WO1993021309A1 - Oligonucleotide sequences and transgenic animals transfected therewith having reduced sensitivity to narcotic analgesics - Google Patents

Abstract

Clones (SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3) have been isolated and sequenced. An antisense construct has been made from part of SEQ ID NO:1 that blocks opioid binding in cells and has been used to produce transgenic animals having a reduced sensitivity to narcotic analgesics. The oligonucleotide sequence introduced into the animals at an embryonic stage was the first about 500 base pairs, but was reversed in direction. Oligonucleotide constructs of the invention are also useful as probes or as therapeutic and diagnostic agents.

Description

OLIGONUCLEOTIDE SEQUENCES AND

TRANSGENIC ANIMALS TRANSFECTED THEREWITH

HAVING REDUCED SENSITIVITY TO NARCOTIC ANALGESICS

Field of the Invention

This invention generally relates to opioid receptors, and more particularly to oligonucleotide constructs that can be used to produce a transgenic non- human animal, such as rodents, with reduced sensitivity to narcotic analgesics, used as probes or used in therapeutic treatment and diagnoses of narcotic addicts. This invention was made with government support under Grant Nos. DA00564, DA02643, DA01583, awarded by the National Institutes of Health. The government has certain rights in this invention.

Background of the Invention

A μ-opioid-specific receptor protein was purified to apparent homogeneity with a molecular weight of 58,000 daltons in 1986. Cho et al., Proc. Natl . Acad. Sci . USA, 83 (1986), pp. 4138-4142. However, this protein from rat brain exhibited high binding activity only when it was reconstituted with certain lipids. The role of the lipids is presumably to stabilize a particular conformation of the protein. Hasegawa et al., J. Neurochem. , 49 : 4 (1987), pp. 1007-1012. Poly- clonal antibodies were reported as having been raised against the purified opioid receptor from bovine brain. Roy et al., Biochem. & Biophy. Res. Comm. , 150 : 1 , (1988) , pp. 237-244. Monoclonal antibodies were raised against the purified opioid receptor protein, and data suggested that the protein may have significant af inity for and 5, and to a certain extent, against K ligands. Roy et al. , Biochem. & Biophy. Res. Comm. , 154 : 2 (1988), pp. 688-693; Roy et al. , in Biochemical Pharmacology — A Tribute to B.B. Brodie, ed. Costa, E. , New York: Raven Press, Ltd. (1989), pp. 177-188.

In 1989, the purified opioid-binding protein from bovine brain was characterized by cDNA cloning. The primary sequence of the protein was deduced from the cDNA clones, with the sequence being found homologous to various members of the immunoglobulin protein super- family, especially to those molecules involved in cell adhesion. Schofield et al. , The EMBO Journal , 8 : 2 (1989) pp. 489-495.

Summary of the Invention

In one aspect of the present invention, a DNA segment corresponds to newly discovered clones. This DNA segment is prepared so as to be in the antisense direction. The product of DNA construct hybridizes with endogenous mRNA for an opioid binding protein, and is useful within a cell (cultured or in a transgenic animal) in reducing sensitivity to narcotic analgesics. Oligonucleotide constructs of the invention can also be RNA. Whether the oligonucleotide constructs are RNA or DNA, they preferably are modified to resist degradation in vivo. Uses of the inventive oligonucleotides include as diagnostic or therapeutic agents or as probes. As therapeutic agents they preferably include a means for therapeutically administrating to a patient. Sequences as small as about 12 bases (when single stranded) or about 12 base pairs (when double stranded) can be used, such as in therapeutic treatments of narcotics addicts. Brief Description of the Drawings

Figure 1 graphically represents the antinoci- ceptive activity of morphine sulfate in the presence of naloxone after control animals (non-transgenic) and inventive animals (transgenic) had received morphine pretreatment, with the inventive transgenic animals showing a marked reduced sensitivity to morphine; and,

Figure 2 graphically represents the cDNA of an inventive gene in which the coding region and the region used for antisense generation is shown (where most of the 5' region shown as "antisense fragment" was ligated

^• into the mammalian expression vector pSVL SV40 and microinjected into mouse zygotes to prepare the transgenic animals to which the Fig. 1 data relates) .

Detailed Description of the Preferred Embodiments

The present invention relates to sensitivity (such as by a cell or an animal) to narcotic analgesics. By "narcotic analgesic" is meant the various alkaloids of opium such as morphine, morphine salts (such as morphine hydrobromide, morphine hydrochloride, morphine muscate, morphine oleate, morphine N-oxide, and morphine sulfate) , and morphine analogs and salts such as normorphine, diacetyldihydromorphine, diacetylmorphine hydrochloride, codeine and diacetylmorphine (heroin) . Other widely used narcotic analgesics include alpha- prodine, methadone, merperidine, levorphanol, propoxy- phene, fentanyl, oxymorphone, anileridine and metopon. Also included as narcotic analgesics are the endogenous opioids (e.g. the endorphins) and the synthetic opioid peptides (e.g. D-Ala-2, D-Leu-5 enkephalin) .

As is well known, continued use of at least many of these narcotic analgesics leads to habituation or addiction. However, despite their abuse potential, these narcotic analgesics have therapeutic uses, for example with patients requiring chronic treatment to ease pain.

Even in such therapeutic uses, though, patients typically develop increasing tolerances to these narcotic analgesics, so that increasingly potent doses are required to achieve relief from pain. Undesired side effects then tend to develop to large, chronic doses of the narcotic analgesics.

The agonistic actions anddependence-producing properties of narcotic analgesics can be, and are, studied in various mammalian species besides humans, since practical and governmental considerations frequently require that studies be first done in small rodents and/or monkeys before the analgesic properties of pharmaceuticals are tested with humans. To the present, however, all drugs that have morphine-like properties in mammals other than man have been found to be morphine-like in man, and a variety of analgesic assays have been developed with animals which have gained widespread acceptance for predicting properties in humans.

The present invention generally relates to several clones pertaining to opioid receptor functions. A particularly preferred, novel clone of this invention (sometimes hereinafter called ^~~DϋZl^~~ , whose sequence is given by SEQ ID N0:1) codes for a protein with opioid receptor function. We have made mice transgenic to a unique region of anti-sense DUZ1 and have demonstrated that these transgenic animals exhibit reduced sensi- tivity to the antinociception effects of morphine. These transgenic animals, and the underlying oligo¬ nucleotide constructs themselves, are useful, for example, in diagnoses of and treatments for addiction to narcotic analgesics. For example, applications of the invention include therapeutic methods in treating addicts who can potentially be cured of their addiction (because the invention can be used in ways that do not lead to tolerance development) . In addition, patients requiring chronic treatment with narcotics to ease pain, such as terminal cancer patients, can be treated in accordance with the invention by administrations of antisense fragments to block the expression of the opioid receptor gene. This means the patient would still have the pain killing benefit of the narcotic analgesic, such as morphine, but that development of tolerance would be blocked. As a consequence, lower doses of the narcotic analgesic could be used and the various, known side effects (such as respiratory depression and constipa¬ tion) , which result form chronic treatment with high dosages of narcotics could be lessened.

Diagnostic kits in accordance with aspects of the invention can be used to determine if a patient is addicted and/or the degree of addiction. For example, cell samples taken from the patient can be screened with oligonucleotides of the invention. Detection of the opioid receptor gene in cells of a patient, such as, for example, blood cells, permits the screening for persons with tolerance to narcotic analgesics (that is, addicts) and differentiates from persons with a narcotic analgesic naivety.

When the invention is used therapeutically by administrating to patients, then single stranded forms tend to be more efficacious; however, double stranded forms tend to be more resistant in vivo (that is, to enzyme degradations and the like) , although the action tends to be not as rapid as when singled stranded forms are used.

The inventive oligonucleotides can also be used as probes for discovering human genomic sequences and for discovering other subunits of opiate receptors. The sequences we have given by SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3 are cDNA. When one wishes to use oligonucleotides of the invention but of a RNA form, then any of the well-known procedures for preparing oligonucleotides with RNA nucleotides (e.g. where the thymines are replaced by uracils) may be utilized.

