SINA MOLECULES, METHODS OF PRODUCTION AND USES THEREOF
20230416754 · 2023-12-28
Inventors
Cpc classification
International classification
C12N15/113
CHEMISTRY; METALLURGY
Abstract
The present disclosure relates to method of producing and using short interfering nucleic acids (siNAs) for preventing or reversing progressive optical neuropathy associated with the elevation of intraocular pressure due to excessive noradrenergic activation. In particular, this disclosure relates to the method of producing and using siNAs for or reversing progressive optical neuropathy, wherein the optical neuropathy is selected from the following list: diabetic retinopathy, infections, inflammation, uveitis and glaucoma, such as open-angle glaucoma, close-angle glaucoma, normal pressure glaucoma, congenital glaucoma, secondary glaucoma, pigmentary glaucoma, pseudoexfoliative glaucoma, traumatic glaucoma, neovascular glaucoma, endothelial iridocorneal syndrome and uveitic glaucoma. The present disclosure is also directed to interfering RNA duplexes and vectors encoding such interfering RNA duplexes.
Claims
1. An isolated or synthetic short interfering nucleic acid(siNA)molecule, wherein said molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID No 144, SEQ ID No 174, SEQ. ID No 233, SEQ ID No 235, SEQ ID No 239, SEQ ID No 250, SEQ ID No 265, SEQ ID No 269, SEQ ID No 281, SEQ ID No 311, SEQ ID No 370, SEQ ID No 372, SEQ ID No 376, SEQ ID No 387, SEQ ID No 402, SEQ ID No 406, and sequences comprising at least 18 contiguous nucleotides differing by no more than 4 nucleotides from the nucleotide sequence, and wherein said siNA molecule reduces expression of the dopamine-beta-hydroxylase(DBH)gene in a cell.
2. The siNA molecule according to claim 1, wherein said molecule comprises a nucleic acid sequence differing by no more than 3 nucleotides from the nucleotide sequence.
3. The siNA molecule according to claim 1, wherein said molecule comprises a nucleic acid sequence differing by no more than 2 nucleotides from the nucleotide sequence.
4. (canceled)
5. The siNA molecule according to claim 1, wherein said molecule is between 19 and 25 base pairs in length.
6. (canceled)
7. The siNA molecule according to claim 1, wherein said molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID No 142, SEQ ID No 144, SEQ ID No 145, SEQ ID No 150, SEQ ID No 151, SEQ ID No 153, SEQ ID No 154, SEQ ID No 157, SEQ ID No 158, SEQ ID No 172, SEQ ID No 174, SEQ ID No 175, SEQ ID No 181, SEQ ID No 183, SEQ ID No 184, SEQ ID No 193, SEQ ID No 195, SEQ ID No 196, SEQ ID No 197, SEQ ID No 200, SEQ ID No 201, SEQ ID No 210, SEQ ID No 211, SEQ ID No 231, SEQ ID No 233, SEQ ID No 234, SEQ ID No 235, SEQ ID No 239, SEQ ID No 248, SEQ ID No 250, SEQ ID No 265, SEQ ID No 269, SEQ. ID No 279, SEQ ID No 281, SEQ ID No 282, SEQ ID No 287, SEQ ID No 288, SEQ ID No 290, SEQ ID No 291, SEQ. ID No 294, SEQ ID No 295, SEQ ID No 309, SEQ ID No 311, SEQ ID No 312, SEQ ID No 318, SEQ ID No 320, SEQ ID No 321, SEQ ID No 330, SEQ ID No 332, SEQ ID No 333, SEQ ID No 334, SEQ ID No 337, SEQ ID No 338, SEQ No 347, SEQ ID No 348, SEQ ID No 368, SEQ ID No 370, SEQ ID No 371, SEQ ID No 372, SEQ ID No 376, SEQ ID No 385, SEQ ID No 387, SEQ ID No 402 and SEQ ID No 406.
8. The siNA molecule according to claim 1, wherein siNA is selected from dsRNA, siRNA or shRNA.
9. (canceled)
10. The siNA molecule according to claim 1, wherein siNA comprises 5 and/or 3 overhangs.
11. The siNA molecule according to claim 1, wherein siNA comprises at least one chemical modification.
12-13. (canceled)
14. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of DBH-gene in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleic acid sequence selected from the group consisting of SEQ ID No 144, SEQ ID No 174, SEQ ID No 233, SEQ ID No 235, SEQ ID No 239, SEQ ID No 250, SEQ ID No 265, SEQ ID No 269, and sequences comprising at least 18 contiguous nucleotides differing by no more than 4 nucleotides from the nucleotide sequence, and wherein the antisense strand comprises a nucleic acid sequence selected from the group consisting of SEQ ID No 281, SEQ. ID No 311, SEQ ID No 370, SEQ ID No 372, SEQ ID No 376, SEQ ID No 387, SEQ ID No 402, SEQ ID No 406, and sequences comprising at least 18 contiguous nucleotides differing by no metre than 4 nucleotides from the nucleotide sequence.
15. The dsRNA agent according to claim 14, wherein said sense strand or antisense strand comprises a nucleic acid sequence differing by no more than 3 nucleotides from the nucleotide sequence.
16. The dsRNA agent according to claim 14, wherein said sense strand or antisense strand comprises a nucleic acid sequence differing by no more than 2 nucleotides from the nucleotide sequence.
17. (canceled)
18. The dsRNA agent according to claim 14, wherein the sense strand is selected from the group consisting of SEQ ID No 142, SEQ ID No 144, SEQ ID No 145, SEQ ID No 150, SEQ ID No 151, SEQ ID No 153, SEQ ID No 154, SEQ ID No 157, SEQ ID No 158, SEQ ID No 172, SEQ ID No 174, SEQ ID No 175, SEQ ID No 181, SEQ ID No 183, SEQ ID No 184, SEQ ID No 193, SEQ ID No 195, SEQ ID No 196, SEQ ID No 197, SEQ ID No 200, SEQ ID No 201, SEQ ID No 210, SEQ ID No 211, SEQ ID No 231, SEQ ID No 233, SEQ ID No 234, SEQ ID No 235, SEQ ID No 239, SEQ No 248, SEQ ID No 250, SEQ ID No 265 and SEQ ID No 269, and wherein the anti-sense strand is selected from the group consisting of SEQ ID No 279, SEQ ID No 281, SEQ ID No 282, SEQ ID No 287, SEQ ID No 288, SEQ ID No 290, SEQ ID No 291, SEQ ID No 294, SEQ ID No 295, SEQ ID No 309, SEQ ID No 311, SEQ ID No 312, SEQ ID No 318, SEQ ID No 320, SEQ ID No 321, SEQ ID No 330, SEQ ID No 332, SEQ ID No 333, SEQ ID No 334, SEQ ID No 337, SEQ ID No 338, SEQ ID No 347, SEQ ID No 348, SEQ ID No 368, SEQ ID No 370, SEQ ID No 371, SEQ ID No 372, SEQ ID No 376, SEQ ID No 385, SEQ ID No 387, SEQ ID No 402 and SEQ ID No 406.
19. A vector comprising a molecule described in claim 1.
20. A liposome, microsphere, nanoparticle or capsule comprising a molecule described in claim 1.
21. A pharmaceutical composition comprising at least one siNA molecule according to claim 1 and a pharmaceutically acceptable carrier.
