EP0906123A1 - OLIGONUCLEOTIDES CIBLES SUR L'ARNm DE L'ANGIOTENSINOGENE - Google Patents

OLIGONUCLEOTIDES CIBLES SUR L'ARNm DE L'ANGIOTENSINOGENE

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Publication number
EP0906123A1
EP0906123A1 EP97916760A EP97916760A EP0906123A1 EP 0906123 A1 EP0906123 A1 EP 0906123A1 EP 97916760 A EP97916760 A EP 97916760A EP 97916760 A EP97916760 A EP 97916760A EP 0906123 A1 EP0906123 A1 EP 0906123A1
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Prior art keywords
oligonucleotide
mrna
angiotensinogen
antisense
human
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EP0906123A4 (fr
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Donna Wielbo
M. Ian Phillips
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University of Florida
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University of Florida
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/311—Phosphotriesters
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3511—Conjugate intercalating or cleaving agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/353—Nature of the modification linked to the nucleic acid via an atom other than carbon
    • C12N2310/3535—Nitrogen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

  • This invention relates to compositions and methods which are useful for reducing hypertension in humans. More particularly, it relates to oligonucleotide compounds capable of binding to angiotensinogen mRNA to inhibit expression of angiotensinogen, and thereby, essential hypertension.
  • Angiotensinogen (AGT) , which is produced largely by the liver, is the substrate for the protein remn, produced by the kidneys. The action of remn on angiotensinogen results in the formation of the decapeptide angiotensm I. Angiotensm I is further converted to the octapeptide angiotensm II, through the action of angiotensm converting enzyme (ACE) as it circulates through the vasculature. Angiotensm II is a potent vasoconstrictor and regulator of blood pressure and volume homeostasis.
  • RAS overactive rerun angiotensm system
  • Hepatic AGT is regulated mainly at the transcriptional level in hepatocytes, by hormones acting at the genomic level such as steroids, thyroid hormones, glucocorticoids and inflammatory cytokines via activation of DNA binding proteins that interact with the appropriate response elements and multi-hormone response sites like the hormone mducible enhancer unit (HIEU) at nucleotides -615 to -440 upstream of the major transcription start site (Peters, J. , supra, 1995; Brashier, A. R. , ej: a . , Kidney International , 46:1564- 1566, 1994) .
  • hormones acting at the genomic level such as steroids, thyroid hormones, glucocorticoids and inflammatory cytokines via activation of DNA binding proteins that interact with the appropriate response elements and multi-hormone response sites like the hormone mducible enhancer unit (HIEU) at nucleotides -615 to -440 upstream of the major transcription start site (Peters, J. , supra,
  • a large number of antihypertensive agents are commercially available, however most have severe side effects which generally require the action of a second group of agents. This, in combination with the nature of the disease, tends to result in poor patient compliance.
  • a number of renin angiotensm system inhibitors presently under use mclude: renin inhibitors, angiotensm converting enzyme inhibitors, and angiotens receptor blockers. These agents also lack specificity, have unwanted side effects, require frequent dosing regimens, and do not completely prevent the formation of angiotensm II.
  • An alternative approach has been proposed to reduce hypertension. A number of researchers have developed antisense sequences for the inhibition of renin- angiotensm system components.
  • Antisense oligodeoxynucleotides have been used to successfully inhibit prote synthesis m a number of biological systems (Yakubov, L. , e_t a_l . , Proc. Natl . Acad . Sci . USA 86:6454-6458, 1989; Wahlstedt, D., et al., Science 259:528-531, 1993; Wahlstedt, D., et al., Na ture 363:260-263, 1993; Wielbo, D., e ⁇ a ., Hypertension 25:314-319, 1995) .
  • Antisense regulation or attenuation of protem synthesis can be applied to any candidate gene with known molecular sequence.
  • Antisense molecules are short strands of DNA or RNA, usually 12-18 bases in length which are synthesized to complement a target region of a candidate gene.
  • the antisense molecule binds to its complementary region and via a number of mechanisms inhibits or attenuates gene expression (Helene, C. C. and Toulme, J. J. , Biochemica et Biophysica Acta 1049:99-125, 1990) .
  • Phosphorothioated ASODN are modified phosphodiester oligonucleotides where one of the non-bridged oxygen atoms of the internucleotide linkage has been replaced with a sulfur. Fully thioated antisense molecules are more resistant to nucleases but also exhibit several non sequence-specific effects m doses generally greater than 1 ⁇ m (Wagner, R. W., JVature 372:333-335, 1994) . Phosphorothioation also stimulates the activity of
  • RNase H This enzyme recognizes the DNA-mRNA duplex as a substrate and cleaves the mRNA portion of the duplex, freeing the DNA antisense molecule to bind to other mRNA strands promoting a type of catalytic effect (Helene, C. C and Toulme, J. J., supra. 1990; Boiziau, C, et . al . , Biochemi cal Soci ety Transactions 20 -. 164 - 161, 1992) .
