gg/ 2 pl antisense oligonucleotides in

the right PVN (coordinates: 1 mm lateral, 1.8 mm caudal to the bregma, 6.5 mm ventral)

resulted in an apparent decrease in AT-1 receptor binding when compared to the contralateral

PVN (Fig. 6)., but the controls showed almost the same change.










































Figure 5. Computer-enhanced images of representative autoradiograms showing reduced
AngIl receptor density in the antisense oligonucleotide treated rat brain. 24 hours after the
i.c.v. oligonucleotide injection the brains were removed, and 20 glm sections were incubated
with 500 pM '2I-Sar-]le-Angl for 2 hours in the presence or absence of excess AngI. The
images show coronal sections of scrambled (SCR), sense (S) and antisense (AS) treated rat
brains in the level of the paraventricular nucleus of the hypothalamus.


S





































Figure 6. 12I-Sar-Ile-Angll binding in the brain injected with oligonucleotides directly into the
tissue. 25 gig antisense oligonucleotide in 2 gl saline was slowly infused into the right
paraventricular nucleus. Bar shows the color code for signal intensity.








Long-term effect ofantisense treatment in SHR

It was noticed upon testing some of the surviving rats that the depressor effect of the

antisense oligonucleotide injection lasted for more than 24 hours. A long-term experiment was

designed where the blood pressure of antisense treated SHRs was monitored daily for 9 days.

A baseline blood pressure recording was taken, and 50 g.g/4 Il antisense or scrambled

oligonucleotide was injected into the lateral brain ventricle. Daily blood pressure recordings

were taken between 10:00-11:00 am. every day for 9 days. There was significant decrease

in the mean arterial pressure (MAP) between days 1 and 7 after antisense oligonucleotide

injection when compared to scrambled oligonucleotide treated rats. MAP decreased to

normotensive levels at day 1 (137.8 + 3.5 mmHg) in the antisense oligonucleotide treated rats,

reached its lowest value on day 2 (133.1 + 7.8 mmHg) and remained below control levels

through day 7 (Fig 7). Blood pressure gradually returned back to hypertensive levels, but it

reached the MAP of scrambled oligonucleotide treated rats only at day 8 after treatment.

Scrambled oligonucleotide injection (50 p.g / 4 p1) did not cause significant changes in MAP of

SHRs throughout the 9-day monitoring period. Heart rate did not show significant changes

from their pre-treatment values (antisense: 435 + 51 /minutes; scrambled: 408 + 12 /minutes).

In the antisense oligonucleotide treated rats, however, there was a tendency of elevated heart

rate when compared to the heart rate of scrambled treated rats (Fig. 8), which might be a

interpreted as a consequence of baroreflex compensation for the lowered blood pressure.

Angiotensin receptor binding on membrane fractions ofhypothalamic cells

The time point when there was the greatest difference in MAP between antisense and

scrambled oligonucleotide treated rats was chosen for receptor binding experiments. At day 3












200 -

190-

Z 180-
E
E
170-

S160- '

150-

140-

| 130-

S120-
** Scrambled
110- H Antisense

100 I I I i 1 I I i
0 1 2 3 4 5 6 7 8 9
Days post-injection


Figure 7. Time course of the blood pressure decrease in SHR following a single injection of
AT-I antisense oligonucleotide. Rats received 50 jig of antisense or scrambled
oligonucleotides in 4 ll saline into the right lateral brain ventricle on day 0. Blood pressure
recordings were made prior and every day after the injection for 9 days between 10 11 am
(*:p<0.05, **:p<0.01, two-way ANOVA, followed by Duncan multiple range test; n=4-1 1).












550-


500 -


450 -


400 -


350-


300 -


250-


200 -


I I I I I I I I I I
0 1 2 3 4 5 6 7 8 9


Days post-injection




Figure 8. Heart rate of SHR after i.c.v. injection of 50 g.g of AS-ODN or 50 jtg of SC-ODN.
Heart rate was measured simultaneously with the daily blood pressure recordings using a
Statham transducer and Gould 2400 recorder (n=4-1 1).


- Antisense
H-M Scrambled







71

post-treatment the rats were sacrificed, the brains were removed, and the hypothalamic

blockcontaining the septum, thalamus and hypothalamus was dissected, homogenized and

centrifuged for radioligand binding. MI-Sar-Ile-AngII binding was determined in the presence

or absence of unlabeled Angl, Losartan or PD123319 to determine nonspecific, total, AT-1

and AT-2 receptor binding, respectively.