22. The composition according to claim 21, comprising a second active ingredient for the treatment of glaucoma.
23. The composition according to claim 22, wherein said second active ingredient is selected from the group consisting of: an alpha adrenoceptor agonist, a beta adrenoceptor blocker, a carbonic anhydrase inhibitor, a muscarinic agonist, a prostaglandin analogue, a rho kinase inhibitor, and mixtures thereof.
24. (canceled)
25. A method for preventing or reversing progressive optical neuropathy in a subject, the method comprising administrating the siNA molecule siRNA according to claim 1 to the subject.
26. The method according to claim 25, wherein the progressive optical neuropathy is selected from the group consisting of diabetic retinopathy, infections, inflammation, uveitis and glaucoma.
27. The method according to claim 25, wherein the progressive optical neuropathy is open-angle glaucoma, close-angle glaucoma, normal pressure glaucoma, congenital glaucoma, secondary glaucoma, pigmentary glaucoma, pseudoexfoliative glaucoma, traumatic glaucoma, neovascular glaucoma, endothelial iridocorneal syndrome, or uveitic glaucoma.
Description
BRIEF OF THE DRAWINGS
[0101] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.
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DETAILED DESCRIPTION
[0108] The present disclosure relates to method of producing and using short interfering nucleic acids (siNAs) for preventing or reversing progressive optical neuropathy associated with the elevation of intraocular pressure due to excessive noradrenergic activation.
[0109] In an embodiment, the present disclosure relates to a method of producing and using siNAs for treating, preventing or reversing progressive optical neuropathy, wherein the optical neuropathy is selected from the following list: diabetic retinopathy, infections, inflammation, uveitis and glaucoma, such as open-angle glaucoma, close-angle glaucoma, normal pressure glaucoma, congenital glaucoma, secondary glaucoma, pigmentary glaucoma, pseudoexfoliative glaucoma, traumatic glaucoma, neovascular glaucoma, endothelial iridocorneal syndrome and uveitic glaucoma.
[0110] In an embodiment, the siNA or vector encoding the siNA, or the medicament comprising the siNA, or vector encoding the siNA, is administered to an individual by topical eye application, subconjunctival injection, intravitreal injection, retrobulbar injection, intracameral injection, subtenon injection or deposition, intravenous injection, intravenous infusion.
[0111] In an embodiment, the present disclosure relates to an in vitro method of inhibiting the expression of dopamine-beta-hydroxylase gene in a cell comprising contacting the cell with siNA that inhibits dopamine-beta-hydroxylase gene expression.
[0112] In an embodiment, said siRNA comprises a sense dopamine-beta-hydroxylase nucleic acid and an anti-sense dopamine-beta-hydroxylase nucleic acid, wherein the sense dopamine-beta-hydroxylase nucleic acid is substantially identical to a target sequence contained within dopamine-beta-hydroxylase mRNA and the anti-sense dopamine-beta-hydroxylase nucleic acid is complementary to the sense dopamine-beta-hydroxylase nucleic acid.
[0113] In an embodiment, the present disclosure also relates to an in vitro method of inhibiting the expression of the dopamine-beta-hydroxylase gene in a cell comprising contacting the cell with a vector encoding a siRNA that inhibits dopamine-beta-hydroxylase gene expression, said siRNA comprises a sense dopamine-beta-hydroxylase nucleic acid and an anti-sense dopamine-beta-hydroxylase nucleic acid, wherein the sense dopamine-beta-hydroxylase nucleic acid is substantially identical to a target sequence contained within dopamine-beta-hydroxylase mRNA and the anti-sense dopamine-beta-hydroxylase nucleic acid is complementary to the sense dopamine-beta-hydroxylase nucleic acid.
[0114] In an embodiment, the expression of the gene is inhibited by introduction of a double stranded ribonucleic acid (dsRNA) molecule into the cell in an amount sufficient to inhibit expression of the dopamine-beta-hydroxylase gene.
[0115] In an embodiment, the siRNAs used in the disclosure cause RNAi-mediated degradation of dopamine-beta-hydroxylase mRNA such that the protein product of the dopamine-beta-hydroxylase gene is not produced or is produced in reduced amounts.
[0116] In an embodiment, the siRNAs of the present disclosure can be used to alter gene expression in a cell in which expression of dopamine-beta-hydroxylase is initiated, e.g., as a result of excessive noradrenergic activation. Binding of the siRNA to a dopamine-beta-hydroxylase mRNA transcript in a cell results in a reduction in dopamine-beta-hydroxylase protein production by the cell.
[0117] The term siRNA is used to mean a double stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into the cell are used, including those in which DNA is a template from which RNA is transcribed. The siRNA that inhibits dopamine-beta-hydroxylase gene expression includes a sense dopamine-beta-hydroxylase nucleic acid sequence and an antisense dopamine-beta-hydroxylase nucleic acid sequence. The siRNA may be constructed such that a single transcript has both the sense and complementary antisense sequences from the target gene, e.g., in the form of a hairpin.
[0118] In an embodiment, the siRNA preferably comprises short double-stranded RNA that is targeted to the target mRNA, i.e., dopamine-beta-hydroxylase protein from dopamine-beta-hydroxylase mRNA. The siRNA comprises a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions (hereinafter base-paired). The sense strand comprises a nucleic acid sequence which is substantially identical to a target sequence contained within the dopamine-beta-hydroxylase mRNA.
[0119] The terms sense/antisense sequences and sense/antisense strands are used interchangeable herein to refer to the parts of the siRNA of the present disclosure that are substantially identical (sense) to the target dopamine-beta-hydroxylase mRNA sequence or substantially complementary (antisense) to the target dopamine-beta-hydroxylase mRNA sequence.
[0120] As used herein, a nucleic acid sequence substantially identical to a target sequence contained within the target mRNA is a nucleic acid sequence which is identical to the target sequence, or which differs from the target sequence by one or more nucleotides. Preferably, the substantially identical sequence is identical to the target sequence or differs from the target sequence by one, two or three nucleotides, more preferably by one or two nucleotides and most preferably by only 1 nucleotide. Sense strands which comprise nucleic acid sequences substantially identical to a target sequence are characterized in that siRNA comprising such a sense strand induces RNAi-mediated degradation of mRNA containing the target sequence. For example, an siRNA of the disclosure can comprise a sense strand comprising a nucleic acid sequence which differs from a target sequence by one, two, three or more nucleotides, as long as RNAi-mediated degradation of the target mRNA is induced by the siRNA.
[0121] The sense and antisense strands of the siRNA can comprise two complementary, single-stranded RNA molecules or can comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded hairpin area. That is, the sense region and antisense region can be covalently connected via a linker molecule. The linker molecule can be a polynucleotide or non-nucleotide linker. The siRNA can also contain alterations, substitutions or modifications of one or more ribonucleotide bases. For example, the present siRNA can be altered, substituted or modified to contain one or more, preferably 0, 1, 2 or 3, deoxyribonucleotide bases. Preferably, the siRNA does not contain any deoxyribonucleotide bases.
[0122] In an embodiment, the siRNA can comprise partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA; modifications that make the siRNA resistant to nuclease digestion (e.g., the use of 2-substituted ribonucleotides or modifications to the sugar-phosphate backbone); or the substitution of one or more, preferably 0, 1, 2 or 3, nucleotides in the siRNA with deoxyribonucleotides.