  • Gyurko was able to show decreases in blood pressure after central administration of ASODN targeted to the ATI receptor (Gyurko, R., et . a_l . , Regula tory Peptides 49:16 ⁇ 174, 1993) and similar effects were observed when Wielbo targeted central AGT (Wielbo, D. , et al., supra, 1995) .
  • Tomita was able to decrease blood pressure in the SHR using three contiguous antisense oligonucleotide sequences to target peripheral AGT and also showed decreases in AGT mRNA (Tomita, N. , et. al.
  • the mvention resides in the discovery that AGT expression can be inhibited m humans by administration of oligonucleotide compounds of Formula I:
  • each X independently is 0, S, or C x 4 alkyl ; each B independently is adenme, guanme, cytosme, or thymme selected such that the oligonucleotide is capable of binding to the sense mRNA strand coding for human angiotensinogen to thereby inhibit the translation thereof; each R independently is H or C 1-4 alkyl or P(0) (0) -substituted ac ⁇ dme; each Y independently is H or OH; and n is 8 to 23.
  • the oligonucleotide compound of Formula I may also be a pharmaceutically acceptable salt or hydrate thereof.
  • B is selected such that the oligonucleotide is antisense to the sense mRNA strand for human angiotensinogen. More preferably, B is selected such that the oligonucleotide is capable of binding to the mRNA base region encompassing the AUG initiation codon.
  • Another aspect of the invention provides a pharmaceutical composition useful for inhibiting expression of angiotensinogen comprising a pharmaceutical carrier and oligonucleotides of the above kind.
  • the pharmaceutical carrier may be a liposome, viral vector, or protem conjugate formulation.
  • Another aspect of the mvention provides a method for treating hypertension m a human comprising administering to a subject an effective amount of oligonucleotides or compositions of the above kind.
  • Fig. 1 is a bar graph depicting the percent expression of angiotensinogen using one antisense oligonucleotide of the present invention
  • Figs. 2a and 2b are micrographs of rat liver tissue one hour after injection of unencapsulated antisense oligonucleotide and liposome encapsulated oligonucleotide, respectively;
  • Fig. 3 is a bar graph depicting the change m mean arterial pressure after treating spontaneously hypertensive rats with liposome encapsulated antisense oligonucleotide and control compositions;
  • Fig. 4 is a bar graph depicting the level of plasma angiotens II of spontaneously hypertensive rats after treatmg them with liposome encapsulated antisense oligonucleotide and control compositions;
  • Fig. 5 is a bar graph depicting the level of plasma angiotensinogen of spontaneously hypertensive rats after treating them with liposome encapsulated antisense oligonucleotide and control compositions;
  • Fig. 6 is a graph showing comparative data using a viral vector delivery system
  • Fig. 7a shows a Northern blot hybridization for AGT and the control gene cathepsm D
  • Fig. 7b is a bar graph showmg the relative intensity of AGT mRNA expression after treatment compared to angiotensinogen expression in untreated control cells;
  • Fig. 8 is a bar graph showmg the effect of cationic liposome conjugated ASODN on angiotensinogen production
  • Fig. 9a shows a Northern blot hybridization for AGT and the control gene cathepsm D;
  • Fig. 9b is a bar graph showing the relative expression of AGT mRNA compared to control treated samples.
  • Fig. 10 is a bar graph showmg the dose dependent decreases in angiotensinogen protem after cationic liposomal delivery of ASODN.
  • AGT AGT
  • the messenger RNA coding for AGT has the same nucleotide sequence as the sense DNA strand except that the DNA thymidme is replaced by undine .
  • synthetic antisense nucleotide sequences should bind with tne DNA and RNA coding for AGT.
  • the oligonucleotide compounds of the mvention bind to the messenger RNA coding for human AGT thereby inhibiting expression of this protem.
  • Preferred compounds of the invention are antisense to the sense DNA sequence coding for human AGT as shown in Fig. 2 of Fukamizu, et al. 265 J ⁇ Biol. Chem. 7576-7582 (1990) .
  • Especially preferred oligonucleotide compounds are those in which B of Formula I is selected such that the base sequence of the oligonucleotide is 5' -CTCGCTTCCGCATACCCT-3' (SEQ ID N0:1) .
  • the letters, A, G, C, T, and U respectively indicate nucleotides in which the nucleoside is Adenosine (Ade) , Guanosine (Gua) , Cytidine (Cyt) , Thymidine (Thy) , and Uridine (Ura) .
  • compounds that are antisense to the AGT DNA or mRNA sense strand are compounds which have a nucleoside sequence complementary to the sense strand. Table 1 shows the four possible sense strand nucleosides and their complements present in an antisense compound.
  • the present invention broadly includes oligonucleotide compounds which are capable of binding to the sense mRNA strand coding for angiotensinogen.
  • the invention includes compounds which are not strictly antisense: the compounds may have some non-complementary bases provided such compounds have sufficient binding affinity for angiotensinogen mRNA to inhibit expression.