Two doses of oligonucleotides were tested. Both with 25 pg and 50 pg

oligonucleotides there was a significant decrease in AT-1 binding in the antisense

oligonucleotide treated rat hypothalami (894 46 cpm/lOO1g tissue homogenate and 850.8 +

46.1 cpm/100gg tissue homogenate for the lower and the higher dose, respectively) when

compared to scrambled oligonucleotide treated rat hypothalami (1044 + 32 cpm/100lg tissue

homogenate and 1039.2 + 50.9 cpm/100gg tissue homogenate). There was no change in AT-

2 binding (Fig. 9).

Oligonucleotide uptake by brain cells

Antisense oligonucleotides were labeled on 5' and 3' ends with fluorescein-

isothiocyanate (FITC). 50 gg/ 4 gl labeled oligonucleotides were injected in the lateral brain

ventricle of SHRs. 1 hour later rats were sacrificed, the brains were removed and cryostat-

sectioned into 20 mm thick slices. The brain slices were mounted onto glass slides, and viewed

with confocal microscope. Fluorescent signal was detected in the lateral brain ventricles on

both sides, in the third ventricle as well as in the fourth ventricle. Fluorescence was also

detected in the cortex. The oligonucleotides diffused well in the cerebrospinal fluid; there was

no apparent difference between the strength of the fluorescent signal in the lateral ventricles or















1200


#A
) 1000-

0)
:=L
o 800-
E
0.
2 600-
0)


400-
C

")
c



a)
,



C4
o1
"? 0-
N
I-


Scrambled (25gg)
Scrambled (50gg)
Antisense (25p g)
Antisense (50gg)


AT-1


AT-2


Receptor subtype


Figure 9. AngIl receptor binding assay on cell membranes prepared from the hypothalamic
region including the hypothalamus, thalamus and septum of SHRs. Binding of 'OI-Sar-Ile-
AngIl was determined in the presence of the AT-1 and AT-2 subtype-specific AngI receptor
antgonists losartan and PD 123319, respectively. The assay was performed 3 days after
injection of two different doses (25 jig or 50 gig) oligonucleotide (*:p<0.05, t-test, n=5).







73
the in third ventricle. Oligonucleotides diffused into the brain tissue in 1-2 mm deep from the

ventricles and from under the dura mater. The highest concentrations of oligonucleotides were

found in the tissue bordering the ventricles, and the signal decreased as the distance from the

ventricles increased (Fig. 10).

When the same amount of FITC-labeled antisense oligonucleotides were injected into

the third ventricle, a similar pattern of oligonucleotide distribution was observed: equal spread

of fluorescent signal in the lateral and in the third ventricle, whereas less signal was detected in

the fourth ventricle and on the surface of the cortex.

Antisense Oligonucleotide Experiments In Vitro

Receptor binding in antisense oligonucleotide treated NG108-15 cells

NG108-15 cells, a rat glioma mouse neuroblastoma cell line was grown to 65-75%

confluence on poly-L-lysine treated 100 mm tissue culture dishes. 3 x 106 cells / dish were

treated with 2.5 or 5 pM sense or antisense oligonucleotides or with saline in serum-free tissue

culture media (DMEM). On the following day, the culture media was replaced with

differentiating media, and the cells were differentiated for 24 hours. At the end of the

differentiation period, cells were harvested, and receptor binding was performed on cell

membrane preparates.

5 pM antisense oligonucleotide treatment resulted in a significant decrease in 1'25I-Sar-

Ile-AngII binding (35% decrease when compared to the saline treated control cells) when

compared to the sense oligonucleotide treated cells (10% decrease compared to control). 2.5

pM antisense oligonucleotide treatment did not cause significant change in receptor binding in

NG108-15 cells (Fig. 11).







































Figure 10. Distribution of FITC-labeled antisense oligonucleotides in the brain of a Sprague-
Dawley rat one hour after injection of 50 jig oligonudcleotide into the right lateral brain
ventricle. The sagittal gap represents the third ventricle in coronal section.














-.. 120-
0
73

0
S100-



4)80 *
0.