[0123] Degradation can be delayed or avoided by a wide variety of chemical modifications that include alterations in the nucleobases, sugars and the phosphate ester backbone of the siRNAs. All of these chemically modified siRNAs are still able to induce siRNA-mediated gene silencing provided that the modifications were absent in specific regions of the siRNA and included to a limited extent. In general, backbone modifications cause a small loss in binding affinity, but offer nuclease resistance. Phosphorothioate (PS)- or boranophosphate (BS)-modified siRNAs have substantial nuclease resistance. Silencing by siRNA duplexes is also compatible with some types of 2-sugar modifications: 2-H,2-O-methyl, 2-O-methoxyethyl, 2-fluoro (2-F), locked nucleic acid (LNA) and ethylene-bridge nucleic acid (ENA). Suitable chemical modifications are well known to those skilled in the art.
[0124] In an embodiment, the siRNA used in the present disclosure is a double-stranded molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a ribonucleotide sequence corresponding to dopamine-beta-hydroxylase protein target sequence, and wherein the antisense strand comprises a ribonucleotide sequence which is complementary to said sense strand, wherein said sense strand and said antisense strand hybridize to each other to form said double-stranded molecule, and wherein said double-stranded molecule, when introduced into a cell expressing the dopamine-beta-hydroxylase gene, inhibits expression of the said gene. As indicated further below, said dopamine-beta-hydroxylase target sequence preferably comprises at least about contiguous, more preferably 19 to 25, and most preferably about 19 to 21 contiguous nucleotides selected from the group consisting of from SEQ ID No 5, SEQ ID No 7, SEQ ID No 8, SEQ ID No 13, SEQ ID No 14, SEQ ID No 16, SEQ ID No 17, SEQ ID No 20, SEQ ID No 21, SEQ ID No 35 SEQ ID No 37, SEQ ID No 38, SEQ ID No 44, SEQ ID No 46, SEQ ID No 47, SEQ ID No 56, SEQ ID No 58, SEQ ID No 59, SEQ ID No 60, SEQ ID No 63, SEQ ID No 64, SEQ ID No 73, SEQ ID No 74, SEQ ID No 94, SEQ ID No 96, S SEQ ID No 97, EQ ID No 98, SEQ ID No 102, SEQ ID No 111, SEQ ID No 113, SEQ ID No 128 and SEQ ID No 132.
[0125] In an embodiment, the siRNA used in the present disclosure can be obtained using a number of techniques known to those of skill in the art. For example, the siRNA can be chemically synthesized or recombinantly produced using methods known in the art, such as the Drosophila in vitro system described in U.S. published application 2002/0086356, the entire disclosure of which is herein incorporated by reference. The siRNA may be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
[0126] In an embodiment, the siRNA can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions. Commercial suppliers of synthetic RNA molecules or synthesis reagents include Biospring (Frankfurt, Germany), ChemGenes (Ashland, Mass., USA), Dharmacon Research (Lafayette, Colo., USA), Glen Research (Sterling, Va., USA), Proligo (Hamburg, Germany), Sigma-Aldrich (St. Louis, MO USA) and Thermo Fisher Scientific (Waltham, MA USA).
[0127] In an embodiment, the siRNA can also be expressed from recombinant circular or linear DNA vectors using any suitable promoter. Suitable promoters for expressing siRNA from a vector include, for example, the U6 or H1 RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art. The vector can also comprise inducible or regulable promoters for expression of the siRNA in a particular tissue or in a particular intracellular environment.
[0128] In an embodiment, the siRNA expressed from a vector can either be isolated from cultured cell expression systems by standard techniques, or can be expressed intracellularly. The vector can be used to deliver the siRNA to cells in vivo, e.g., by intracellularly expressing the siRNA in vivo. siRNA can be expressed from a vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions. Selection of vectors suitable for expressing the siRNA, methods for inserting nucleic acid sequences for expressing the siRNA into the vector, and methods of delivering the vector to the cells of interest are well known to those skilled in the art.
[0129] In an embodiment, the siRNA can also be expressed from a vector intracellularly in vivo.
[0130] As used herein, the term vector means any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid. Any vector capable of accepting the coding sequences for the siRNA molecule(s) to be expressed can be used, including plasmids, cosmids, naked DNA, optionally condensed with a condensing agent, and viral vectors. Suitable viral vectors include vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. When the vector is a lentiviral vector it is preferably pseudotyped with surface proteins from vesicular stomatitis virus, rabies virus, Ebola virus or Mokola virus.
[0131] In an embodiment, vectors are produced, for example, by cloning the dopamine-beta-hydroxylase target sequence into an expression vector so that operatively-linked regulatory sequences flank the dopamine-beta-hydroxylase sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands (Lee et al., 2002). An RNA molecule that is antisense to dopamine-beta-hydroxylase mRNA is transcribed by a first promoter (e.g., a promoter sequence 3 of the cloned DNA) and an RNA molecule that is the sense strand for the dopamine-beta-hydroxylase mRNA is transcribed by a second promoter (e. g., a promoter sequence 5 of the cloned DNA). The sense and antisense strands hybridize in vivo to generate siRNA constructs for silencing of the dopamine-beta-hydroxylase gene. Alternatively, two vectors are utilized to create the sense and anti-sense strands of a siRNA construct. Cloned dopamine-beta-hydroxylase can encode a construct having secondary structure, e. g., hairpins, wherein a single transcript has both the sense and complementary antisense sequences from the target gene. Such a transcript encoding a construct having secondary structure, will preferably comprises a single-stranded ribonucleotide sequence (loop sequence) linking said sense strand and said antisense strand.
[0132] In an embodiment, the siRNA is preferably isolated.
[0133] As used herein, isolated means synthetic, or altered or removed from the natural state through human intervention. For example, a siRNA naturally present in a living animal is not isolated, but a synthetic siRNA, or a siRNA partially or completely separated from the coexisting materials of its natural state is isolated. An isolated siRNA can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the siRNA has been delivered. By way of example, siRNA which are produced inside a cell by natural processes, but which are produced from an isolated precursor molecule, are themselves isolated molecules. Thus, an isolated dsRNA can be introduced into a target cell, where it is processed by the Dicer protein (or its equivalent) into isolated siRNA.
[0134] As used herein, inhibit means that the activity of the dopamine-beta-hydroxylase gene expression product or level of the dopamine-beta-hydroxylase gene expression product is reduced below that observed in the absence of the siRNA molecule of the disclosure. The inhibition with a siRNA molecule preferably is significantly below that level observed in the presence of an inactive or attenuated molecule that is unable to mediate an RNAi response. Inhibition of gene expression with the siRNA molecule is preferably significantly greater in the presence of the siRNA molecule than in its absence. Preferably, the siRNA inhibits the level of dopamine-beta-hydroxylase gene expression by at least 10%, more preferably at least 50% and most preferably at least 75%.
[0135] Preferably the siRNA molecule inhibits dopamine-beta-hydroxylase gene expression so that the protein product of the dopamine-beta-hydroxylase gene is not produced or is produced in reduced amounts. By inhibiting dopamine-beta-hydroxylase expression for is meant that the treated cell produces at a lower rate or has decreased the dopamine-beta-hydroxylase protein that allows the prevention or reversion of progressive optical neuropathy associated with the elevation of intraocular pressure due to excessive noradrenergic activation. The dopamine-beta-hydroxylase is measured by mRNA or protein assays known in the art.