  • C ⁇ 4 alkyl means a branched or unbranched hydrocarbon having 1 to 4 carbon atoms .
  • Formula I compounds also may be substituted at the 3' and/or 5' ends by a substituted acridme derivative.
  • substituted acridme means any acridme derivative capable of intercalating nucleotide strands such as DNA.
  • Preferred substituted acrid es are 2- methoxy-6-chloro-9-pentylammoacr ⁇ dme, N- (6-chloro-2- methoxyacndmyl) -O-methoxydiisopropylammophosphmyl-3- ammopropanol, and N- (6-chloro-2-methoxyacridmyl) -O- methoxyd ⁇ sopropylammophosphmyl-5-ammopentanol .
  • Other suitable acridme derivatives are readily apparent to persons skilled m the art.
  • P(0) (0) -substituted acridme means a phosphate covalently linked to a substitute acridme.
  • Formula I compounds also may mclude ribozyme sequences inserted into their nucleotide sequence.
  • the ribozyme sequences are inserted mto Formula I compounds such that they are immediately preceded by AUC, UUC, GUA, GUU, GUC, or, preferably, CUC.
  • the ribozyme sequence is any sequence which can be inserted and causes self- cleavage of messenger RNA.
  • the sequence CUG AUG AGU CCG UGA CGA A is preferred.
  • Other such sequences can be prepared as described by Haseloff and Gerlach, 334 Nature 585-591 (1988) .
  • the compounds of Formula I have about 10 to 25 nucleotides.
  • nucleotides includes nucleotides which the phosphate moiety is replaced by phosphorothioate or alkylphosphonate and the nucleotides may be substituted by substituted acridmes.
  • Preferred Formula I compounds have 13 to 22 nucleotides. More preferred are compounds havmg 16 to 20 nucleotides. Most preferred are compounds havmg 18 nucleotides. Compounds havmg fewer than 10 nucleotides are less desirable because they generally have less specificity and compounds having greater than 25 nucleotides are less desirable because their physical size and charge will attenuate the crossing of the lipophilic cell membrane. Thus, they are less likely to enter cells.
  • Formula I includes nucleotide compounds which lack a complement for each nucleotide in a segment of the mRNA sense strand provided such compounds have sufficient binding affinity for human AGT mRNA to inhibit expression.
  • the procedures of Examples 1 and 5 are useful for screening whether specific oligonucleotides of the present invention are effective in inhibiting angiotensinogen expression.
  • R is H
  • R can be C j 4 alkyl provided the resulting compounds retains sufficient binding affinity for the AGT mRNA sense strand to inhibit expression of AGT.
  • Formula I compounds in which at least one X is S are prepared by the following published procedures: W.J. Stec, e_t al. , 106 J ⁇ Am. Che . Soc . 6077-6079 (1984) ; S.P. Adams, et al., 105 J_ Am. Chem. Soc. 661 (1983) ;
  • the reaction scheme involves lH-tetrazole-catalyzed coupling of phosphoramidites to give phosphate intermediates which are reacted with sulfur in 2,6- lutidme to give phosphate compounds.
  • Oligonucleotide compounds are prepared by treating the phosphate compounds with thiophenoxide (1:2:2 thiophenol/triethylamine/tetra-hydrofuran, room temperature, 1 hour) . The reaction sequence is repeated until an oligonucleotide compound of the desired length has been prepared (Formula 1) .
  • Formula I compounds are cleaved from the support by treating with ammonium hydroxide at room temperature for 1 hour and then are further deprotected by heating at about 50°C overnight to yield Formula I compounds.
  • Formula I compounds m which at least one X is oxygen are prepared by substituting I 2 - H 2 0 for the sulfur in 2,6-lut ⁇ dme.
  • Formula I compounds in which at least X is CH 3 or other Cj 4 alkyl are prepared by the following published procedure: K.L. Aqarwal and F. Riftma, 6 Nucl . Acids Res. 3009-3023 (1979) .
  • the reaction sequence is conducted on a solid support.
  • the reaction procedure involves phosphorylation of the 3'-hydroxyl group of a 5' -protected nucleoside using methylphosphonoditriazolide as the phosphorylatmg reagent followed by benzene sulfonyl-catalyzed coupling of the methylphosphonates to yield the methyl phosphonate oligonucleotide.
  • Methylphosphonoditriazolide is prepared m situ from equimolar quantities of methylphosphono-dichloridate, triethylamme, and triazole. Benzene sulfonyl tetrazole also was prepared m situ from pyrid e, benezene sulfonic acid and triethylamme. Repeating this reaction sequence followed by cleavage from the support and deprotection yield Formula I compounds.
  • Formula I compounds in which R is C x 4 alkyl are prepared by replacing the DMT-protected compounds with C j 4 alkylethers.
  • Formula I compounds in which R is P(0) (0) - substituted acridme also are prepared by the following published procedures.