DNA or RNA constructs of the invention may have at least a portion of the normal phosphodiester backbone modified in order to limit degradation in vivo or to convey resistance to protease or other enzymes. This is because even though antisense oligonucleotides have been shown to act as specific inhibitors of gene expression in a variety of in vitro systems, one of the major problems encountered is the rapid degradation in cells or culture media of the naturally occurring phosphodiester backbone (see, for example, Hoke et al. , Nucl. Acids Res. , 19 (1991), pp. 5743-5748. Methods to replace all or part of the sugar-phosphate backbone of a stretch of DNA and of RNA are being reported, such as by Nielsen et al. , Science, 254 (1991) pp. 1497-1500.

These chimeric oligonucleotides can form stable heteroduplexes with complementary single-stranded DNA.

Constructs of the invention thus usefully can have all or a portion of the normal phosphodiester backbone replaced with a variety of synthetic or semi- synthetic analogues capable of mimicking the distance and orientation of the native nucleobase substituents. Such modifications may be by means of a methylphos- phonate, a phosoramidate, a phosphorothioate, a 2'-o- alkyl, a polya ide (2-aminoethylgylcine) or a modification of ribose to a cycloalkane or partially unsaturated cycloalkane.

Portions of clones described in an 1989 article from work conducted in our laboratory (Schofield et al. , supra) were used to prepare antisense cDNA. A plasmid was then constructed containing the insert of rat OBCAM cDNA using the eukaryotic expression vector, pSVL. As illustrated by Example 1, when cells used as a model system containing a homogenous population of δ- opioid receptor were transfected with the plasmid, then opioid binding was blocked.

Clones of the invention have some coding regions common to the previously published work of Schofield et al., supra , but the inventive clones have unique regions. These unique regions are preferred in preparing the probes, DNA or RNA constructs, the therapeutic and/or diagnostic agents, and in providing transgenic animals with an altered response to narcotic analgesics. Example 2 describes our isolation of three clones. The largest clone we sometimes call ^~~DUZ1^~~ (the sequence of which is given by SEQ ID NO:l) with a reading frame comparable to OBCAM in its C-terminal 318 amino acids, but differing in its N-terminal amino acids, differing in all of its 5'-noncoding region, and differing in some of the 3'-noncoding region.

Most of this unique 5'-noncoding region was then used as described by Example 3 (in the antisense orientation) in raising transgenic mice having a dramatically altered response to morphine (taking about three times as much morphine to kill pain as with control animals) . These transgenic animals apparently do not develop tolerance to morphine.

The clone we sometimes call "SG13" (the sequence of which is given by SEQ ID NO:2) and the third clone we sometimes call "SG8" (the sequence of which is given by SEQ ID NO:3) are also useful in antisense constructs and applications. The SEQ ID NO:3 clone also has a unique 5'-noncoding region. The inventive transgenic animals, for example, can also be used to provide cell cultures where somatic cells from the animals are cultured, and then can be used in a variety of diagnostic techniques and assays. Transgenes introduced into animals in order to make transgenic, non-human animals of the invention contain an oligonucleotide sequence introduced into the animals, or an ancestor of the animal, at an embryonic stage. The oligonucleotide sequence is expressible in the animals and is effective to alter the animal's response to a narcotic analgesic, as will be exemplified by Example 3. The oligonucleotide sequence introduced into the animals forms a transcriptional unit expressible in the animals as a messenger RNA compound capable of hybridizing to endogenous mRNA for an opioid binding protein. An altered response by the animal to a narcotic analgesic is, for example, a reduced sensi¬ tivity to narcotic analgesic (e.g., such as morphine, morphine analogues, endogenous opioids, and synthetic opioid peptides) . For example, the oligonucleotide sequence introduced into the animals, is preferably selected from all or part of the sequences illustrated by SEQ ID N0:1, SEQ ID NO:2, SEQ ID NO:3, but wherein the sequence selected (or its portion) is read in the antisense direction. Thus, Example 3 illustrates such a use where the first 500 base pairs of SEQ ID NO:l (a novel portion of SEQ ID NO:l) was used, but used in the antisense direction in constructing the transgene.

Thus, aspect of the invention is the use of DNA or RNA constructs comprising a segment corresponding to all or part of SEQ ID NO:l, SEQ ID NO:2 and SEQ ID NO:3, but reversed with respect to the direction shown

(and having RNA bases, if the construct is RNA) . The segment preferably is sufficient in size to hybridize with endogenous mRNA for an opioid binding protein. We believe a sufficient size is as short as 12 bases (when single stranded) or 12 base pairs (when double stranded) . Shorter sequences than this lose specificities.

The inventive constructs may be operatively linked to an expression vector, such as a mammalian expression vector or a retroviral expression vector, or not. That is, in many therapeutic applications an expression vector is believed not necessary, and the construct can be, for example, directly administered (in single or double stranded form) to a patient by a variety of administration means. However, as earlier mentioned, constructs of the invention preferably have been modified for resistance to degradation, such as where at least a portion of the normal phosphodiester backbone is modified with an analogue that is effective to limit degradation in vivo .

We believe that therapeutic uses of our DNA constructs include administration to patients who are addicted to narcotic analgesics in order to assist these patients in curing their addiction while avoiding development of tolerance. For example, the smaller inventive antisense constructs can be used to suppress expression of the gene.

We anticipate administration in amounts of 10 nmol or greater will be useful with the actual dosages and the duration of therapy to be determined by the treating physician and in accordance with developing oligonucleotide therapies. We believe a variety of administration means and methods will be useful, including i.v., transdermal, transnasal, and patch techniques, as well as microinjection. Thus, the invention includes diagnostic or therapeutic agents comprising an oligonucleotide with at least about 12 bases when single stranded and about 12 base pairs when double stranded and having a backbone, the oligonucleo- tide substantially corresponding to DNA bases (or two RNA analogous thereto) of all or a portion of SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3, but in the antisense direction thereof, and including a means for therapeu¬ tically administrating the oligonucleotide to a patient. Such therapeutic administrating means includes patches and physiologically acceptable solutions and carriers. Aspects of the invention will now be illustrated by Examples 1-3 and with reference to Figs. 1 and 2, which are meant to illustrate but not limit the invention. Abbreviations used are: OBCAM, opioid-binding cell adhesion molecule; cDNA OBCAM, DNA copy of gene encoding OBCAM; bOBCAM, bovine gene encoding opioid- binding cell adhesion molecule; rOBCAM, rat OBCAM; pBOM, cDNA for bovine opioid-binding molecule; pROM, cDNA for rat opioid-binding molecule; bp, base pairs; PCR, polymerase chain reaction; RT, reverse transcriptions; HEPES, 4-(2-hydroxy)ethyl-1-piperazineethanesulfonic acid; aa, amino acid(s) ; DUZ2 , clone 2 from UZ library; kb, kilobase(s) or 1000 bp; nt, nucleotide(s) ; UZ, cDNA library created using rat brain poly(A) RNA (Clontech) and Stratagene's Uni-Zap XR.

EXAMPLE 1

Plasmid Construction for Cell Line Transfection

Plasmids were constructed using standard recombinant techniques. The eukaryotic expression vector, pSVL was used. The rat OBCAM cDNA was digested with Sma∑, and a 649-bp fragment was isolated on agarose gel. This SmaZ fragment bracketed the AUG codon of rat OBCAM cDNA, containing 272 bp upstream of the AUG and 377 bp downstream. This fragment was ligated to a Smal- cleaved preparation of pSVL, followed by transformation of Escherichia coli DH5α competent cells. Plasmid DNAs from twenty of these ampicillin-resistant colonies were isolated and nine out of twenty colonies were shown to contain the 649-bp insert. The orientation of the insert was verified by the cleavage of internal Xhol site. Two colonies which carried either sense or antisense orientation were selected and named "pSVL sense" or "pSVL antisense", respectively. To ensure that the orientations of pSVL sense and pSVL antisense were correct, the plasmid DNAs were digested by the enzymes Kpnl and PvuII; the products corresponded in size to those expected from pROM sequences.