[0136] As used herein, an isolated nucleic acid is a nucleic acid removed from its original environment (e. g., the natural environment if naturally occurring) and thus, synthetically altered from its natural state. In the present disclosure, isolated nucleic acid includes DNA, RNA, and derivatives thereof. When the isolated nucleic acid is RNA or derivatives thereof, base T should be replaced with U in the nucleotide sequences.
[0137] As used herein, the term complementary refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a polynucleotide, and the term binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof.
[0138] As used herein, the phrase highly conserved sequence region means a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.
[0139] As used herein, the term complementarity or complementary means that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types of interaction. In reference to the present disclosure, the binding free energy for a siRNA molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. For example, the degree of complementarity between the sense and antisense strand of the siRNA molecule can be the same or different from the degree of complementarity between the antisense strand of the siRNA and the target RNA sequence.
[0140] A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. Preferably the term complementarity or complementary means that at least 90%, more preferably at least 95% and most preferably 100% of residues in a first nucleic acid sense can form hydrogen binds with a second nucleic acid sequence.
[0141] Complementary nucleic acid sequences hybridize under appropriate conditions to form stable duplexes containing few (one or two) or no mismatches. Furthermore, the sense strand and antisense strand of the siRNA can form a double stranded nucleotide or hairpin loop structure by the hybridization.
[0142] In an embodiment, such duplexes contain no more than 1 mismatch for every 10 matches.
[0143] In an embodiment, the sense and antisense strands of the duplex are fully complementary, i.e., the duplexes contain no mismatches.
[0144] As used herein, the term cell is defined using its usual biological sense. The cell can be present in an organism, e.g., mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be eukaryotic (e.g., a mammalian cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell. Preferably the cell is in eye, eye cornea, eye ciliary body, eye trabecular mesh, eye retina, brain, colon, head and neck, kidney, liver, lung, or lymph.
[0145] As used herein, the term RNA means a molecule comprising at least one ribonucleotide residue. By ribonucleotide is meant a nucleotide with a hydroxyl group at the 2 position of a beta-D-ribo-furanose moiety. The term includes double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant disclosure can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogues of naturally-occurring RNA. Preferably the term RNA consists of ribonucleotide residues only.
[0146] As used herein, the term organism refers to any living entity comprised of at least one cell. A living organism can be as simple as, for example, a single eukaryotic cell or as complex as a mammal, including a human being.
[0147] As used herein, the term subject means an organism, which is a donor or recipient of explanted cells or the cells themselves. Subject also refers to an organism to which the nucleic acid molecules of the disclosure can be administered. The subject is preferably a mammal, e.g., a human, non-human primate, mouse, rat, dog, cat, horse, or cow. Most preferably the subject is a human.
[0148] As used herein, the term biological sample refers to any sample containing polynucleotides. The sample may be a tissue or cell sample, or a body fluid containing polynucleotides (e.g., blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). The sample may be a homogenate, lysate, extract, cell culture or tissue culture prepared from a whole organism or a subset of its cells, tissues or component parts, or a fraction or portion thereof. Lastly, the sample may be a medium, such as a nutrient broth or gel in which an organism, or cells of an organism, have been propagated, wherein the sample contains polynucleotides.
[0149] In an embodiment, the disclosure relates to methods of producing and using short interfering nucleic acids (siNAs) for preventing or reversing progressive optical neuropathy associated with the elevation of intraocular pressure due to excessive noradrenergic activation.
[0150] In an embodiment, this disclosure relates to a method of producing and using siNAs for preventing or reversing progressive optical neuropathy, wherein the optical neuropathy is selected from the following list: diabetic retinopathy, infections, inflammation, uveitis and glaucoma, such as open-angle glaucoma, close-angle glaucoma, normal pressure glaucoma, congenital glaucoma, secondary glaucoma, pigmentary glaucoma, pseudoexfoliative glaucoma, traumatic glaucoma, neovascular glaucoma, endothelial iridocorneal syndrome and uveitic glaucoma. The cell may be further contacted with a transfection-enhancing agent to enhance delivery of the siRNA or siRNA encoding vector to the cell. Depending on the specific method of the present disclosure, the cell may be provided in vitro, in vivo or ex vivo.
[0151] Sequence information regarding the dopamine-beta-hydroxylase protein gene (GenBank accession NM_000787.4) was extracted from the NCBI Entrez nucleotide database. Up to 137 mRNA segments were identified. See for example, U.S. Pat. No. 6,506,559, and Elbashir et al., 2001, herein incorporated by reference in its entirety.
[0152] Selection of siRNA target sites can be performed as follows: [0153] i) Beginning with the ATG start codon of the transcript, scan downstream for AA dinucleotide sequences. Record the occurrence of each AA and the 3 adjacent 19 nucleotides as potential siRNA target sites. Tuschl et al. (Tuschl, Sharp & Bartel, 1998) recommend against designing siRNA to the 5 and 3 untranslated regions (UTRs) and regions near the start codon (within 75 bases) as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex. [0154] ii) Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences. We suggest using BLAST, which can be found on the NCBI server at: www.ncbi.nlm.nih.gov/BLAST/ [0155] iii) Select qualifying target sequences (i.e., sequences having over 45% GC content) for synthesis.
[0156] In an embodiment, the length of the sense nucleic acid is at least 10 nucleotides and may be as long as the naturally-occurring dopamine-beta-hydroxylase transcript.
[0157] In an embodiment, the length of the sense nucleic acid is preferably less than 75 nucleotides in length, preferably 50 nucleotides in length, more preferably 25 nucleotides in length.
[0158] In an embodiment, the length of the sense nucleic acid is at least 19 nucleotides, preferably, 19-25 nucleotides in length.
[0159] Examples of dopamine-beta-hydroxylase target siRNA sense nucleic acids of the present disclosure which inhibit dopamine-beta-hydroxylase expression in mammalian cells include oligonucleotides comprising any one of the following target sequences of the dopamine-beta-hydroxylase gene: SEQ ID No 5, SEQ ID No 7, SEQ ID No 8, SEQ ID No 13, SEQ ID No 14, SEQ ID No 16, SEQ ID No 17, SEQ ID No 20, SEQ ID No 21, SEQ ID No 35 SEQ ID No 37, SEQ ID No 38, SEQ ID No 44, SEQ ID No 46, SEQ ID No 47, SEQ ID No 56, SEQ ID No 58, SEQ ID No 59, SEQ ID No 60, SEQ ID No 63, SEQ ID No 64, SEQ ID No 73, SEQ ID No 74, SEQ ID No 94, SEQ ID No 96, S SEQ ID No 97, EQ ID No 98, SEQ ID No 102, SEQ ID No 111, SEQ ID No 113, SEQ ID No 128 and SEQ ID No 132.
[0160] One hundred and thirty-seven sequences, which set forth the sequence for one strand of the double stranded is RNA, were identified and isolated for dopamine-beta-hydroxylase (Table 1).