  • These published procedures include synthesis of a nucleoside phosphoramidite-bearmg ac ⁇ dme derivative which then is reacted with 2, 2'- dithiodiethanol attached to a support. The elongation chain then is carried out on an automatic solid-phase DNA synthesized as described above.
  • nucleoside phosphoramidite-bearmg acridme derivatives by reacting substituted 9- (3-hydroxypropyl) amino acridmes with N- ethyldiisopropylamme followed by N,N- diisopropylmethylphosphonamidic chloride.
  • Formula I compounds in which R is P(0) (0) -substituted acridme are prepared by an extra round of synthesis using the ac ⁇ dinyl phosphoramidites in acetonitrile.
  • the compounds of Formula I can be incorporated into convenient pharmaceutical dosage forms such as capsules, tablets, or mjectable preparations.
  • Solid or liquid pharmaceutical carriers can be employed.
  • Solid carriers mclude starch, lacrose, calcium sulfate dehydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid.
  • Liquid carriers mclude syrup, peanut oil, olive oil, saline and water.
  • Liposomal, viral vector, and prote conjugate preparations can also be used as carriers.
  • the carrier or diluent may include any prolonged release material, such as glyceryl monosteararate of glyceryl disteararate, alone or with a wax.
  • the amount of solid carrier varies widely but, preferably, will be from about 25 mg to about 1 g per dosage unit.
  • a liquid carrier When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile mjectable liquid such as an ampoule, or an aqueous or nonaqueous liquid suspension.
  • a liquid carrier When a liquid carrier is used it will most often be a saline solution or phosphate buffered saline solution.
  • the pharmaceutical preparations are made following conventional techniques of a pharmaceutical chemist mvolv g mixing, granulating and compressing, when necessary, for tablet forms, or mixing, filling, and dissolving the ingredients, as appropriate, to give the desired oral or parenteral products.
  • Doses of the present Formula I compounds (a pharmaceutical dosage unit as described above) will be an efficacious, nontoxic quantity selected from the range of 1 ng/kg to 500 mg/kg of active compound, preferably less than 1 mg/kg.
  • the selected dose is administered to a human patient in need of inhibition of AGT expression from 1-6 or more times daily, orally, rectally, by injection, or continuously by infusion.
  • Oral formulations would generally require somewhat larger dosages to overcome the effects of gastric decomposition.
  • Intravenous or mtraarterial administration would generally require minimum doses since the drug is placed directly into the systemic circulation. Therefore, the dose will depend on the actual route of administration.
  • peripheral administration we mean by any other route of delivery apart from oral or central (into the brain) .
  • Peripherally administering the oligonucleotide compounds of the present invention via an artery (carotid) , vein (tail vein in rats or arm vein m humans) , or mtrape ⁇ toneally (m mammals) allows delivery to the liver without nav g to surgically open the abdominal cavity for injection mto the hepatic vein or artery as in Tomita, _t al . . , supra.
  • This invention relates to an oligonucleotide compound which binds to a region of angiotensinogen mRNA preventing the production of angiotensinogen protem which is involved m the development and maintenance of hypertensive blood pressure.
  • encapsulating the antisense molecules -in pharmaceutical carriers, such as liposomes it is possible to obtain compositions which can be used in a method to target the delivery of the antisense molecules directly to the liver, the major site of angiotensinogen production in the body. Liver targeting can also be accomplished by packaging the antisense molecules with viral vectors and protein conjugates .
  • the compounds and compositions of the present invention are unique because there is no commercially available agent which specifically inhibits the action of angiotensinogen. Additionally, due to the chemical nature of antisense molecules the action is highly specific, allowing the administration of small doses, with potentially fewer side effects than conventional antihypertensive agents.
  • the liposome encapsulated oligonucleotide compounds of the present invention were demonstrated using an antisense oligodeoxynucleotide for inhibiting the angiotensinogen mRNA of spontaneously hypertensive rats.
  • the sequence for rat mRNA is disclosed in Fig. 3 of Ohkubo, e al . , 80 PNAS USA 2196-2200 (1983) .
  • One version of the invention is an 18 base oligomer, synthesized to complement the -5 to +13 base region of rat angiotensinogen mRNA, which encompasses the AUG translation initiation codon, the oligomer being composed of the following base sequence: 5' -CCGTGGGAGT CATCACGG-3' (SEQ ID NO:2) .
  • a phosphorothioated backbone modification can be included on every base to confer nuclease resistance.
  • compositions of the present invention comprise novel DNA sequences capable of binding to angiotensinogen mRNA, preferably encapsulated in a pharmaceutical delivery system, which results in the ASODN being delivered directly to the liver, the major site of angiotensinogen production. No viral antigen modification of the antisense oligonucleotide is necessary.
  • Liposomes (80% phosphatidylcholme, 20% cholesterol) are prepared using a rotary evaporator apparatus for drying and rehydration of the lipid film.
  • the liposomes are subjected to multiple freeze thaw cycles which enhances the entrapment of the antisense molecules and are then passed through an extruder (0.1 ⁇ m filter) m order to reduce their size. Size can then be determined by dynamic light scattering.