Cell Culture and DNA Transfection

NG108-15 neuroblasto a x glioma cells were grown in tissue cultures. Tissue culture medium components were purchased from GIBCO. Fetal calf serum was obtained through Hyclone. Calcium phosphate-mediated DNA transfection was carried out as follows. On day 1, cells were plated at a density of approximately 1.2 x 10 cells/10 cm dish. On day 2, 3 hours before transfection, all the medium was removed and replaced with 10 ml of fresh medium. Twenty to 30 μg of plasmid DNA (in the ratio 1:50 of pSVL sense or pSVL antisense plus selectable plasmid pSVNeo) were transfected/plate. The plate was incubated overnight at 37°C in a 5% C0₂ atmosphere. On day 3, the medium was removed, and cells were washed twice with phosphate-buffered saline, 20 ml of fresh medium was added, and incubation was shifted to a 10% C0₂ atmosphere at 37°C overnight. On day 4, the cells were divided into an appropriate ratio (1:3 to 1:5) and incubated in fresh medium in a 10% C0₂ atmosphere, 37°C for 24 hours before selection was started. On day 5, the medium was removed, and the selection medium (containing G418 sulfate, actual concentration 400 μg/ml) was used for the next 4 weeks. The medium was changed twice/week, until resistant colonies were visualized. Generally speaking, in each 10-cm dish 10- 30 colonies were observed, and a single colony was isolated and expanded to an individual cell line.

Nucleic Acid Preparation

Poly(A)⁺ RNA from these clones was isolated from cultures in 10. cm dishes. Poly(A)⁺ RNA of rat brain was purchased from CLONTECH. Genomic DNA was isolated from rat brain, NG108-15, ST8-4, and ST7-3 cells.

Polv erase Chain Reaction Oligodeoxynucleotide primers (synthesized by

Northern Biosciences Incorporated, Hamel, MN) were prepared (with numbers in brackets indicating the positions in the sequence of pROM) :

RPJ1, [nucleotides 482-499]; RPJ2, [nucleotides 620-603];

RPJ3, [nucleotides 158-172];

RPJ4, [nucleotides 760-743];

mRNA PCR Reverse Transcription. Ten ng of poly(A)⁺ RNA were added in 20 μl of reaction solution containing 50 mM Tris-HCL (pH 8.3), 3 mM MgCl₂, 75 mM KC1, 2.5 mM dithiothreitol, 1 mM of each dNTP, 20 units of RNase inhibitor (Promega Biotec) , 200 units of Moloney murine leukemia virus reverse transcriptase (GIBCO/Bethesda Research Laboratories) , and 50 pmol of random hexamer, and incubated for 1 hour at 42°C. The reaction was stopped by heating at 99°C for 5 minutes.

Amplification. To 20 μl of mRNA/cDNA hybrid, 80 μl of PCR solution containing 15 pmol of each primer and 2.5 units of Taq DNA Polymerase were added, and the mixture was overlaid with 60 μl of mineral oil (Perkin- El er Cetus) . Final concentration of MgCl₂ was 3.8 mM. After the reaction mixture was denatured by heating at 94°C for 5 minutes, cDNA was amplified in a Perkin-Elmer DNA thermal cycler for 35 cycles, with a denaturation step at 94°C for 30 seconds, an annealing step at 55°C for 30 seconds, and an extension step at 72°C for 30 seconds.

DNA PCR

DNA PCR was performed in 100 μl containing 50 mg of genomic DNA, 15 pmol of each primer, 20 nmol of each dNTP in 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, and 0.001% (w/v) gelatin. The thermal cycling condition was as described above.

These PCR products were electrophoresed on the agarose gel and transferred to the MagnaGraph nylon membranes. The membranes were subjected to hybridiza- tion using P-labeled Smal fragment.

Northern Blot Analysis Seven μg of poly(A)⁺ RNA were size-frac¬ tionated on a denaturing formaldehyde/agarose (1%) gel and transferred to a MagnaGraph nylon membrane (Micron Separations Inc.) . The Smal fragment of pROM and 138 bp of PCR product between RPJ1 and RPJ2 were labeled using [α- 32P]dCTP and random hexamers (Femberg and Vogelstein,

1983) . Hybridization was carried out as described previously (Thomas, 1980), using P-labeled probes. The nylon membrane was washed with 40 minutes incubations at

68°C in 1 x SSC and 0.5% (w/v) sodium dodecyl sulfate

(Ann et al., 1987). The SSC composition had 0.5 M sodium chloride and 45 mM sodium citrate.

Sequencing of PCR Product PCR products between RPJ1 and RPJ2 were extracted from the reaction solution. For making blunt ends, the extracted DNA was incubated with 2 units of T4

DNA polymerase (Promega) in the presence of 0.1 mg/ml of bovine serum albumin and 100 μM of each dNTP at 37°C for 5 minutes. The reaction was stopped by heating at 75°C for 10 minutes. The cDNA containing blunt ends was collected by using spin column (Boehringer Mannheim) and cloned into the Smal site of pUC19 vector (GIBCO/BRL) . Supercoiled plasmid DNA was sequenced with Sequenase (United States Biochemical) .

Membrane Preparation Cells were harvested in phosphate-buffered saline/EDTA (phosphate buffer + 1 mM EDTA) , homogenized. in 25 mM HEPES (pH 7.4) , 0.32 M Sucrose, and centrifuged at 1,000 x g. The resulting supernatant was centrifuged at 100,000 x g, and the pellet resuspended in 25 mM HEPES (pH 7.4) .

Receptor Binding Assay

Cell membranes (about 300 μg) were incubated for 90 minutes at room temperature with labeled ligand (2 nMunless otherwise noted) in the presence or absence of 1 μM unlabeled ligands, in 25 mM HEPES (pH 7.4) . The ligands used for the binding assays were tritiated diprenorphine (opioid) , scopolamine (muscarinic) , rauwolscme (α₂-adrenergιc) , and 125I-ιnsulιn. The unlabeled ligands used for opioid, muscarinic, α₂- adrenergic, and insulin binding assays were D-Ala -D- Leu⁵-enkephalin (DADLE) or various other opioid ligands, atropine, phentolamine, and insulin, respectively. The reaction was terminated by filtration on GF/B filters

(Whatman) , the filters washed three times with the HEPES buffer, and allowed to stand with Scintiverse-BD (Fisher) overnight before counting in a scintillation counter. Results Use of RT-PCR for the Detection of OBCAM cDNA in NG108-15 Cells. Before transfecting NG108-15 cells with sense and antisense cDNA derived from pROM, it was essential to demonstrate that the pROM sequence, or a highly similar homolog, was present in these cells. Northern blot analysis, using pROM as probe, indicated that transcripts for OBCAM were barely detectable in 7 μg of poly(A)⁺ RNA prepared from NG108-15 cells (data not shown) . This observation was unexpected in light of the fact that NG108-15 cells are derived from rat and mouse cell lines and would thus be expected to contain either pROM itself, from rat, or a highly homologous molecule that is presumably present in mouse. Northern blot is a relatively insensitive assay system. As a more sensitive approach, we used RT- PCR. Two pairs of primers were designed and synthesized from this experiment. RPJ1 and 2 consisted of pROM nucleotides 682-499 and 603-620, respectively, and RPS5 and 6 contained pROM nucleotides 158-172 and 760-743, respectively. The latter two were chosen to span most

^• of the region used for sense and antisense cDNA; however, this region contained some non-coding sequence, which might be expected to be of lower homology between different species. Thus, we also used RPJ1 and 2 , which spanned a short region, completely within the reading frame.

Products using RPS5 and 6 of the expected size of approximately 600 bp were detected using sense- and anti-sense-transfected cells, but not non-transfected cells. Using RPJ1 and 2, however, the expected product of about 140 bp was detected in both control and transfeeted cells.