TABLE-US-00001 TABLE1 5sensedopamine-beta-hydroxylasetargetprotein. SEQIDNo 5DNAsense SEQIDNo1 GTCCCTGGAGCTCTCATGGAA SEQIDNo2 AATGTCAGCTACACCCAGGAG SEQIDNo3 GCTCCTGGTGCGGAGGCTCAA SEQIDNo4 AAGGCTGGCGTCCTGTTTGGG SEQIDNo5 TGTCCGACCGTGGCGAGCTTGAGAA SEQIDNo6 TCCGACCGTGGCGAGCTTGAGAA SEQIDNo7 CGACCGTGGCGAGCTTGAGAA SEQIDNo8 ACCGTGGCGAGCTTGAGAA SEQIDNo9 AACGCAGATCTCGTGGTGCTC SEQIDNo10 GGACGCCTGGAGTGACCAGAA SEQIDNo11 AAGGGGCAGATCCACCTGGAT SEQIDNo12 CAGGTGCAGAGGACCCCAGAA SEQIDNo13 AAGGCCTGACCCTGCTTTTCA SEQIDNo14 AAGGCCTGACCCTGCTTTT SEQIDNo15 AAGGCCTGACCCTGCTTTTCAAG SEQIDNo16 AAGGCCTGACCCTGCTTTTCAAGAG SEQIDNo17 AGGCCTGACCCTGCTTTTCAA SEQIDNo18 AAGAGGCCCTTTGGCACCTGC SEQIDNo19 CTTTGGCACCTGCGACCCCAA SEQIDNo20 CCCAAGGATTACCTCATTGAA SEQIDNo21 AAGGATTACCTCATTGAAGAC SEQIDNo22 AAGACGGCACTGTCCACTTGG SEQIDNo23 CCGGTCACTGGAGGCCATCAA SEQIDNo24 AACGGCTCGGGCCTGCAGATG SEQIDNo25 GCAGAGGGTGCAGCTCCTGAA SEQIDNo26 GGTGCAGCTCCTGAAGCCCAA SEQIDNo27 CTGAAGCCCAATATCCCCGAA SEQIDNo28 AAGCCCAATATCCCCGAACCG SEQIDNo29 AATATCCCCGAACCGGAGTTG SEQIDNo30 AACCGGAGTTGCCCTCAGACG SEQIDNo31 GCGTGCACCATGGAGGTCCAA SEQIDNo32 CATGGAGGTCCAAGCTCCCAA SEQIDNo33 AAGCTCCCAATATCCAGATCC SEQIDNo34 AATATCCAGATCCCCAGCCAG SEQIDNo35 AGACCACGTACTGGTGCTACATTAA SEQIDNo36 ACCACGTACTGGTGCTACATTAA SEQIDNo37 CACGTACTGGTGCTACATTAA SEQIDNo38 CGTACTGGTGCTACATTAA SEQIDNo39 GCTACATTAAGGAGCTTCCAA SEQIDNo40 CTACATTAAGGAGCTTCCAAA SEQIDNo41 AAGGAGCTTCCAAAGGGCTTC SEQIDNo42 AAAGGGCTTCTCTCGGCACCA SEQIDNo43 AAGGGCTTCTCTCGGCACCAC SEQIDNo44 GCTTCTCTCGGCACCACATTATCAA SEQIDNo45 TTCTCTCGGCACCACATTATCAA SEQIDNo46 CTCTCGGCACCACATTATCAA SEQIDNo47 CTCGGCACCACATTATCAA SEQIDNo48 AAGTACGAGCCCATCGTCACC SEQIDNo49 GTACGAGCCCATCGTCACCAA SEQIDNo50 GCCCATCGTCACCAAGGGCAA SEQIDNo51 AAGGGCAATGAGGCCCTTGTC SEQIDNo52 AATGAGGCCCTTGTCCACCAC SEQIDNo53 GCCCTTGTCCACCACATGGAA SEQIDNo54 AAGTCTTCCAGTGCGCCCCCG SEQIDNo55 CAGCGGGCCCTGCGACTCCAA SEQIDNo56 CGGGCCCTGCGACTCCAAGATGAAA SEQIDNo57 GGCCCTGCGACTCCAAGATGAAA SEQIDNo58 GCCCTGCGACTCCAAGATGAA SEQIDNo59 CCCTGCGACTCCAAGATGAAA SEQIDNo60 CTGCGACTCCAAGATGAAA SEQIDNo61 AAGATGAAACCCGACCGCCTC SEQIDNo62 GATGAAACCCGACCGCCTCAA SEQIDNo63 AAACCCGACCGCCTCAACTAC SEQIDNo64 AACCCGACCGCCTCAACTACT SEQIDNo65 AACTACTGCCGCCACGTGCTG SEQIDNo66 CGCCTGGGCCCTGGGTGCCAA SEQIDNo67 AAGGCATTTTACTACCCAGAG SEQIDNo68 GCATTTTACTACCCAGAGGAA SEQIDNo69 AAGCCGGCCTTGCCTTCGGGG SEQIDNo70 TCCAGATATCTCCGCCTGGAA SEQIDNo71 CCTGGAAGTTCACTACCACAA SEQIDNo72 AAGTTCACTACCACAACCCAC SEQIDNo73 CACAACCCACTGGTGATAGAA SEQIDNo74 AACCCACTGGTGATAGAAGGA SEQIDNo75 CACTGGTGATAGAAGGACGAA SEQIDNo76 ACTGGTGATAGAAGGACGAAA SEQIDNo77 AAGGACGAAACGACTCCTCAG SEQIDNo78 AAACGACTCCTCAGGCATCCG SEQIDNo79 AACGACTCCTCAGGCATCCGC SEQIDNo80 CCGCTTGTACTACACAGCCAA SEQIDNo81 AGCCAAGCTGCGGCGCTTCAA SEQIDNo82 AAGCTGCGGCGCTTCAACGCG SEQIDNo83 AACGCGGGGATCATGGAGCTG SEQIDNo84 CACTGGCTACTGCACGGACAA SEQIDNo85 AAGTGCACCCAGCTGGCACTG SEQIDNo86 ACACACACCTGACTGGGAGAA SEQIDNo87 CACACACCTGACTGGGAGAAA SEQIDNo88 AAAGGTGGTCACAGTGCTGGT SEQIDNo89 AAGGTGGTCACAGTGCTGGTC SEQIDNo90 CCGGGAGTGGGAGATCGTGAA SEQIDNo91 GGAGATCGTGAACCAGGACAA SEQIDNo92 AACCAGGACAATCACTACAGC SEQIDNo93 AATCACTACAGCCCTCACTTC SEQIDNo94 ACTTCCAGGAGATCCGCATGTTGAA SEQIDNo95 TTCCAGGAGATCCGCATGTTGAA SEQIDNo96 CCAGGAGATCCGCATGTTGAA SEQIDNo97 AGGAGATCCGCATGTTGAA SEQIDNo98 GGAGATCCGCATGTTGAAGAA SEQIDNo99 AAGAAGGTCGTGTCGGTCCAT SEQIDNo100 AAGGTCGTGTCGGTCCATCCG SEQIDNo101 CATCACCTCCTGCACGTACAA SEQIDNo102 TCCTGCACGTACAACACAGAA SEQIDNo103 AACACAGAAGACCGGGAGCTG SEQIDNo104 AAGACCGGGAGCTGGCCACAG SEQIDNo105 CCTGGAGGAGATGTGTGTCAA SEQIDNo106 AACTACGTGCACTACTACCCC SEQIDNo107 GACGCAGCTGGAGCTCTGCAA SEQIDNo108 AAGAGCGCTGTGGACGCCGGC SEQIDNo109 GGACGCCGGCTTCCTGCAGAA SEQIDNo110 GAAGTACTTCCACCTCATCAA SEQIDNo111 AAGTACTTCCACCTCATCAAC SEQIDNo112 CCACCTCATCAACAGGTTCAA SEQIDNo113 CCTCATCAACAGGTTCAACAA SEQIDNo114 AACAGGTTCAACAACGAGGAT SEQIDNo115 AACAACGAGGATGTCTGCACC SEQIDNo116 AACGAGGATGTCTGCACCTGC SEQIDNo117 GTTCACCTCTGTTCCCTGGAA SEQIDNo118 TGTTCCCTGGAACTCCTTCAA SEQIDNo119 AACTCCTTCAACCGCGACGTA SEQIDNo120 CTTCAACCGCGACGTACTGAA SEQIDNo121 AACCGCGACGTACTGAAGGCC SEQIDNo122 AAGGCCCTGTACAGCTTCGCG SEQIDNo123 GCCCATCTCCATGCACTGCAA SEQIDNo124 CATCTCCATGCACTGCAACAA SEQIDNo125 AACAAGTCCTCAGCCGTCCGC SEQIDNo126 AAGTCCTCAGCCGTCCGCTTC SEQIDNo127 GCCGTCCGCTTCCAGGGTGAA SEQIDNo128 CCGCTTCCAGGGTGAATGGAA SEQIDNo129 AATGGAACCTGCAGCCCCTGC SEQIDNo130 GAACCTGCAGCCCCTGCCCAA SEQIDNo131 AACCTGCAGCCCCTGCCCAAG SEQIDNo132 AAGGTCATCTCCACACTGGAA SEQIDNo133 AAGAGCCCACCCCACAGTGCC SEQIDNo134 GCCCCACCAGCCAGGGCCGAA SEQIDNo135 AAGCCCTGCTGGCCCCACCGT SEQIDNo136 TGTCAGCATTGGTGGGGGCAA SEQIDNo137 GTCAGCATTGGTGGGGGCAAA
[0161] The dopamine-beta-hydroxylase gene specificity was confirmed by searching NCBI BlastN database. The siRNAs were chemically synthesized.