  • extruder 0.1 ⁇ m filter
  • liposomal encapsulation facilitates cellular uptake of the antisense molecules resulting in an increased efficiency of delivery. This has allowed us to administer the antisense composition peripherally mto the blood stream and obtain physiological responses with doses which produced no physiological response when previously tested (see Gyurko, et al . , and Wielbo, et al . , supra) .
  • the antisense molecule Once the antisense molecule enters the cells it binds to the targeted region of angiotensinogen mRNA forming an mRNA/DNA duplex. This duplex formation serves to prevent the assembly of ribosomal sub units and the subsequent reading of the protem message, thereby inhibiting angiotensinogen production.
  • the phosphorothioated form of the antisense molecule has enhanced nuclease resistance and additionally stimulates the action of RNase H, an enzyme which cleaves the mRNA portion of the duplex, subsequently freeing the antisense molecule to bind to another target mRNA.
  • hypertensive blood pressure m animal models of hypertension can be decreased with a single, mtra-arterial dose of a composition comprising a liposo e encapsulated antisense DNA fragment targeted to angiotensinogen.
  • a composition comprising a liposo e encapsulated antisense DNA fragment targeted to angiotensinogen.
  • biochemical data shows a subsequent decrease m angiotens II m the periphery at the time of blood pressure decreases.
  • Our previous studies have also shown that a single dose of the antisense molecule into the brain of hypertensive animals will also decrease blood pressures for up to seven days.
  • FIG. 6 shows the effect of a viral vector antisense delivery system on angiotensm II type 1 receptor expression in a neuroma-glioma brain tumor cell line.
  • antisense the entire length of the protem sequence
  • Cells were either treated with transfectam--an agent available to enhance the uptake of the viral vector; an empty, mock plasmid viral vector; paAT, the adeno-associated virus (AAV) vector containing the antisense sequence; and paAT + Ad, the adeno-associated viral vector containing the antisense and a helper virus for adeno-associated virus which enhances the AAV's p40 promoter which drives the expression of the antisense mRNA. Substantially less receptor binding was observed m the paAT + Ad treated cells. This indicates that mRNA expression is attenuated with the paAT + Ad treatment.
  • Another delivery system which can be used in the present invention is a protem conjugate oligonucleotide composition.
  • This technique is based on the construction of DNA-protem complexes that are recognized by the liver specific asialoglycoprote receptor. Binding of polyd- lysme) -asialoorosomucoid (ASoR) protem conjugates with phosphorothioated antisense can facilitate cellular uptake m the liver.
  • the conjugates use receptor mediated endocytotic mechanisms for delivering genes mto cells.
  • a specific receptor ligand such as asialoorosomucoid or transferrm
  • Fig. 1 demonstrates the specificity of the target antisense molecule (SEQ ID NO:2) to the chosen target, the rat protein angiotensinogen.
  • SEQ ID NO:2 target antisense molecule
  • cDNA complimentary DNA
  • the m vi tro reactions were carried out in the presence and absence of antisense molecules and scrambled or sense control oligonucleotide molecules. These m vi tro reactions can also be employed to evaluate the ability of the oligonucleotides of the present invention to inhibit expression of human angiotensinogen.
  • Figs. 2a and 2b show two confocal micrographs of rat liver tissue, one hour after injection of 50 ⁇ g of (a) unencapsulated, fluorescently labeled antisense (SEQ ID NO:2) , or (b) liposome encapsulated fluorescently labeled antisense (SEQ ID NO:2) directly mto the carotid artery.
  • Micrograph (a) shows little or no distribution of fluorescent signal within the liver tissue.
  • Micrograph (b) shows an intense and evenly distributed fluorescent signal throughout the tissue, indicating that liposome encapsulation facilitates the delivery of the antisense to the target organ.
  • peripherally administered compositions comprising liposome encapsulated mRNA, antisense oligonucleotide targeted to liver angiotensinogen mRNA, groups of male, spontaneously hypertensive rats (250- 275 g) were catheterized via the carotid artery and allowed 24 hours to recover from surgery.
  • Baseline mean arterial pressure (MAP) was then measured via direct blood pressure transducer, attached to the indwelling arterial catheter.
  • This example demonstrates the effect of liposome encapsulated ASODN (SEQ ID NO:2) on mean arterial pressure (MAP) in spontaneously hypertensive rats.
  • Baseline MAP was established in groups of rats. Then 50 ⁇ g of liposome encapsulated (25 mg lipid) ASODN (AS/L) ; liposome encapsulated ScrODN (Scr/L) ; empty liposomes (25 mg) (Lipo) ; or 50 ⁇ g unencapsulated ASODN was administered mtra-arterially.
  • Fig. 3 shows the blood pressure changes observed 24 hours after each treatmen .
  • Fig. 4 shows the effect of liposome encapsulated antisense (AS/L) treatment on plasma angiotensm II levels 24 hours after administration.