Two clones, NGJ2 and NGJ6, from PCR products between RPJ1 and RPJ2 were sequenced. These sequences differed in only a few nucleotides from pROM or pBOM, and these differences in nucleotide sequence did not affect the derived amino acid sequences, which were identical to bovine and rat OBCAM. The slight differences between the two PCR-derived clones, on the other hand, might have resulted from either hetero¬ geneity in the NG108-15 cells or from errors introduced by the Taq polymerase. This conservation in the derived amino acid sequences suggested that these RT-PCR products were the corresponding cDNAs of NG108-15 cells. Identification of Clones Successfully

Transfected with Sense and Antisense DNA. To study the function of OBCAM further, we have attempted to generate stably transfected sense and antisense pSVL pROM in NG108-15 cells. Twelve individual clones from pSVL sense and nine clones from pSVL antisense transfection were selected on the basis of G418 resistance and expanded into 10 cm culture dishes. Clones containing the appropriate RNA could be identified by Northern blot analysis using the Smal fragment as probe. However, 7 μg of poly(A)⁺ RNA from NG108-15 cells not transfected with rat OBCAM DNA were not hybridized with P-labeled Smal fragment of pROM.

Determination of the Copy Number of pROM Inserts in the Two Transfected Cell Lines. Genomic DNA was isolated from NG108-15, ST8-4, and ST7-3 cell lines, and the pROM inserts were amplified by PCR an analyzed on 2% agarose gel. An estimate of the number of ST7-3 antisense and ST8-4 sense inserts was obtained by using densitometry scanning to compare the number of antisense sequences in known amounts of clone pSVL antisense DNA to antisense or sense sequence present in 50 ng of either ST7-3 or ST8-4 genomic DNA. An estimate of the copy number was achieved by comparing the relative intensity of PCR amplification products from ST7-3 and ST8-4 cells to the intensities of the identically sized PCR products of a known copy number of pSVL antisense DNA. This analysis indicated the presence of approxi¬ mately 12 copies of pSVL antisense in ST7-3 genome and four copies pSVL sense in ST8-4 genomes.

Receptor Binding of Opioids and Other Ligands to Transfected Cell Lines. The OBCAM-transfected cell lines ST8-4 (sense) and ST7-3 (antisense) were tested for the presence of receptors for opioids as well as for several other classes of ligands. Binding of the opioid [ H] diprenorphine was reduced by 80% in the antisense- transfected cells, as compared to non-transfected cells. [ H] Diprenorphine binding to sense-transfected cells was reduced about 10%, which was statistically nonsignificant. In contrast, binding of the α₂- adrenergic ligand [ H]rauwolscine, the muscarinic ligand

3H scopolami.ne, and 125I-insulm were not affected by antisense (or sense) transfection.

The properties of the opioid receptors in the sense- and antisense-transfected cell lines were further tested. Scatchard analysis indicated that essentially all of the 80% reduction of [³H] diprenorphine binding in the antisense-transfected cells was due to a decrease in B_mx, while K_d was unaffected. The opioid receptors in both cell lines retained the δ characteristics of non-transfected NG108-15 cells, as indicated by the rank order of affinity of various opioid ligands: DADLE > β- endorphin > morphine > U-50,488H. In addition, the opioid receptors present in either transfected cell retained their stereoselectivity.

Finally, the effect of chronic opioid agonist treatment on the transfected cell lines was examined. Incubation of sense-transfected cells with 100 nM DADLE for 24 hours induced a reduction of 80% of opioid receptors, essentially the same as that observed in non- transfected cells. However, the same treatment did not further reduce the opioid receptor binding of antisense- transfected cells. These experiments show that cells transfected with antisense cDNA to OBCAM can block opioid binding.

EXAMPLE 2

This example illustrates our having conducted a search for cDNAs coding for an OBCAM-like protein, but with one or more additional regions (such as regions coding for a transmembrane and/or intracellular domain, which domains are missing in OBCAM) . We found a clone about 3.0 kb in length that was highly homologous to bOBCAM throughout the latter¹s open reading frame, but with unique regions.

Genera1 Approach. Two cDNA libraries were screened, one purchased from stratagene (SG; amplified one time) , and one created in our laboratory using rat brain poly(A)⁺ RNA (Clontech) and Stratagene's Uni-Zap XR (UZ) . The library was screened with probes repre¬ senting two different regions of OBCAM, one from the bovine clone (bOBCAM) and one from the partial rat clone (rOBCAM) . Comparison of these two clones revealed that the coding region was highly conserved, so both probes were generated from within this region.

Screening of the UZ Library. Screening of the UZ library (of which 1/5 was packaged and plated) produced only two clones, DϋZl (SEQ ID NO:l) and DUZ2 , both about 3.0 kb in length. From terminal sequencing of the 5' and 3' ends, in addition to restriction maps, it was concluded that they were identical. Hence, only DϋZl (SEQ ID N0:1) was subjected to complete sequencing. The resulting sequence was highly homologous to bOBCAM throughout most of the latter's open reading frame but had no homology in a portion of the 5'-noncoding region. DϋZl (SEQ ID N0:l) contained a slightly shorter putative reading frame, of 338 aa; the C-terminal 318 aa were virtually identical to those of OBCAM, including the same stop codon, but the N-terminal 20 aa were unique. Screening of the SG Library. Using high stringency conditions, 18 clones were obtained from the SG library, ranging in size from 0.6 kb to 3.5 kb. Only those clones greater than 1.0 kb were further character¬ ized, and terminal sequencing quickly identified which clones most likely contained an entire coding sequence. Most of these clones appeared to be identical or highly homologous with DUZ1 (SEQ ID NO:l) in the 3' untrans¬ lated region and the 3' portion of the coding region. These clones contained a stop codon in the same position as found in bOBCAM. However, two of these clones, SG8 (SEQ ID NO:3) and SG13 (SEQ ID NO:2), proved to be of particular interest.

SG13 (SEQ ID NO:2) was identical to rOBCAM in the area where the two clones overlapped; however, unlike rOBCAM, SG13 (SEQ ID NO:2) contained a complete reading frame, a short 3' non-coding region, and a 5* non-coding region that extended upstream further than that of rOBCAM. We believe that SG13 (SEQ ID NO:2) represents a sequence extended with respect to rOBCAM, and which contains an additional unique 5' region.

SG8 (SEQ ID NO:3) also had a complete reading frame, identical to that of SG13 (SEQ ID NO:2), and was also identical to the latter throughout its somewhat shorter 3' non-coding region and in the 200 or so nucleotides immediately upstream of the putative start codon. However, the 5' non-coding region further upstream had no significant sequence homology with SG13 (SEQ ID NO:2), suggesting that it may involve a region regulating translation.

Physiological Significance of Clones. Both ΩUZ1 (SEQ ID NO:l) and SG13 (SEQ ID NO:2) have been found to be located on mouse chromosome 9. Preliminary in situ hybridization studies, using unique regions of these two clones, reveal that their cDNAs are distri¬ buted differently in different brain regions, suggesting they are under different transcriptional regulation. One or more of the unique regions in these new clones may be involved in negatively regulating gene expression.