[0162] All of the purified siRNA duplexes were complexed with lipofectamine and added to the cells for up to 12 h in serum-free medium. Thereafter, cells were cultured for 24-96 h in serum-supplemented medium, which was replaced by serum-free medium 24 h before the experiments. A scrambled negative siRNA duplex was used as control.
[0163] The dopamine-beta-hydroxylase-siRNA is directed to a single target dopamine-beta-hydroxylase gene sequence. Alternatively, the siRNA is directed to multiple target dopamine-beta-hydroxylase gene sequences. For example, the composition contains dopamine-beta-hydroxylase-siRNA directed to two, three, four, five or more dopamine-beta-hydroxylase target sequences. By dopamine-beta-hydroxylase target sequence is meant a nucleotide sequence that is identical to a portion of the dopamine-beta-hydroxylase gene. The target sequence can include the 5 untranslated (UT) region, the open reading frame (ORF) or the 3 untranslated region of the dopamine-beta-hydroxylase gene. Alternatively, the siRNA is a nucleic acid sequence complementary to an upstream or downstream modulator of dopamine-beta-hydroxylase gene expression. Examples of upstream and downstream modulators include a transcription factor that binds the dopamine-beta-hydroxylase gene promoter, a kinase or phosphatase that interacts with the dopamine-beta-hydroxylase polypeptide, a dopamine-beta-hydroxylase promoter or enhance.
[0164] In an embodiment, the dopamine-beta-hydroxylase-siRNA hybridize to target mRNA and decrease or inhibit production of the dopamine-beta-hydroxylase polypeptide product encoded by the dopamine-beta-hydroxylase gene by associating with the normally single-stranded mRNA transcript, thereby interfering with translation and thus, expression of the protein.
[0165] Exemplary nucleic acid sequence for the production of dopamine-beta-hydroxylase-siRNA include the sequences of nucleotides SEQ ID No 5, SEQ ID No 7, SEQ ID No 8, SEQ ID No 13, SEQ ID No 14, SEQ ID No 16, SEQ ID No 17, SEQ ID No 20, SEQ ID No 21, SEQ ID No 35 SEQ ID No 37, SEQ ID No 38, SEQ ID No 44, SEQ ID No 46, SEQ ID No 47, SEQ ID No 56, SEQ ID No 58, SEQ ID No 59, SEQ ID No 60, SEQ ID No 63, SEQ ID No 64, SEQ ID No 73, SEQ ID No 74, SEQ ID No 94, SEQ ID No 96, S SEQ ID No 97, EQ ID No 98, SEQ ID No 102, SEQ ID No 111, SEQ ID No 113, SEQ ID No 128 and SEQ ID No 132 as the target sequence. In a further embodiment, in order to enhance the inhibition activity of the siRNA, nucleotide U can be added to 3 end of the antisense strand of the target sequence. Preferably at least 2, more preferably 2 to 10, and most preferably 2 to 5 U's are added. The added U's form single strand at the 3 end of the antisense strand of the siRNA.
[0166] In an embodiment, the dopamine-beta-hydroxylase-siRNA is directly introduced into the cells in a form that is capable of binding to the mRNA transcripts. Alternatively, a vector encoding the dopamine-beta-hydroxylase-siRNA can be introduced into the cells.
[0167] A loop sequence consisting of an arbitrary nucleotide sequence can be located between the sense and antisense sequence in order to form a hairpin loop structure.
[0168] In an embodiment, the present disclosure also relates to siRNA having the general formula 5-[A]-[B]-[A]-3, wherein [A] is a ribonucleotide sequence corresponding to a target sequence of the spike (S) glycoprotein gene.
[0169] In an embodiment, preferably [A] is a sequence selected from the group consisting of nucleotides SEQ ID No 5, SEQ ID No 7, SEQ ID No 8, SEQ ID No 13, SEQ ID No 14, SEQ ID No 16, SEQ ID No 17, SEQ ID No 20, SEQ ID No 21, SEQ ID No 35 SEQ ID No 37, SEQ ID No 38, SEQ ID No 44, SEQ ID No 46, SEQ ID No 47, SEQ ID No 56, SEQ ID No 58, SEQ ID No 59, SEQ ID No 60, SEQ ID No 63, SEQ ID No 64, SEQ ID No 73, SEQ ID No 74, SEQ ID No 94, SEQ ID No 96, S SEQ ID No 97, EQ ID No 98, SEQ ID No 102, SEQ ID No 111, SEQ ID No 113, SEQ ID No 128 and SEQ ID No 132; [B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides; and [A] is a ribonucleotide sequence consisting of the complementary sequence of [A]. The region [A] hybridizes to [A], and then a loop consisting of region [B] is formed. The loop sequence may be preferably 3 to 23 nucleotides in length. Suitable loop sequences are described at http://www.ambion.com/techlib/tb/tb_506.html. Furthermore, loop sequence consisting of 23 nucleotides also provides active siRNA (Jacque et al., 2002).
[0170] In an embodiment, the 5 sense siRNA sequences against dopamine-beta-hydroxylase target sequences were identified. The 5 anti-sense siRNA sequences against dopamine-beta-hydroxylase were then designed and produced. Sense and anti-sense siRNA sequences have a length of 19 to 25 nucleotides. Table 2 shows 5 sense and anti-sense siRNA sequences against dopamine-beta-hydroxylase. siRNA sequences have a length of 19 to 25 nucleotides.