  • This example demonstrates the effect of liposome encapsulated ASODN (SEQ ID NO:2) on angiotensin II.
  • Animals were sacrificed 24 hours after treatment. Plasma angiotensin II levels were measured by radioimmunoassay. As shown in Fig.
  • This data suggest that the antisense effects are mediated via the proposed mechanism of action, attenuating angiotensinogen production with a subsequent decrease in plasma angiotensin II.
  • This example demonstrates the effect of liposome encapsulated ASODN (SEQ ID NO:2) on plasma AGT. Animals were sacrificed 24 hours after treatment and plasma AGT levels were measured by radioimmunoassay. As shown in
  • Liposomes are drug vesicles in which an aqueous phase is enclosed in a membrane of phospholipid molecules, which form spontaneously when the lipids are dispersed in an aqueous medium. These vesicles range in size from nanometers to microns, and can be constructed to entrap quantities of materials in both the aqueous compartment and within the membrane. Advances in targeted drug delivery now enable liposomal encapsulation of drug molecules to improve protection, sustained release and more efficient cellular uptake. Phosphatidylcholme liposomes have been shown to be efficient carriers of oligonucleotides, increasing efficiency of cellular uptake and also increasing the stability of the oligonucleotides in culture medium.
  • Cationic liposomes have been shown to be especially efficient in cellular uptake, due to complexmg of the negatively charged oligonucleotides on the liposome surface via electrostatic interactions (Ahktar, S. and
  • Antisense Synthesis Using the AGT sequence established by Ohkubo
  • Cationic liposomes composed of dimethyldiactadecylamonium bromide (DDAB) and dioleoylphosphatidylethanolamme (DPOE) (2:5, w/w) (Avanti Polar Lipids Inc., Alabaster, Alabama) were dissolved in 30 mL chloroform and the solvent evaporated by heating at 55°C under partial vacuum. Liposomes were prepared by resuspendmg the lipids m 1 mL sterile deionized water and somcation on ice until the solution was almost clear. ASODN or ScrODN were then added to give a concentration of 1 uM.
  • DDAB dimethyldiactadecylamonium bromide
  • DPOE dioleoylphosphatidylethanolamme
  • the -/+ charge ratios of ODS/cationic lipids was 0.18, giving a net positive charge to the ODN-liposome conjugates to allow fusion between the cell membranes and ODN-liposome conjugates.
  • five different oligonucleotide concentrations were prepared with different concentrations of DDAB to make ODN/cationic lipid complexes with the same-/+ charge ratios.
  • H-4-II E Hepatoma, Reuber H35, rat cells were purchased from ATCC (Rockville, Maryland) . Cells were grown m monolayer culture in 12 mL of Eagles Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum, 10% calf serum and mcubated m 95% air - 5% C0 2 at 37°C. Cultures were fed everyday and passaged when confluent at a ratio of 1:6 usmg 1.0 mL of 0.25% tryps -EDTA. Cells were grown on 10 cm pet ⁇ dishes until experimentation. Cultures were then washed with EMEM to remove any serum supplemented media and then treated with varying concentrations of oligonucleotides or liposome complexed oligonucleotides and control treatments.
  • EMEM Eagles Minimum Essential Medium
  • AGT sample content was measured from a standard curve of pure rat AGT diluted in the same cell culture medium as the medium as the sample.
  • the assay sensitivity was 0.3 ng/tube, and an ter-assay and intra-assay variability of 14% and 9% respectively.
  • H4 Hepatoma cell cultures were grown to confluence then treated with cationic liposome conjugated ASODN and ScrODN (1 ⁇ M) or 1 ⁇ M naked ASODN or cationic lipid control solutions. Cultures were mcubated for 24 hours. Media was decanted and after 100 ⁇ l aliquots were collected for AGT assay, cells were recovered from the petri dishes. Combing two treatment plates to ensure sufficient cells, mRNA was extracted and analyzed by Northern blot to determine AGT mRNA levels. Northern blots were quantified by densitometry. Media aliquots were lyophilized and assayed for AGT levels by RIA. Six, 100 mm plates were combined for each sample assay.
  • H4 Hepatoma cell cultures were grown to confluence then treated with cationic liposome complexed ASODN at the following concentrations: 0, 0.01, 0.05, 0.1, 0.5 and 1.0 ⁇ M.
  • Control groups consisted of increasing amounts of cationic lipid in amounts necessary to maintain an ASODN lipid ratio of -/+. ODN/cationic lipid, 0.18.
  • Cultures were cubated for 24 hours. Media was decanted and 100 ⁇ l aliquots were lyophilized and assayed for AGT levels by RIA. Cells were recovered from petri dishes after collection of the media, combining two treatment plates to ensure sufficient cells, mRNA was extracted and analyzed by Northern blot analysis to determine ASODN dose effects on AGT mRNA. Northern blots were quantified by densitomet ⁇ c methods.
  • Fig. 7a shows the result of Northern hybridization for AGT (1.9 Kb) and the control gene cathepsm (2.1 Kb) .