EXAMPLE 3

Transgenic mice containing antisense oriented region from DϋZl (SEQ ID NO:l) (prepared in a double- stranded construct) exhibited reduced sensitivity to the antinociceptive eff cts of morphine. The transgene used in these studies was the first 500 base pairs as a fragment of the clone DUZ1 (SEQ ID NO:l), but constructed to be in the antisense orientation. Transgenicmice (founders) were identified and non-transgenic littermates were used as controls for antinociceptive tests. Of 33 DϋZl (SEQ ID NO:l) founders, 18 (54%) contained the transgene. One of the transgenic males (#1355) was mated with females of an inbred strain (C57BL/10) to produce Nl generation mice; of 29 offspring, 8 (28%) contained the transgene. Six transgenic (two males, four females) and six non- transgenic littermates (two males, four females) of similar age were grouped for antinociceptive tests. Control mice, lacking the transgene, had an

AD₅₀ for morphine as shown in Table 1. These values were similar whether non-transgenic littermates of transgenic animals or age- and sex-matched naive C57BL/10 mice were used as a control group, as illustrated by the data of Table 1. TABLE 1

Antinociceptive Activity (AD₅₀) of Morphine Sulfate in Transgenic Mice

Fertilized hybrid zygotes were obtained approximately 12 hours postcoitus from the mating of (C57BL/6 female x SJL male) F2 hybrid mice. Zygote isolation, injection, and reimplantation into the oviducts of pseudopregnant Swiss-Webster recipient females. Transgenic offspring were identified by using the polymerase chain reaction on mouse tail DNA samples. Approximately 1 cm of tail was removed and incubated overnight at 55°C in 200 μl of 50 mM Tris, pH 8, 100 mM EDTA, 100 mM NaCl, 1% sodium dodecyl sulfate, and 2.5 mg/ml proteinase K. The digested solution was precipi- tated with isopropanol, spooled, transferred into 50 μl water, and boiled for 15 minutes.

Antinociceptive activity of morphine sulphate was determined by the tail flick method. Morphine sulphate was injected s.c. Because of the small number of animals available (6 in each group) , the up-and-down method was used to determine AD₅₀. (Dixon, Amer. Statistical J . , (1965), 967-978.)

Manipulation of zygotes had no effect on morphine sensitivity of the animals as non-transgenic adults. In contrast, founder mice, and first generation containing the antisense construct, had a morphine AD₅₀ of 11.22±1.32, while the Nl generation mice, the second generation, had a morphine AD₅₀ of 22.4±1.22. Thus the presence of the transgene in mice was correlated with a reduced sensitivity to morphine, and this reduction in sensitivity was increased in the next generation. Nl generationmice derived from founder male 1355 contained the antisense transgene in another inbred strain (DBA/2J) show significantly less sensitivity to morphine. This observation, taken with the difference in sensitivity to morphine in founders and C57BL/10 Nl offspring suggests the expression of the transgene is influenced by the genetic background of the mouse strain harboring it.

In addition, Nl generation of transgenic and non-transgenic animals were tested for acute tolerance. Animals were first assayed for tail-flick latency in the absence of any drug, to generate baseline data. Thirty minutes later, the mice were pretreated with a fixed dose of morphine given s.c. in 0.1 ml physiological saline (see below) , and tail flick latencies monitored again until they had returned to baseline. Morphine antinociception was then determined in the presence of 125 μg/kg s.c. naloxone, given ipsilateral to the morphine sulfate administration at the same time. As in the initial antinociceptive tests, the morphine AD₅₀s were determined by the up-and-down method.

Four sets of acute tolerance tests were carried out, employing morphine pretreatment doses of 0, 10, 30 and 100 mg/kg. Because of the small number of transgenic mice available, the same animals were used for these studies as for the initial tests of antinoci¬ ception. To be certain there were no residual drug effects from the earlier tests, the first acute tolerance studies were begun two weeks after the initial antinociceptive tests, and each subsequent study (employing a higher pretreatment dose of morphine) was carried out two weeks after the preceding one. Finally, two weeks after the last acute tolerance study, the morphine AD₅₀s of animals were again determined (no naloxone) , to confirm that no changes in this parameter had occurred over the course of the experiments.

As shown in Figure 1, the AD_S0 of morphine was increased in the acute tolerance studies, and the higher the pretreatment dose of morphine, the higher the AD₅₀. With a maximum pretreatment dose of 100 mg/kg, the AD₅₀ in the nontransgenic animals reached a maximum of 67.45 mg/kg, a nearly 10-fold increase over the control mice not pretreated with morphine. In contrast, the AD₅₀ of morphine in the transgenic animals (Nl generation) increased relatively slightly with morphine pretreatment dose, with a maximum value of 35.53 mg/kg in transgenic animals pretreated with 100 mg/kg morphine. Since the control AD₅₀ in these animals was elevated to 22.44 mg/kg, this represented an increase of only about 50%.

By this test, too, mice containing the transgene showed a reduced sensitivity to morphine. In this case, this reduced sensitivity was manifested in less of an effect of morphine pretreatment in raising the AD₅₀.

These data show that we have successfully transfected mice with antisense cDNA coding for a portion of an opioid binding protein. The presence of this cDNA results in reduced sensitivity to morphine, by two different tests. The line of transfected animals should be useful in a wide variety of applications relating to opioid function.

It is to be understood that while the invention has been described above in conjunction with preferred specific embodiments, the description and examples are intended to illustrate and not limit the

' scope of the invention, which is defined by the scope of the appended claims. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Lee, Nancy M. Loh, Horace H. Lippman, David

(ii) TITLE OF INVENTION: Oligonucleotide Sequences and Transgenic Animals Transfected Therewith Having Reduced Sensitivity to Narcotic Analgesics (iϋ) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: J. Suzanne Siebert

(B) STREET: Four Embarcadero Center

Suite 1450 (C) CITY: San Francisco

(D) STATE: California

(E) COUNTRY: USA

(F) ZIP: 94111

(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0,

Version #1.25 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 10-APR-1992

(C) CLASSIFICATION: (viϋ) ATTORNEY/AGENT INFORMATION: (A) NAME: Siebert, J. Suzanne

(B) REGISTRATION NUMBER: 28,758

(C) REFERENCE/DOCKET NUMBER: 2983.1 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (415) 362-5556 (B) TELEFAX: (415) 362-5418

(C) TELEX: 278638 MGPS

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3069 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iϋ) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (Vi) ORIGINAL SOURCE:

(A) ORGANISM: Rattus rattus (D) DEVELOPMENTAL STAGE: Adult (F) TISSUE TYPE: brain

(vϋ) IMMEDIATE SOURCE:

(A) LIBRARY: library of Lee, Loh, Lippman (genebank accession #M88709)

(B) CLONE: clone DUZ1 (viii) POSITION IN GENOME:

(B) MAP POSITION: on mouse chromosome 9

(C) UNITS: bp

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CGGGGCGAAG CTGGCTGCAT CCTGGGCTGG AGACCAGGAA GGCGGAATGA TAAGGCTTTG 60

GCATGGGTAT TAGTTAATTT CTGCCAGGAA GCAGCCTTTT CCTGTTCCTT GTGTCCTGAG 120

ACCTGCTAGG GTAAGATTTA GGGAAGGGCA TTAGACCCCT CCCGCTGCCC ACCCAGGAGC 180 GGGTGTGCCC ATGCGAGTCA CACTGGCATA CTCACACAAA TCCCAGCAGT ACTGCTGCGC 240

GGTGAGGGGG TGCGCAAGGG ACAGGCTTCA ACTCCCCTTG GAGTGGAGCC TTCCCAACAG 300

GAAAGTCTCC CGGTGTCGCT CAGGAGAGTG GAGCCAATGT GTGGCTTGGA GGTGGTCTGC 360

TGTGCTCCCA GTGAAGATTG GTGCTGATGA TTCTTACCAG TTGTGTGTAT GTGCAAGGGG 420

GAAAAAATCA GAGACGCTGT CTGCCTGCTC CCATCTCGCG CGCGCTCTCT CTCTTCTGCT 480

CTCTCCCTCC CTTTGCAAAC ATTGGATTTA AACCTGCTCA GAATTCAGTA CAGAGGAAGC 540

AGCCTCGGTG GTAGCAGCAG CAGCACCAGC AGCAGCAGCA CCAGCAGCAC CAGCACCAGC 600

AGGAGCTCGC CGGGCCGCCG CGCACCACAG CCTCGAGATG TACCATCCCG CCTACTGGAT 660

CGTCTTCTCG GCCACCACTG CCCTGCTCTT CATCCCAGGA GTGCCGGTGC GCAGCGGAGA 720

TGCCACCTTT CCCAAAGCTA TGGACAACGT GACGGTCCGG CAGGGAGAGA GCGCCACCCT 780

CAGGTGTACC ATAGATGACC GGGTCACCAG AGTAGCCTGG CTAAACCGCA GCACAATCCT 840

CTACGCTGGG AATGACAAGT GGTCCATAGA CCCTCGAGTG ATCATCTTGG TCAACACGCC 900 TACCCAGTAC AGTATCATGA TCCAGAATGT GGATGTTTAT GACGAAGGTC CGTACACCTG 960