TABLE-US-00002 TABLE2 5senseandanti-sensesiRNAsequencesofdopamine-beta-hydroxylase-19to25 nucleotides. SEQIDNo 5RNAsense SEQIDNo 5RNAantisense SEQIDNo138 GUCCCUGGAGCUCUCAUGGAA SEQIDNo275 UUCCAUGAGAGCUCCAGGGAC SEQIDNo139 AAUGUCAGCUACACCCAGGAG SEQIDNo276 CUCCUGGGUGUAGCUGACAUU SEQIDNo140 GCUCCUGGUGCGGAGGCUCAA SEQIDNo277 UUGAGCCUCCGCACCAGGAGC SEQIDNo141 AAGGCUGGCGUCCUGUUUGGG SEQIDNo278 CCCAAACAGGACGCCAGCCUU SEQIDNo142 UGUCCGACCGUGGCGAGCUUGAGAA SEQIDNo279 UUCUCAAGCUCGCCACGGUCGGACA SEQIDNo143 UCCGACCGUGGCGAGCUUGAGAA SEQIDNo280 UUCUCAAGCUCGCCACGGUCGGA SEQIDNo144 CGACCGUGGCGAGCUUGAGAA SEQIDNo281 UUCUCAAGCUCGCCACGGUCG SEQIDNo145 ACCGUGGCGAGCUUGAGAA SEQIDNo282 UUCUCAAGCUCGCCACGGU SEQIDNo146 AACGCAGAUCUCGUGGUGCUC SEQIDNo283 GAGCACCACGAGAUCUGCGUU SEQIDNo147 GGACGCCUGGAGUGACCAGAA SEQIDNo284 UUCUGGUCACUCCAGGCGUCC SEQIDNo148 AAGGGGCAGAUCCACCUGGAU SEQIDNo285 AUCCAGGUGGAUCUGCCCCUU SEQIDNo149 CAGGUGCAGAGGACCCCAGAA SEQIDNo286 UUCUGGGGUCCUCUGCACCUG SEQIDNo150 AAGGCCUGACCCUGCUUUUCA SEQIDNo287 UGAAAAGCAGGGUCAGGCCUU SEQIDNo151 AAGGCCUGACCCUGCUUUU SEQIDNo288 AAAAGCAGGGUCAGGCCUU SEQIDNo152 AAGGCCUGACCCUGCUUUUCAAG SEQIDNo289 CUUGAAAAGCAGGGUCAGGCCUU SEQIDNo153 AAGGCCUGACCCUGCUUUUCAAGAG SEQIDNo290 CUCUUGAAAAGCAGGGUCAGGCCUU SEQIDNo154 AGGCCUGACCCUGCUUUUCAA SEQIDNo291 UUGAAAAGCAGGGUCAGGCCU SEQIDNo155 AAGAGGCCCUUUGGCACCUGC SEQIDNo292 GCAGGUGCCAAAGGGCCUCUU SEQIDNo156 CUUUGGCACCUGCGACCCCAA SEQIDNo293 UUGGGGUCGCAGGUGCCAAAG SEQIDNo157 CCCAAGGAUUACCUCAUUGAA SEQIDNo294 UUCAAUGAGGUAAUCCUUGGG SEQIDNo158 AAGGAUUACCUCAUUGAAGAC SEQIDNo295 GUCUUCAAUGAGGUAAUCCUU SEQIDNo159 AAGACGGCACUGUCCACUUGG SEQIDNo296 CCAAGUGGACAGUGCCGUCUU SEQIDNo160 CCGGUCACUGGAGGCCAUCAA SEQIDNo297 UUGAUGGCCUCCAGUGACCGG SEQIDNo161 AACGGCUCGGGCCUGCAGAUG SEQIDNo298 CAUCUGCAGGCCCGAGCCGUU SEQIDNo162 GCAGAGGGUGCAGCUCCUGAA SEQIDNo299 UUCAGGAGCUGCACCCUCUGC SEQIDNo163 GGUGCAGCUCCUGAAGCCCAA SEQIDNo300 UUGGGCUUCAGGAGCUGCACC SEQIDNo164 CUGAAGCCCAAUAUCCCCGAA SEQIDNo301 UUCGGGGAUAUUGGGCUUCAG SEQIDNo165 AAGCCCAAUAUCCCCGAACCG SEQIDNo302 CGGUUCGGGGAUAUUGGGCUU SEQIDNo166 AAUAUCCCCGAACCGGAGUUG SEQIDNo303 CAACUCCGGUUCGGGGAUAUU SEQIDNo167 AACCGGAGUUGCCCUCAGACG SEQIDNo304 CGUCUGAGGGCAACUCCGGUU SEQIDNo168 GCGUGCACCAUGGAGGUCCAA SEQIDNo305 UUGGACCUCCAUGGUGCACGC SEQIDNo169 CAUGGAGGUCCAAGCUCCCAA SEQIDNo306 UUGGGAGCUUGGACCUCCAUG SEQIDNo170 AAGCUCCCAAUAUCCAGAUCC SEQIDNo307 GGAUCUGGAUAUUGGGAGCUU SEQIDNo171 AAUAUCCAGAUCCCCAGCCAG SEQIDNo308 CUGGCUGGGGAUCUGGAUAUU SEQIDNo172 AGACCACGUACUGGUGCUACAUUAA SEQIDNo309 UUAAUGUAGCACCAGUACGUGGUCU SEQIDNo173 ACCACGUACUGGUGCUACAUUAA SEQIDNo310 UUAAUGUAGCACCAGUACGUGGU SEQIDNo174 CACGUACUGGUGCUACAUUAA SEQIDNo311 UUAAUGUAGCACCAGUACGUG SEQIDNo175 CGUACUGGUGCUACAUUAA SEQIDNo312 UUAAUGUAGCACCAGUACG SEQIDNo176 GCUACAUUAAGGAGCUUCCAA SEQIDNo313 UUGGAAGCUCCUUAAUGUAGC SEQIDNo177 CUACAUUAAGGAGCUUCCAAA SEQIDNo314 UUUGGAAGCUCCUUAAUGUAG SEQIDNo178 AAGGAGCUUCCAAAGGGCUUC SEQIDNo315 GAAGCCCUUUGGAAGCUCCUU SEQIDNo179 AAAGGGCUUCUCUCGGCACCA SEQIDNo316 UGGUGCCGAGAGAAGCCCUUU SEQIDNo180 AAGGGCUUCUCUCGGCACCAC SEQIDNo317 GUGGUGCCGAGAGAAGCCCUU SEQIDNo181 GCUUCUCUCGGCACCACAUUAUCAA SEQIDNo318 UUGAUAAUGUGGUGCCGAGAGAAGC SEQIDNo182 UUCUCUCGGCACCACAUUAUCAA SEQIDNo319 UUGAUAAUGUGGUGCCGAGAGAA SEQIDNo183 CUCUCGGCACCACAUUAUCAA SEQIDNo320 UUGAUAAUGUGGUGCCGAGAG SEQIDNo184 CUCGGCACCACAUUAUCAA SEQIDNo321 UUGAUAAUGUGGUGCCGAG SEQIDNo185 AAGUACGAGCCCAUCGUCACC SEQIDNo322 GGUGACGAUGGGCUCGUACUU SEQIDNo186 GUACGAGCCCAUCGUCACCAA SEQIDNo323 UUGGUGACGAUGGGCUCGUAC SEQIDNo187 GCCCAUCGUCACCAAGGGCAA