"6 60=
C
:3

40-


20-
ID
N
VL 0
S2.5uM A2.5uM S 5uM A5uM


oligonucleotide treatment




Figure 11. Receptor binding on membrane preparates of NG108-15 cells shows 35% decrease
in '2I-Sar-lIe-AngI binding in cells treated with 5 pM antisense ODNs for 24 hours when
compared to sense ODN treated cells. The cells were differentiated for another 24 hours after
oligonucleotide treatment, then processed for receptor binding. 100 jgg of the membrane
homogenates was incubated with 200 pM '2I-Sar-IIe-AnglI in the presence or absence of 1
pM unlabeled AngI for 2 hours. Membrane-bound ligand was separated using a Brandel cell
harvester. Radioactivity was measured with a Beckman gamma counter. Values are shown as
percent of control (1885359 cpm/100 pig cell homogenate), where control cells received
0.9% saline treatment (*:p<0.05, t-test, n=3-4).








Detection of AT-1 mRNA with Northern blot after oligonucleotide treatment

In order to detect any possible changes in the amount or length of the mRNA of the

AT-1 receptor after antisense treatment, we treated NG108-15 cells with 5 AM sense or

antisense oligonucleotides or saline vehicle, and after 24 hours of cell differentiation total

cellular RNA was extracted, run on 1.2 % agarose gel, blotted onto nylon membrane and

hybridized overnight with a 32P-labeled riboprobe. The membranes were washed and exposed

with X-ray films for 3-7 days. The resulting autoradiograms were analyzed with a digital

densitometer system.

The pooled results of three such experiments showed that there was no significant

difference in the amount of AT-1 mRNA detected in the control, sense or antisense treated

cells (Fig 12). The probe hybridized with a single band of RNA in all treatment groups. The

estimated size of the RNA in this band is in good agreement with the expected size of the AT-1

mRNA (2.3 kb).

Cellular uptake of oligonucleotides

To determine the transfection efficiency and the intracellular localization of

oligonucleotides, we administered 5 or 25 pM FITC-labeled sense or antisense

oligonucleotides to NG108-15 cells and incubated them for 15 minutes, 30 minutes, 1 hour, 6

hours and 24 hours on 37 C. At the end of the incubation period the cells were fixed,

mounted onto glass slides, and examined with laser-scanning confocal microscope.

Examination of the cells with laser-scanning confocal microscope showed ring-shaped

intracellular localization of the fluorescent labeled oligonucleotides after 15 minutes and 1 hour






77






2.4-
0
0
2.2 OD
r 0 0
oW
1.8
e 2.0 C
> 1.86



1.4-

1.2-

1.0-

0.8-
0
IV 0.6-
0.
0 0.4-

0.2


Control Sense Antisense

Oligonucleotide treatment


Figure 12. Computer-aided densitometric readings of the pooled results of three Northern
blots. NG108-15 cells were treated with 5 gM sense or antisense ODNs for 24 hours, then
differentiated for an additional 24 hours. Cells were harvested and RNA was extracted using
TRI-REAGENT (Molecular Research Center, Inc.). Twelve microgram of the extracted RNA
was ran on agarose-formaldehyde gel and transferred to nylon membrane. A radiolabeled
runoff transcript of the plasmid pKSCal8b carrying the full length AT, receptor cDNA was
used for Northern hybridization. Control cells received 0.9% saline treatment (n=3). A
representative Northern blot is also shown.







78
incubation. After 6 hour incubation, however, the fluorescent signal was detected almost

exclusively in the center of the cells (Fig. 13). As it was confirmed with light microscopy, this

spherical image corresponded well to the shape and localization of the nucleus of NG108-15

cells, and the ring-shaped signal detected in the first 1 hour represented cytoplasmic

localization. The nuclear signal was maintained through 24 and 48 hours. Based on the

number of cells showing fluorescent staining, we estimate that the transfection efficiency is 75-

95% inNG108-15 cells.

To examine whether base sequence influences cellular trafficking of oligonucleotides,

both sense and antisense oligonucleotides were incubated with NG108-15 cells for the time

periods described above. Both sense and antisense oligonucleotides were taken up by the cells

with similar efficiency, and both oligonucleotides were transported through the cytoplasm to

the nucleus with similar rate. There was no apparent difference in the strength or the duration

of the fluorescence between sense and antisense oligonucleotide treated cells.

Antisense Expression Vector Experiments

Inhibition of AT-1 receptor with antisense expression vector

AT-1 antisense expression vector was constructed by inserting 855 bp of the AT-1

receptor cDNA (29) into the AAV-derived expression vector pJDT95 downstream to the

AAV promoter p40 in antisense direction (Fig. 14). The resulting plasmid paATI was

analyzed by digesting it with NdeI restriction enzyme to confirm the insertion and the antisense

orientation of the AT-1 fragment.