CTCTGTGCAG ACAGACAATC ACCCCAAAAC CTCCCGGGTC CACCTCATAG TGCAAGTTCC 1020

TCCCCAGATA ATGAACATCT CTTCAGACAT TACTGTGAAT GAGATAAGCA GTGTGACCTT 1080

GTTATGTCTC GCAATTGGCA GACCAGAACC AACAGTGACA TGGCGACACC TGTCAGTCAA 1140

GGAAGGCCAG GGCTTTGTGA GTGAAGATGA ATACCTGGAA ATCTCAGACA TCAAACGCGA 1200

CCAATCTGGA GAGTATGAGT GCAGCGCCTT GAATGATGTC GCTGCACCTG ATGTTCGGAA 1260

AGTAAAAATC ACTGTAAACT ATCCTCCCTA TATCTCAAAA GCGAAGAACA CTGGCGTTTC 1320

AGTAGGCCAG AAGGGCATCC TGAGCTGTGA AGCCTCTGCT GTCCCCATGG CTGAATTCCA 1380

GTGGTTCAAG GAAGATACCA GGTTAGCCAC TGGCCTGGAT GGCGTGAGAA TTGAGAACAA 1440

AGGCCGCATA TCCACTTTGA CTTTCTTCAA TGTCTCAGAG AAGGATTATG GGAACTATAC 1500

CTGTGTGGCC ACAAACAAGC TTGGGAACAC CAATGCCAGC ATCACCCTGT ATGGGCCTGG 1560

AGCAGTCATT GATGGTGTAA ACTCGGCCTC TAGAGCACTG GCTTGTCTCT GGCTCTCAGG 1620 GACCTTCTTT GCCCACTTCT TCATCAAGTT TTGATAAGAA ACCTTAGGTC CTCTGAGCAT 1680

CGCCTGCTTC TCCATATCAC AGACTTTAAT CTACACTGCG GAGGGGCAAA CCAGTTTGGG 1740

CTTTCTTTTG GTTATTTTTT TGTTCTTCTT GACTGTTTAG TTTTTTGGTT TGATTTCTGG 1800

GATTTTCAAT TTGATTTGTT TTTCTTTTTT CGTTTGAATG GACCGGGGTT GGGGGTTGGG 1860

ATGGGCAGGG TTCTACCACG AGTAGGATAA TCAGGTATTG GTGGGCCCCC AAATGGAATA 1920

TATTCCTGCT ACCTTGGCCT TCCTTTTCTC TACTTCTCTT CCTACCACCA TTAACACACA 1980

AACACACAAG CACACGCCCT AAAGATGGCC TAAAAAATGT CCCATGACAC GCACCCTGAA 2040

GGTACAACTT GGCCCACAGT GCAGTACACA ATAAGAGTTG CATCTACATT TCCTGTTTTC 2100

TTTGTCCTTT AAGTTTTCAA TAAGACAGTT TAAAAAGAGC ACATCCTTAT CCCTATGTTT 2160

GTATCACCTA TCCCATTAAG CTGCACACCT TTTCTAAGAA ACTTTCTTAC TACATCCTCA 2220

ATGTGCACAC ATGTACATTC TCATAAAAAT TTTACCATCT TCTCTGGCCA TTCTGCTTAA 2280

TCTGCATTTT CCACTAGCAG TAAGACTTAC AGGCTTGATG AAATATACAT GTATCCAAAG 2340 CTACAAATTT AGAAGTCACG AGGGAAGTCT ATCTAGGGGT AGTACTTCCC TTAAAATGCT 2400

AATGCAACTC ATAAAAAAGT GATCAATAGC TAGCTAATTA TATCAAGCTA TCAAAGCAAT 2460

CATATTATTA TCCAGTTACT CAATTGATTT CTCTCTCCAT CTCCTTCTAC ATACCTATCT 2520

TTCTTATAAT CTATTTGTTC AATTTATCTA ACTACTCTTT TTCTCTTCCC ACTGCACTAT 2580

CCATTCATCT CATTTAAATC TCTAATCATA TTGTGACTGT GCCCTTATAT TGTGTCTCTG 2640

CACTACCACC AAGACATTGC CACCTTCAAA ATTGCCTAGC AACTTCTTAT GTGAAAGCCA 2700

GTAATGTCTC GGGCTAACTA CCCAGAAGAA CAATTTCCTA TACCATGGAT CCTTGGGAAT 2760

GCACTAACCC ATCCACCCAA ATTAGGCCTT GTGAACAGAT GGACCAAAGT AGCAATCTAT 2820

GGATCAGTCA CTCATGATTT TCCGAGAGAC TGTTCCCTTA GCCACCTTCT CAGGAGGCAG 2880

GTCAGCCCCG AAGAAGCCTT GATTATGCTG AATTTCTCCT TTAACAGCTG GAAAATTAAG 2940

GTACCAACCC TGTGCTTCTC TCAGCCTTTC AAGAAAAGTA CATGTCAGGA ACTTGGAGAA 3000

ACTTCTTCAT GGCTGGCTTT TATTAGCAGA AAGAACCTGA CCAAAAAAAA AACCAAAAAA 3060 AAAAAAAAA

3069

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2337 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iϋ) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE:

(A) ORGANISM: Rattus rattus (D) DEVELOPMENTAL STAGE: Adult (F) TISSUE TYPE: brain

(Vii) IMMEDIATE SOURCE:

(A) LIBRARY: stratagene (genebank accession #M88711)

(B) CLONE: SG13 (Viii) POSITION IN GENOME:

(B) MAP POSITION: on mouse chromosome 9

(C) UNITS: bp

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

CTTTTTTTTA TTTTTTATTT TTTTTACGAC ATCTCCTTTC TGTCAGAGAC ACTTGCCTTC 60

TTTGGGTTAT AAACTTTTGA TGCCAGACCT CTGCAGAATC GCCCAACTGA ATCTTAAAGT 120

AGCTTCACCG AGAGAGGCAG AGAACGCACA ATCTGTGACC TTCGTCCCTG CTCCTTTTTT 180 GTGTTCTCCC TCCTCCCGGG CTCCCTCCGC CCCAGGGAGC GTTGAGAAAG CTCTTTTTTG 240

GATGCAGGAG GGGGCATCTG GTTCTGCCAG GCTGGAAAGC TGAGGCTGGA GCAGAGGAAG 300

ACTTATTAGA CTCCGGAGAG CCTGGACTCA GCCTTGCCTT CTCCCGCTCC CAGCTCCTGG 360

TTTGCTCTCT GTGTGCTTTC GTCCCTCAAC ATTCCGGCTA TTCTGAGAGG GCAGGGGACA 420

AGGACCGTGC AGCTGCAAGA GTTCTAGGAA GTTGTGGCTG TCGAGAATGG GGGTCTGTGG 480

GTACCTGTTC CTGCCCTGGA AGTGCCTCGT GGTCGTGTCT CTGAGGCTGC TGTTCCTTGT 540

ACCCACAGGA GTGCCGGTGC GCAGCGGAGA TGCCACCTTT CCCAAAGCTA TGGACAACGT 600

GACGGTCCGG CAGGGAGAGA GCGCCACCCT CAGGTGTACC ATAGATGACC GGGTCACCAG 660

AGTAGCCTGG CTAAACCGCA GCACAATCCT CTACGCTGGG AATGACAAGT GGTCCATAGA 720

CCCTCGAGTG ATCATCTTGG TCAACACGCC TACCCAGTAC AGTATCATGA TCCAGAATGT 780

GGATGTTTAT GACGAAGGTC CGTACACCTG CTCTGTGCAG ACAGACAATC ACCCCAAAAC 840

CTCCCGGGTC CACCTCATAG TGCAAGTTCC TCCCCAGATA ATGAACATCT CTTCAGACAT 900 TACTGTGAAT GAGATAAGCA GTGTGACCTT GTTATGTCTC GCAATTGGCA GACCAGAACC 960