  • Samples 1-5 correspond to the following treatments: 1. Naked antisense (ASODN) ; 2. Empty cationic liposome control (CL) ; 3. Cationic liposome complexed ScrODN control (Scr/CL) ; 4. Cationic liposome complexed ASODN (AS/CL) and 5.
  • Non-treated control CRL
  • Fig. 7b corresponds to the relative intensity of AGT mRNA expression after treatment compared to AGT expression in untreated control cells. Expression is presented as percent control.
  • Cells treated with naked ASODN ASODN
  • Control treatments of uncomplexed liposomes (CL) and liposome complexed ScrODN (Scr/CL) resulted in little change from baseline levels with approximately 90% expression.
  • cells treated with liposome complexed ASODN AS/CL only show 22% expression.
  • Fig. 8 shows the amount of AGT produced by Hepatoma cell culture after control and ASODN treatments. This graphs shows the effect of cationic liposome complexed ASODN on AGT production.
  • Cells were incubated with 1 ⁇ M of ASODN in the presence or absence of cationic liposomes and appropriate controls. In untreated controls (CTRL) the baseline AGT level was 52.0 ⁇ 2.46 ng/mL.
  • Fig. 9a and 9b show a dose dependant attenuation of AGT mRNA using cationic liposomes as delivery mechanisms.
  • Fig. 9a shows the result of Northern hybridization for AGT and the control gene cathepsm D.
  • Samples 1-5 shows representative blots of cells treated with only cationic liposomes. Each oligonucleotide concentration was accompanied by a separate control of uncomplexed liposomes as the concentration of liposome per treatment increased proportionally with each increase in oligonucleotide concentration.
  • Samples 6-10 correspond to cells treated with 0.01, 0.05, 0.1, 0.5 and 1.0 ⁇ M cationic liposome complexed ASODN.
  • Sample 11 shows AGT mRNA expression in untreated control cells, which is used as the baseline expression of AGT for the comparison of attenuation after test ASODN treatments.
  • Fig. 9b graphs the relative expression of the mRNA compared to the control treated samples. The average expression for cells treated with control cationic liposomes was 92%. Cells treated with 0.01 and 0.05 ⁇ M ASODN had approximately 92% expression which was similar to the control cationic liposome treatments.
  • Fig. 10 shows dose dependant decreases in AGT protem after cationic liposomal delivery of ASODN.
  • each oligonucleotide concentration was accompanied by a separate control of uncomplexed liposomes as the concentration of liposome per treatment increased proportionally with each increase in oligonucleotide concentration.
  • AGT protem levels were 47.6+7.4 and 40.29 ⁇ 4.0 ng/mL, respectively, although a decreasing trend is apparent.
  • uptake efficiency is poor with as little as 2% of ASODN entering the cells and as few as 1% of cells being transfected (Ahktar, S. and Juliano, R. L., supra, 1992) .
  • the amount of naked oligonucleotides taken up by viable cells ranges from 1-10% with the rate of transfection being variable between cell types.
  • Liposomal delivery systems have been shown to facilitate and increase the efficiency of cellular uptake of ASODN, by protecting the antisense from degradation and by bringing the antisense molecules into closer proximity to the cells thereby facilitating the uptake process.
  • cationic liposomes have been shown to increase uptake efficiency by 30% (Lappalainen, K. , et, al . , Biochimica et Biophysica Acta 1196:201-208, 1994) . Consequently due to the expected enhanced delivery and cellular transfection of cationic liposomes we expected that AGT protein and mRNA levels would be decreased accordingly.
  • cationic liposomes are efficient delivery systems for an antisense molecule targeted to angiotensinogen mRNA.
  • Our data shows that at 1 ⁇ M doses, liposome complexed ASODN specifically decreases target protein and mRNA more profoundly than naked ASODN alone. These observations can be attributed to the specific inhibition of target mRNA as no significant decreases m target protein were observed with liposome complexed ScrODN or control cationic lipids.
  • the same dose of liposome complexed ASODN also resulted m significant decreases m target mRNA from baseline levels. This suggests that the mode of oligonucleotide action may be through the stimulation of RNase H.
  • This enzyme recognizes the mRNA-DNA double- strand as a substrate and subsequently degrades the mRNA portion of the duplex. Hence the observed decrease m angiotensinogen mRNA although this particular oligonucleotide was designed to inhibit translational processes and ribosome binding at the AUG initiation codon.
  • Cationic liposomes may be utilized to substantially increase the delivery efficiency of oligonucleotides m cell culture to the extent that intracellular ASODN levels elicit both attenuation of target protem and target mRNA.
  • This delivery system offers a simple approach for the determination of basic ASODN properties, enablmg sufficient mtracellular levels of ASODN for activity without the need for viral vector delivery or targeted cellular delivery.
  • baby SH rats which are normotensive until about 8 weeks of age, were injected with a single dose (50 ⁇ g oligo) of liposome encapsulated oligonucleotide (the base sequence of the oligonucleotide being in accordance with SEQ ID NO: 2) via the tail vein, once a week for four weeks. This was done after weaning from age 4 weeks to 8 weeks.