NG108-15 cells were grown on poly-L-lysine coated plates to 65-75% confluence. 1

day after plating the cells, the growth medium was removed, the cells were washed 3 times







































Figure 13. Laser scanning confocal microscopic images ofNG108-15 cells after 15 minutes, 1
hour, 6 hours, and 24 hours of incubation with FITC-labeled antisense ODNs. Cells were
grown to 40-50% confluence on glass coverslips coated with poly-L-Lysine. Following the
incubation with 25 pM F1TC-labeled ODNs the tissue culture medium was removed, and the
cells on the coverslips were mounted on microscope slides using paraphynyline polyvinyl
alcohol-glycerol mounting medium. Images were acquired using a BioRad MRC-600 laser
scanning confocal microscope.















polyA

1
HindMll, KpnI digest



T4 li


polyA


KpnI, EcoRI digest
I
HindIM end
generated by PCR

/
;ase

B



p40 0

K ATH antisense

K AT, antisense


Figure 14. Strategy for antisense expression vector cloning targeting angiotensin type-1
receptor. The plasmid pJDT95 carrying recombinant adeno-associated virus (AAV) DNA was
linearized by digestion with HindUI and KpnI. The plasmid carrying the full length AT-1
cDNA was digested with KpnI and EcoRI, which released a 855 bp long fragment of the 5' end
of the AT-1 cDNA including the translation initiation site. The EcoRI end was modified to
HindlIl by performing polymerase chain reaction with one primer containing the HindII site.
The resulting fragment was ligated in the antisense direction into the linearized pJDT95.







81
with DMEM and incubated with 15 gg ofpaATI completed with 30 W1 Transfectam in 4.5 ml

DMEM. The next day the transfection medium was removed and differentiation medium was

added for 48 hours. At the end of this incubation period, NG108-15 cells were harvested, and

membrane receptor binding was performed. Control cells received either no treatment, or 30

Jl Transfectam alone, or 15 gg control vector containing no antisense sequence (pJDT95dlk)

completed with 30 gl Transfectam.

NG108-15 cells transfected with paATI showed 20% decrease in I-Sar-]le-Angll

binding when compared to mock-treated cells (p<0.05). Transfectamn treatment alone, or

pJDT95dlk control vector treatment resulted in no difference in receptor binding when

compared to control cells (Fig. 15).

Coinfection of AAV-transduced cells with adenovirus activated AAV, resulting in an

enhanced antisense effect. Immediately after transfecting NG108-15 cells as described above,

5X106 adenovirus (tsl149) was added to the transfection medium of paATI treated cells.

Differentiation and receptor binding was performed as described above. '22-Sar-Ile-AngII

binding decreased by 63 % compared to control vector treated levels in the paATI plus

adenovirus treated cells. Adenoviral enhancement of antisense activity was observed only in

the AT-1 subtype of AngII receptors, whereas the AT-2 subtype remained unaffected (Fig.

16).

Immunocytochemical detection of AAV transcripts

Besides making AT-i antisense RNA from its p40 promoter, paATI also produces

viral rep proteins, albeit in smaller amounts. The rep proteins are not essential for the antisense












I 8000-


L 7000-
a
C
S6000-
0.
U
c)5000-


"5 4000-


1 3000-
0
'U2000
*0
1000-



Control Transfectam Mock Antisense

vector treatment



Figure 15. The AT-1 antisense expression vector paAT produces 20%/ decrease in receptor
binding in NG108-15 cells when compared to mock vector treated cells. A glioma-
neuroblastoma hybrid cell line, NG108-15 was grown to 65-75 % confluence and was
transfected with the angiotensin type-1 receptor antisense vector paATI using the synthetic
transfection agent Transfectam. 20 hours later the cells were placed on differentiating medium,
which induced receptor synthesis. 48 hours later the cells were harvested, homogenized and
centrifuged to separate the membrane fraction. Radioligand binding assay was performed
using '2I-Sar-Ile-Angll. The membrane bound ligand was separated by filtering the reaction
through a Whatman filter paper (*:p<0.05, one-way ANOVA, Duncan multiple range test;
n=5).