AACAGTGACA TGGCGACACC TGTCAGTCAA GGAAGGCCAG GGCTTTGTGA GTGAAGATGA 1020

ATACCTGGAA ATCTCAGACA TCAAACGCGA CCAATCTGGA GAGTATGAGT GCAGCGCCTT 1080

GAATGATGTC GCTGCACCTG ATGTTCGGAA AGTAAAAATC ACTGTAAACT ATCCTCCCTA 1140

TATCTCAAAA GCGAAGAACA CTGGCGTTTC AGTAGGCCAG AAGGGCATCC TGAGCTGTGA 1200

AGCCTCTGCT GTCCCCATGG CTGAATTCCA GTGGTTCAAG GAAGATACCA GGTTAGCCAC 1260

TGGCCTGGAT GGCGTGAGAA TTGAGAACAA AGGCCGCATA TCCACTTTGA CTTTCTTCAA 1320

TGTCTCAGAG AAGGATTATG GGAACTATAC CTGTGTGGCC ACAAACAAGC TTGGGAACAC 1380

CAATGCCAGC ATCACCCTGT ATGGGCCTGG AGCAGTCATT GATGGTGTAA ACTCGGCCTC 1440

TAGAGCACTG GCTTGTCTCT GGCTCTCAGG GACCTTCTTT GCCCACTTCT TCATCAAGTT 1500

TTGATAAGAA ACCTTAGGTC CTCTGAGCAT CGCCTGCTTC TCCATATCAC AGACTTTAAT 1560

CTACACTGCG GAGGGGCAAA CCAGTTTGGG CTTTCTTTTG GTTATTTTTT TGTTCTTCTT 1620 GACTGTTTAG TTTTTTGGTT TGATTTCTGG GATTTTCAAT TTGATTTGTT TTTCTTTTTT 1680

CGTTTGAATG GACCGGGGTT GGGGGTTGGG ATGGGCAGGG TTCTACCACG AGTAGGATAA 1740

TCAGGTATTG GTGGGCCCCC AAATGGAATA TATTCCTGCT ACCTTGGCCT TCCTTTTCTC 1800

TACTTCTCTT CCTACCACCA TTAACACACA AACACACAAG CACACGCCCT AAAGATGGCC 1860

TAAAAAATGT CCCATGACAC GCACCCTGAA GGTACAACTT GGCCCACAGT GCAGTACACA 1920

ATAAGAGTTG CATCTACATT TCCTGTTTTC TTTGTCCTTT AAGTTTTCAA TAAGACAGTT 1980

TAAAAAGAGC ACATCCTTAT CCCTATGTTT GTATCACCTA TCCCATTAAG CTGCACACCT 2040

TTTCTAAGAA ACTTTCTTAC TACATCCTCA ATGTGCACAC ATGTACATTC TCATAAAAAT 2100

TTTACCATCT TCTCTGGCCA TTCTGCTTAA TCTGCATTTT CCACTAGCAG TAAGACTTAC 2160

AGGCTTGATG AAATATACAT GTATCCAAAG CTACAAATTT AGAAGTCACG AGGGAAGTCT 2220

ATCTAGGGGT AGTACTTCCC TTAAAATGCT AATGCAACTC ATAAAAAAGT GATCAATAGC 2280

TAGCTAATTA TATCAAGCTA TCAAAGCAAT CATATTATTA TCCAGTTACT CAATTGA 2337 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2179 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ϋi) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE:

(A) ORGANISM: Rattus rattus (D) DEVELOPMENTAL STAGE: Adult (F) TISSUE TYPE: brain

(vii) IMMEDIATE SOURCE: (A) LIBRARY: stratagene (genebank accession

#M88710)

(B) CLONE: SG8 (viii) POSITION IN GENOME:

(C) UNITS: bp

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

CGTGTGATCA ATGGTCTTAG AAGGCTTGTT AGACAGCTGT GGGGCTCAGT CTTCTCGCAG 60

AATGTTAACA GTGGGAGCAG GGGCTGTCTC TGACTCTGTT GCCTGCCGGC AGTGAGCAAT 120

GGTCTTAGAA GACTTATTAG ACTCCGGAGA GCCTGGACTC AGCCTTGCCT TCTCCCGCTC 180

CCAGCTCCTG GTTTGCTCTC TGTGTGCTTT CGTCCCTCAA CATTCCGGCT ATTCTGAGAG 240

GGCAGGGGAC AAGGACCGTG CAGCTGCAAG AGTTCTAGGA AGTTGTGGCT GTCGAGAATG 300 GGGGTCTGTG GGTACCTGTT CCTGCCCTGG AAGTGCCTCG TGGTCGTGTC TCTGAGGCTG 360

CTGTTCCTTG TACCCACAGG AGTGCCGGTG CGCAGCGGAG ATGCCACCTT TCCCAAAGCT 420

ATGGACAACG TGACGGTCCG GCAGGGAGAG AGCGCCACCC TCAGGTGTAC CATAGATGAC 480

CGGGTCACCA GAGTAGCCTG GCTAAACCGC AGCACAATCC TCTACGCTGG GAATGACAAG 540

TGGTCCATAG ACCCTCGAGT GATCATCTTG GTCAACACGC CTACCCAGTA CAGTATCATG 600

ATCCAGAATG TGGATGTTTA TGACGAAGGT CCGTACACCT GCTCTGTGCA GACAGACAAT 660

CACCCCAAAA CCTCCCGGGT CCACCTCATA GTGCAAGTTC CTCCCCAGAT AATGAACATC 720

TCTTCAGACA TTACTGTGAA TGAGATAAGC AGTGTGACCT TGTTATGTCT CGCAATTGGC 780

AGACCAGAAC CAACAGTGAC ATGGCGACAC CTGTCAGTCA AGGAAGGCCA GGGCTTTGTG 840

AGTGAAGATG AATACCTGGA AATCTCAGAC ATCAAACGCG ACCAATCTGG AGAGTATGAG 900

TGCAGCGCCT TGAATGATGT CGCTGCACCT GATGTTCGGA AAGTAAAAAT CACTGTAAAC 960

TATCCTCCCT ATATCTCAAA AGCGAAGAAC ACTGGCGTTT CAGTAGGCCA GAAGGGCATC 1020 CTGAGCTGTG AAGCCTCTGC TGTCCCCATG GCTGAATTCC AGTGGTTCAA GGAAGATACC 1080

AGGTTAGCCA CTGGCCTGGA TGGCGTGAGA ATTGAGAACA AAGGCCGCAT ATCCACTTTG 1140

ACTTTCTTCA ATGTCTCAGA GAAGGATTAT GGGAACTATA CCTGTGTGGC CACAAACAAG 1200

CTTGGGAACA CCAATGCCAG CATCACCCTG TATGGGCCTG GAGCAGTCAT TGATGGTGTA 1260

AACTCGGCCT CTAGAGCACT GGCTTGTCTC TGGCTCTCAG GGACCTTCTT TGCCCACTTC 1320

TTCATCAAGT TTTGATAAGA AACCTTAGGT CCTCTGAGCA TCGCCTGCTT CTCCATATCA 1380

CAGACTTTAA TCTACACTGC GGAGGGGCAA ACCAGTTTGG GCTTTCTTTT GGTTATTTTT 1440

TTGTTCTTCT TGACTGTTTA GTTTTTTGGT TTGATTTCTG GGATTTTCAA TTTGATTTGT 1500

TTTTCTTTTT TCGTTTGAAT GGACCGGGGT TGGGGGTTGG GATGGGCAGG GTTCTACCAC 1560

GAGTAGGATA ATCAGGTATT GGTGGGCCCC CAAATGGAAT ATATTCCTGC TACCTTGGCC 1620

TTCCTTTTCT CTACTTCTCT TCCTACCACC ATTAACACAC AAACACACAA GCACACGCCC 1680

TAAAGATGGC CTAAAAAATG TCCCATGACA CGCACCCTGA AGGTACAACT TGGCCCACAG 1740 TGCAGTACAC AATAAGAGTT GCATCTACAT TTCCTGTTTT CTTTGTCCTT TAAGTTTTCA 1800