  • the present invention includes oligonucleotide compounds which lack a complement for each nucleotide in a particular segment of the angiotensinogen mRNA, provided such compounds have sufficient binding affinity for the AGT mRNA to inhibit expression thereof.
  • the materials and methods of Examples 6-9, supra are useful m determmmg whether specific oligonucleotides are effective in inhibiting angiotensinogen expression.
  • the above methods would be modified when evaluating specific compounds for the inhibition of human angiotensinogen.
  • human cells such as human embryonic cell lines or human hepotoma cell lines could be used for the cell culture.
  • the present mvention is intended to mclude mixtures of oligonucleotide compounds, each compound being capable of binding to angiotensinogen mRNA. Therefore, the scope of the claims is not limited to the specific examples of the preferred versions herein. Rather, the claims should be looked to m order to judge the full scope of the invention.
  • oligonucleotides disclosed herein are useful for inhibiting the expression of angiotensinogen to thereby control angiotensinogen induced hypertension.
  • MOLECULE TYPE Other nucleic acid
  • MOLECULE TYPE Other nucleic acid (XI) SEQUENCE DESCRIPTION: SEQ ID NO:2: CCGTGGGAGT CATCACGG 18

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Abstract

L'invention concerne des oligonucléotides et des compositions de ces derniers qui peuvent être administrés à l'homme pour inhiber l'expression de l'angiotensinogène. Cela permet ainsi de combattre l'hypertension induite par l'angiotensinogène.
EP97916760A 1996-03-15 1997-03-14 OLIGONUCLEOTIDES CIBLES SUR L'ARNm DE L'ANGIOTENSINOGENE Withdrawn EP0906123A4 (fr)

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WO1999015643A2 (fr) * 1997-09-25 1999-04-01 University Of Florida COMPOSITIONS OLIGONUCLEOTIDIQUES ANTISENSE CIBLEES SUR L'ARNm DE L'ENZYME DE CONVERSION DE L'ANGIOTENSINE ET METHODES D'UTILISATION
US20150297629A1 (en) 2012-07-27 2015-10-22 Isis Pharmaceuticals, Inc. Modulation of renin-angiotensin system (ras) related diseases by angiotensinogen
WO2014170786A1 (fr) 2013-04-17 2014-10-23 Pfizer Inc. Dérivés de n-pipéridin-3-ylbenzamide dans le traitement des maladies cardiovasculaires
BR122020023854B1 (pt) * 2015-10-08 2022-11-29 Ionis Pharmaceuticals, Inc Composto compreendendo oligonucleotídeo modificado e seu uso, uso do oligonucleotídeo modificado, composição e seu uso
JP2023549563A (ja) 2020-11-18 2023-11-27 アイオーニス ファーマシューティカルズ， インコーポレーテッド アンジオテンシノーゲン発現を調節するための化合物及び方法
CN113862268A (zh) * 2021-10-20 2021-12-31 厦门甘宝利生物医药有限公司 Agt抑制剂及其用途
CN116940682A (zh) * 2021-11-16 2023-10-24 上海舶望制药有限公司 抑制血管紧张素原（agt）蛋白表达的组合物和方法

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WO1995030330A1 (fr) * 1994-05-10 1995-11-16 Dzau Victor J Procede pour l'administration in vivo d'agents therapeutiques par des liposomes

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WO1995030330A1 (fr) * 1994-05-10 1995-11-16 Dzau Victor J Procede pour l'administration in vivo d'agents therapeutiques par des liposomes

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COOK, J. ET AL.: "Identification and antisense inhibition of a renin-angiotensin system in transgenic cardiomyocytes" AM. J. PHYSIOL. (1995), 268(4, PT. 2), H1471-H1482, XP002098211 *
PHILLIPS, I. ET AL.: "Antisense inhibition of hypertension: a new strategy for renin-angiotensin candidate genes." KIDNEY INT., vol. 46, 1994, pages 1554-1556, XP002098212 *
PHILLIPS, I.: "Antisense inhibition and adeno-associated viral vector delivery for reducing hypertension" HYPERTENSION, vol. 29, January 1997, pages 177-187, XP002098214 *
See also references of WO9733623A1 *
UHLMANN E ET AL: "ANTISENSE OLIGONUCLEOTIDES: A NEW THERAPEUTIC PRINCIPLE" CHEMICAL REVIEWS, vol. 90, no. 4, 1 June 1990, pages 543-584, XP000141412 *
WIELBO, D. ET AL.: "Antisense inhibition of hypertension in the spontaneously hypertensive rat" HYPERTENSION, vol. 25, 1995, pages 314-319, XP002098210 *
WIELBO, D. ET AL.: "Inhibition of hypertension by peripheral administration of antisense oligodeoxynucleotides" HYPERTENSION, vol. 28, July 1996, pages 147-151, XP002098213 *

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