~*1


Em
^m
I^^


Angll


AT1


Transfectam
Mock
paAT
paAT -lAd


100 -



80 -


60-


40-



20-


0-


Receptor subtype


Figure 16. A representative figure showing that infection of paATl-transfected cells with
adenovirus further decreased AnglI receptor binding. Adenovirus (2-3/cell) was added to the
differentiating medium of NG108-15 cells for 48 hours following transfection with paAT1.
Adenovirus (Ad), a helper virus for adeno-associated virus (AAV) enhances the activity of
AAVs p40 promoter which drives the expression of the antisense mRNA. Receptor binding
was performed as described above, using Losartan and PD123319, the AT-1 and AT-2 specific
receptor inhibitors to differentiate between AnglI receptor subtypes.


AT2







84
effect, but we used them as markers for monitoring AAV transcriptional activity. Following

transfection with paATI and adenovirus as described above, NG108-15 cells were incubated

with the rep antibody IF811 and stained with diaminobenzidine (DAB). For controls,

uninfected cells, Transfectamn-only treated cells, control plasmid plus Transfectam treated cells

and paATI transfected cells without adenovirus were also stained. As a control vector, pTR-

UF, an AAV-derived expression vector lacking the rep gene was used.

Cells infected with paATI and adenovirus showed strong immunoreactivity in the

nucleus. Approximately 10-20% of the cells were stained with the rep antibody (Fig. 17).

Uninfected controls were devoid of any staining, whereas Transfectam-treated or control

vector treated cells showed uniform, light nonspecific staining. Cells transfected with paATI

but without adenovirus did not show specific staining.

Inhibition of angiotensinogen synthesis by antisense vector

It was shown in our laboratory that antisense oligonucleotides targeted to

angiotensinogen mRNA decrease angiotensinogen synthesis in the hypothalamus and decrease

the mean arterial pressure (190). An antisense expression vector was constructed by inserting

the complete cDNA of the angiotensinogen gene into the AAV-derived expression vector

pJDT95 in the antisense direction (Fig 18). The resulting construct (paAo) was tested on the

hepatoma cell line H-4 for inhibition of angiotensinogen production. 1 gg or 2 pgg of the

vector DNA was mixed with 10 1t or 20 jgl Lipofectin, a cationic transfection agent and

incubated with the H-4 cells for 20 hours. After the transfection medium was removed, cells















,Aw












4JT%


0^e


J.-~j.
-
* .1......


^ ~'tril ,*



Ek A<


Figure 17. Immunocytochemical staining of the viral rep protein in NG108-15 cells following
liposomal gene delivery. NG108-15 cells were transfected with 15 gg paATI antisense vector
DNA mixed with 30 gl Transfectamn and tsl49 adenovirus (2-3 adenovirus/cell). Two days
after the transfection the cells were fixed and stained with the rep antibody IF811.


I


t*














B polyA

Hind III, Acc65 (Kpn I)
Hind III, Acc65 I (Kpn 1)


n K


Hind III, Acc65 I (Kpn I)
/


T4 ligase

I
,B


H

antisense Ao


polyA


Figure 18. Construction of the angiotensinogen antisense expression vector paAo
(pJDT95aAo). The adeno-associated virus derived expression vector pJDT95 and the plasmid
pGEM4 carrying the complete angiotensinogen cDNA was digested with Hindll and Acc65I.
The digestion products were separated on low melting gel and the larger fragment of pJDT95
and the smaller fragment ofpGEM4Ao were in-gel ligated with T4 ligase.







87
were incubated for an additional 72 hours in EMEM supplemented with insulin, transferrin and

selenium. At the end of the incubation period the medium was collected for radioimmunassay.

The amount of secreted angiotensinogen decreased in the cells transfected with 2 jig

paAo plus 20 jig Lipofectin to 15+2.2 pg/mg protein whereas the cells transfected with similar

amounts of Lipofectin alone secreted 22.9+5 pg/mg protein. 1 jig paAo completed with 10 g1

Lipofectin showed less inhibition in angiotensinogen production (Fig 19).

Transfection of SHR brains with antisense vector

SHRs were equipped with indwelling guide cannulas to the median preoptic area of the

anterior hypothalamus. After baseline blood pressure recordings, one rat was injected with 1

gg paATI mixed with 2 jil Transfectam and 105 adenovirus (ts149). The control rats received

the same dose of Transfectam and ts149 with or without control plasmid DNA (pJDT95dlk).