ATAAGACAGT TTAAAAAGAG CACATCCTTA TCCCTATGTT TGTATCACCT ATCCCATTAA 1860

GCTGCACACC TTTTCTAAGA AACTTTCTTA CTACATCCTC AATGTGCACA CATGTACATT 1920

CTCATAAAAA TTTTACCATC TTCTCTGGCC ATTCTGCTTA ATCTGCATTT TCCACTAGCA 1980

GTAAGACTTA CAGGCTTGAT GAAATATACA TGTATCCAAA GCTACAAATT TAGAAGTCAC 2040

GAGGGAAGTC TATCTAGGGG TAGTACTTCC CTTAAAATGC TAATGCAACT CATAAAAAAG 2100

TGATCAATAG CTAGCTAATT ATATCAAGCT ATCAAAGCAA TCATATTATT ATCCAGTTAC 2160

TCAATTGATT TCTCTCTCC

2179

Claims

It is Claimed:

1. A transgenic, non-human animal all of whose germ cells and somatic cells contain an oligo¬ nucleotide sequence introduced into said animal, or an ancestor of said animal, at an embryonic stage, said oligonucleotide sequence expressible in the animals and effective to alter the animal's response to a narcotic analgesic.

2. The animal as in claim 1 wherein the altered response to a narcotic analgesic is a reduced sensitivity to the narcotic analgesic.

3. The animal as in claim 1 or 2 wherein the narcotic analgesic is morphine, a morphine analogue, an endogenous opioid, or a synthetic opioid peptide.

4. The animal as in claim 1 or 2 wherein the oligonucleotide sequence forms a transcriptional unit expressible in the animal as a messenger RNA compound capable of hybridizing to endogenous mRNA for an opioid binding protein.

5. The animal as in claim 4 wherein the oligonucleotide sequence is double or single stranded and is selected from all or a portion of the sequences of the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, but wherein the selected sequence, or sequence portion, is reversed with respect thereto.

6. The animal as in claim 4 wherein the oligonucleotide sequence selected has at least about 12 bases. 7. The animal as in claim 4 wherein the oligonucleotide sequence is selected from all or a portion of the first 500 bases of SEQ ID NO:l.

8. A method of providing a cell culture comprising: providing a transgenic, non-human animal all of whose germ cells and somatic cells contain an oligonucleotide sequence introduced into said animal, or an ancestor of said animal, at an embryonic stage, said oligonucleotide sequence expressible in the animal and effective to alter the animal's response to a narcotic analgesic; and culturing one or more somatic cells from said animal.

9. An oligonucleotide construct comprising a segment corresponding to all or part of the first about 500 bases of SEQ ID N0:l, but reversed with respect thereto, the segment being sufficient in size to be capable of hybridizing with endogenous mRNA for an opioid binding protein.

10. The construct as in claim 9 wherein the segment has at least about 12 bases.

11. The construct of claim 9 wherein said segment is operatively linked to an expression vector.

12. The construct as in claim 11 wherein the expression vector is a mammalian or retroviral expression vector.

13. The construct as in claim 9 wherein at least a portion of the normal phosphodiester backbone has been modified with an analogue effective to limit degradation in vivo.

14. The construct as in claim 13 wherein the modification is by means of a methylphosphonate, a phosoramidate, a phosphorothioate, a 2'-o-alkyl, a polyamide (2-aminoethylgylcine) or a modification of ribose to a cycloalkane or partially unsaturated cycloalkane.

15. The construct as in claim 9 wherein the segment has a complementary strand associated therewith.

16. The construct as in claim 9 or 15 wherein the bases of the segment correspond to RNA.

17. A diagnostic or therapeutic agent comprising an oligonucleotide with at least about 12 bases when single stranded and about 12 base pairs when double stranded and having a backbone, the oligonucleo¬ tide defining a sequence of DNA bases or RNA bases analogous to all or a portion of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, but in the antisense direction thereof.

18. The agent as in claim 17 wherein at least a portion of the backbone has been modified effective to limit enzymatic degradation.

19. The agent as in claim 17 wherein the fragment is capable of hybridizing to mRNA for an opioid binding protein.

20. The agent as in claim 17 including a means for therapeutically administrating the oligo¬ nucleotide to a patient. AMENDED CLAIMS

[received by the International Bureau on 30 August 1993 (30.08.93); original claims 1,4,9 and 10 amended; other claims unchanged (3 pages)]

1. A transgenic, non-human animal all of whose germ cells and somatic cells contain an oligo¬ nucleotide sequence introduced into said animal, or an ancestor of said animal, at an embryonic stage, wherein expression of the oligonucleotide sequence in the animals affects the animal's response to a narcotic analgesic.

2. The animal as in claim 1 wherein the altered response to a narcotic analgesic is a reduced sensitivity to the narcotic analgesic.

3. The animal as in claim 1 or 2 wherein the narcotic analgesic is morphine, a morphine analogue, an endogenous opioid, or a synthetic opioid peptide.

4. The animal as in claim 1 or 2 wherein the oligonucleotide sequence forms a transcriptional unit in the animal as a messenger RNA compound which hybridizes to endogenous mRNA for an opioid binding protein.

5. The animal as in claim 4 wherein the oligonucleotide sequence is double or single stranded and is selected from all or a portion of the sequences of the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, but wherein the selected sequence, or sequence portion, is reversed with respect thereto.

6. The animal as in claim 4 wherein the oligonucleotide sequence selected has at least about 12 bases.

7. The animal as in claim 4 wherein the oligonucleotide sequence is selected from all or a portion of the first 500 bases of SEQ ID NO:l.

8. A method of providing a cell culture comprising: providing a transgenic, non-human animal all of whose germ cells and somatic cells contain an oligonucleotide sequence introduced into said animal, or an ancestor of said animal, at an embryonic stage, said oligonucleotide sequence expressible in the animal and effective to alter the animal's response to a narcotic analgesic; and culturing one or more somatic cells from said animal.

9. An oligonucleotide construct comprising a segment corresponding from about 12 to about 500 bases of SEQ ID NO:l, but reversed with respect thereto, the segment being sufficient in size to hybridize with endogenous mRNA for an opioid binding protein.

10. The construct as in claim 9 wherein the segment is effective to suppress expression of an opioid receptor component gene.

11. The construct of claim 9 wherein said segment is operatively linked to an expression vector.

12. The construct as in claim 11 wherein the expression vector is a mammalian or retroviral expression vector.

13. The construct as in claim 9 wherein at least a portion of the normal phosphodiester backbone has been modified with an analogue effective to limit degradation in vivo .

14. The construct as in claim 13 wherein the modification is by means of a methylphosphonate, a phosoramidate, a phosphorothioate, a 2'-o-alkyl, a polyamide (2-aminoethylgylcine) or a modification of ribose to a cycloalkane or partially unsaturated cycloalkane.

15. The construct as in claim 9 wherein the segment has a complementary strand associated therewith.

16. The construct as in claim 9 or 15 wherein the bases of the segment correspond to RNA.

17. A diagnostic or therapeutic agent comprising an oligonucleotide with at least about 12 bases when single stranded and about 12 base pairs when double stranded and having a backbone, the oligonucleo¬ tide defining a sequence of DNA bases or RNA bases analogous to all or a portion of SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3, but in the antisense direction thereof.

18. The agent as in claim 17 wherein at least a portion of the backbone has been modified effective to limit enzymatic degradation.

19. The agent as in claim 17 wherein the fragment is capable of hybridizing to mRNA for an opioid binding protein.

20. The agent as in claim 17 including a means for therapeutically administrating the oligo¬ nucleotide to a patient.