Systolic blood pressure was measured again three days after treatment with tail cuff blood

pressure recorder. After sacrificing the rats, the brains were removed and processed for

immunohistochemistry using the rep antibody. A limited number of cells along the cannula

tract in the cortex and the septum of the vector-treated (paATI and pJDT95dlk) rat showed

positive immunostaining (Fig. 20). There was no change in systolic blood pressure in the

antisense vector treated rat (181.5 mmHg pre-treatment vs. 185 mmHg post-treatment).













35 -


30-

C
*25-
0

0.
CL
20-



Is 15
0
C
C 10-
2
0




0
control Lipo (10) Lipo (20) aAo (1) aAo (2)




Figure 19. Transfection of H-4 hepatoma cells with the angiotensinogen antisense expression
vector paAo resulted in 30-34% decrease in angiotensinogen production. Hepatoma cells were
grown to 50-60%o confluence in EMEM+10%FBS+10%/oCS. 1 jg or 2 gg vector DNA was
mixed with 10 pIl or 20 gl Lipofectin and overlaid onto cells in serum-free EMEM for 20
hours. After the incubation period the cells were maintained in EMEM+ITS (insulin-
transferrin-sodium-selenite serum supplement) for 72 hours, after which the medium was
collected for angiotensinogen radioimmunoassay.













































Figure 20. Immunohistochemical localization of the rep protein in SHR brains transfected with
paATl. 1 gg of the AT-1 antisense expression vector paATI was completed with 2 pl
Transfectam and mixed with 105 adenovirus. SHRs were injected in the median preoptic area.
Three days later the animals were sacrificed, and 40 pm thick brain section were processed for
rep immunohistochemistry.








Summary of the Results

1. Both phosphodiester and phosphorothioate antisense oligonucleotides effectively

decreased high blood pressure in SHRs when injected i.c.v. The effect of a single dose of

phosphorothioate oligonucleotide lasted for 7 days.

2. Membrane receptor binding revealed significant decrease in AT-1 receptor binding in

the hypothalamic region, whereas AT-2 receptor binding remained unaffected.

Autoradiography showed that the most affected areas were in the anterior hypothalamus: the

paraventricular nucleus, suprachiasmatic nucleus and the median preoptic nucleus.

3. Antisense oligonucleotide treatment markedly inhibited drinking response of AT-I

receptors to i.c.v. AnglI injection.

4. Oligonucleotides diffused throughout the ventricular system and into the brain tissue

from the ventricles within 1 hour.

5. Antisense oligonucleotides decreased receptor binding in cultured NG108-15 cells.

6. There was no change in the amount or in the length of the AT-1 mRNA in NG108-15

cells following antisense oligonucleotide treatment at doses which were effective in decreasing

receptor binding.

7. Oligonucleotides are taken up by NG108-15 cells rapidly, and the majority of them are

transported to the nucleus within 6 hours.

8. Antisense expression vectors can decrease AT-1 receptor and angiotensinogen

synthesis in cultured cells.

9. Liposomal delivery of the antisense vector to the rat brain was insufficient in eliciting

any change in systolic blood pressure.












CHAPTER 6

CONCLUSIONS

Antisense Oligonucleotide Design

At the time of the writing of this manuscript the theory of antisense oligonucleotide

design has not been completely worked out (191). This is probably a direct consequence of the

fact that the question of the mechanism of action of antisense oligonucleotides is also a matter

of debate: several potential action sites exist for antisense, and it is not unlikely that different

mechanisms will be active under different circumstances.

When our project started, however, even less was known about how to use antisense

molecules, and to our knowledge, there was but one published article on applying antisense

oligonucleotides to living animals (177). Therefore we started out testing different

oligonucleotides, including the one which followed the most common example, the antisense

oligonucleotide targeting the initiation codon AUG and neighboring nucleotides on the AT-1

mRNA, as well as the one which is used in most of the experiments and targeted bases +63 to

+77 downstream to the initiation codon.

Even though our initial screening of antisense oligonucleotides was not entirely

systematic, the picture emerged that multiple injections of the phosphodiester antisense

oligonucleotide was needed to elicit a response similar to that of a single injection of the

phosphorothioated oligonucleotide. This effect was most likely to be due to the shorter half-

life of the phosphodiester oligonucleotide (141). On the other hand, the two antisense oligos

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targeting different areas on the AT-1 mRNA appeared to be similar in their ability to decrease

high blood pressure.

Lowering Blood Pressure with Antisense Oligonucleotides

Blood pressure measurements have been a favorite subject for physiologists, since it

provides numerical data on an essential life function, and it is the outcome of the interplay

between several biological systems. Our system of interest was the brain and its influence on

hypertension.

Our result of lowering the blood pressure of SHRs by i.c.v. injection lends itself to the

explanation that the AT-1 antisense oligonucleotides entered brain cells, bound to AT-1

mRNA which resulted in decreased AT-1 receptor synthesis, the consequence of which was a

decreased effectiveness of brain AngII and, eventually, lower blood pressure.

Let's examine this proposed chain of events step-by-step:

Oligonucleotide Uptake

Our results show that 1 hour after the injection of 50 gg/4 gl FITC-labeled

oligonucleotide into the right lateral ventricle there was fluorescent signal in the wall of both

lateral ventricles as well as around the third ventricle, and the signal could be followed to about

2 mm into the tissue. We detected signal around the fourth ventricle as well as in the cerebral

and cerebellar cortex, although with decreased density.

This spread of the oligonucleotides seems to be sufficient, since most of the AT-1-

containing brain nuclei are located around the ventricular system. We have also identified

individual cells showing fluorescent signal by confocal microscopy (190). That the FITC label

remains attached to the oligonucleotide after entering the cell has been shown by others in our







93
laboratory (B. Kimura, personal communication). Other investigators using biotin-labeled

oligonucleotides reported similar distribution in the brain after i.c.v. injection (192).

Inhibition of AT-i mRNA

Our investigations of the effects of antisense on its target mRNA focused on

measurements of the amount and the size of AT-1 mRNA. Our northern blots did not show

decrease in the amount or in the size of the AT-1 mRNA. This suggested to us that the target

mRNA was unlikely to be degraded or cleaved upon antisense treatment, but probably other

mechanisms were involved in inhibiting normal translation (see below). Direct measurements

of the binding of antisense oligonudcleotide to AT-1 mRNA (e.g. nuclease protection assay)

were not conducted.

Inhibition of AT-1 Protein Synthesis

Receptor binding on freshly prepared hypothalamic-thalamic cell membranes supported

the notion that the number of AT-1 binding sites was decreased after i.c.v. antisense injection.

The effect appeared to be specific to the AT-1 receptor subtype since the AT-2 receptors

remained unaffected. The single-point binding assays performed do not typically allow one to

draw numerical conclusions about the number of binding sites, but since it was not anticipated

that antisense treatment would in any way change receptor affinity, we concluded that the

decreases seen in our assay were proportional to the decrease of the number of AT-1

receptors.

We employed autoradiography to localize the brain areas affected by the antisense

injections. Besides lateral ventricular injections we tested the effect of antisense

oligonucleotides injected into the third ventricle. Although not statistically significant, the







94
results suggested that the decreases were localized in the anterior hypothalamus and

anteroventral third ventricle (AV3V), the most prominent decrease being detected in the PVN.

Whereas both sets of experiments, the one using third ventricle and the other using

lateral ventricle injections, suggested a decrease in the AV3V region, they differ in that after

third ventricular injections the receptor number in the OVLT tended to be decreased, whereas

after lateral ventricular injections a decrease was seen in the MnPO. A possible explanation for

this difference might be that the oligonucleotides had better access to the OVLT after injection

in the third ventricle than after lateral ventricular injections. An even more striking difference

occurred in another circumventricular organ, the SFO, where all injected oligonucleotides

tended to increase the AT-1 receptor number after being injected into the third ventricle. Since

this tendency for increase was seen with all oligonucleotides including antisense, sense and

scrambled, it appeared to be non-specific. Finally, the results of the autoradiography suggested

that some decrease, although to a variable extent, may have also occurred in the SCN and

SON.

AT-1 receptors in sites that were farther away from the ventricles, such as piriform

cortex or NTS in the brainstem showed no tendency to be decreased by antisense, thus

suggesting a rather localized action of antisense oligonucleotides. This is in agreement with the

experiments showing relatively restricted distribution and tissue penetration of fluorescent

labeled oligonucleotides. In nuclei containing exclusively AT-2 receptors (mediodorsal

thalamic nucleus, lateral septum or inferior olive), there was also no sign of decrease by

antisense treatment. This might be attributed to the specificity of the antisense inhibition to the