SEQIDNo324 UUGCCCUUGGUGACGAUGGGC SEQIDNo188 AAGGGCAAUGAGGCCCUUGUC SEQIDNo325 GACAAGGGCCUCAUUGCCCUU SEQIDNo189 AAUGAGGCCCUUGUCCACCAC SEQIDNo326 GUGGUGGACAAGGGCCUCAUU SEQIDNo190 GCCCUUGUCCACCACAUGGAA SEQIDNo327 UUCCAUGUGGUGGACAAGGGC SEQIDNo191 AAGUCUUCCAGUGCGCCCCCG SEQIDNo328 CGGGGGCGCACUGGAAGACUU SEQIDNo192 CAGCGGGCCCUGCGACUCCAA SEQIDNo329 UUGGAGUCGCAGGGCCCGCUG SEQIDNo193 CGGGCCCUGCGACUCCAAGAUGAAA SEQIDNo330 UUUCAUCUUGGAGUCGCAGGGCCCG SEQIDNo194 GGCCCUGCGACUCCAAGAUGAAA SEQIDNo331 UUUCAUCUUGGAGUCGCAGGGCC SEQIDNo195 GCCCUGCGACUCCAAGAUGAA SEQIDNo332 UUCAUCUUGGAGUCGCAGGGC SEQIDNo196 CCCUGCGACUCCAAGAUGAAA SEQIDNo333 UUUCAUCUUGGAGUCGCAGGG SEQIDNo197 CUGCGACUCCAAGAUGAAA SEQIDNo334 UUUCAUCUUGGAGUCGCAG SEQIDNo198 AAGAUGAAACCCGACCGCCUC SEQIDNo335 GAGGCGGUCGGGUUUCAUCUU SEQIDNo199 GAUGAAACCCGACCGCCUCAA SEQIDNo336 UUGAGGCGGUCGGGUUUCAUC SEQIDNo200 AAACCCGACCGCCUCAACUAC SEQIDNo337 GUAGUUGAGGCGGUCGGGUUU SEQIDNo201 AACCCGACCGCCUCAACUACU SEQIDNo338 AGUAGUUGAGGCGGUCGGGUU SEQIDNo202 AACUACUGCCGCCACGUGCUG SEQIDNo339 CAGCACGUGGCGGCAGUAGUU SEQIDNo203 CGCCUGGGCCCUGGGUGCCAA SEQIDNo340 UUGGCACCCAGGGCCCAGGCG SEQIDNo204 AAGGCAUUUUACUACCCAGAG SEQIDNo341 CUCUGGGUAGUAAAAUGCCUU SEQIDNo205 GCAUUUUACUACCCAGAGGAA SEQIDNo342 UUCCUCUGGGUAGUAAAAUGC SEQIDNo206 AAGCCGGCCUUGCCUUCGGGG SEQIDNo343 CCCCGAAGGCAAGGCCGGCUU SEQIDNo207 UCCAGAUAUCUCCGCCUGGAA SEQIDNo344 UUCCAGGCGGAGAUAUCUGGA SEQIDNo208 CCUGGAAGUUCACUACCACAA SEQIDNo345 UUGUGGUAGUGAACUUCCAGG SEQIDNo209 AAGUUCACUACCACAACCCAC SEQIDNo346 GUGGGUUGUGGUAGUGAACUU SEQIDNo210 CACAACCCACUGGUGAUAGAA SEQIDNo347 UUCUAUCACCAGUGGGUUGUG SEQIDNo211 AACCCACUGGUGAUAGAAGGA SEQIDNo348 UCCUUCUAUCACCAGUGGGUU SEQIDNo212 CACUGGUGAUAGAAGGACGAA SEQIDNo349 UUCGUCCUUCUAUCACCAGUG SEQIDNo213 ACUGGUGAUAGAAGGACGAAA SEQIDNo350 UUUCGUCCUUCUAUCACCAGU SEQIDNo214 AAGGACGAAACGACUCCUCAG SEQIDNo351 CUGAGGAGUCGUUUCGUCCUU SEQIDNo215 AAACGACUCCUCAGGCAUCCG SEQIDNo352 CGGAUGCCUGAGGAGUCGUUU SEQIDNo216 AACGACUCCUCAGGCAUCCGC SEQIDNo353 GCGGAUGCCUGAGGAGUCGUU SEQIDNo217 CCGCUUGUACUACACAGCCAA SEQIDNo354 UUGGCUGUGUAGUACAAGCGG SEQIDNo218 AGCCAAGCUGCGGCGCUUCAA SEQIDNo355 UUGAAGCGCCGCAGCUUGGCU SEQIDNo219 AAGCUGCGGCGCUUCAACGCG SEQIDNo356 CGCGUUGAAGCGCCGCAGCUU SEQIDNo220 AACGCGGGGAUCAUGGAGCUG SEQIDNo357 CAGCUCCAUGAUCCCCGCGUU SEQIDNo221 CACUGGCUACUGCACGGACAA SEQIDNo358 UUGUCCGUGCAGUAGCCAGUG SEQIDNo222 AAGUGCACCCAGCUGGCACUG SEQIDNo359 CAGUGCCAGCUGGGUGCACUU SEQIDNo223 ACACACACCUGACUGGGAGAA SEQIDNo360 UUCUCCCAGUCAGGUGUGUGU SEQIDNo224 CACACACCUGACUGGGAGAAA SEQIDNo361 UUUCUCCCAGUCAGGUGUGUG SEQIDNo225 AAAGGUGGUCACAGUGCUGGU SEQIDNo362 ACCAGCACUGUGACCACCUUU SEQIDNo226 AAGGUGGUCACAGUGCUGGUC SEQIDNo363 GACCAGCACUGUGACCACCUU SEQIDNo227 CCGGGAGUGGGAGAUCGUGAA SEQIDNo364 UUCACGAUCUCCCACUCCCGG SEQIDNo228 GGAGAUCGUGAACCAGGACAA SEQIDNo365 UUGUCCUGGUUCACGAUCUCC SEQIDNo229 AACCAGGACAAUCACUACAGC SEQIDNo366 GCUGUAGUGAUUGUCCUGGUU SEQIDNo230 AAUCACUACAGCCCUCACUUC SEQIDNo367 GAAGUGAGGGCUGUAGUGAUU SEQIDNo231 ACUUCCAGGAGAUCCGCAUGUUGAA SEQIDNo368 UUCAACAUGCGGAUCUCCUGGAAGU SEQIDNo232 UUCCAGGAGAUCCGCAUGUUGAA SEQIDNo369 UUCAACAUGCGGAUCUCCUGGAA SEQIDNo233 CCAGGAGAUCCGCAUGUUGAA SEQIDNo370 UUCAACAUGCGGAUCUCCUGG SEQIDNo234 AGGAGAUCCGCAUGUUGAA SEQIDNo371 UUCAACAUGCGGAUCUCCU SEQIDNo235 GGAGAUCCGCAUGUUGAAGAA SEQIDNo372 UUCUUCAACAUGCGGAUCUCC SEQIDNo236 AAGAAGGUCGUGUCGGUCCAU SEQIDNo373 AUGGACCGACACGACCUUCUU SEQIDNo237 AAGGUCGUGUCGGUCCAUCCG SEQIDNo374 CGGAUGGACCGACACGACCUU SEQIDNo238 CAUCACCUCCUGCACGUACAA SEQIDNo375 UUGUACGUGCAGGAGGUGAUG SEQIDNo239 UCCUGCACGUACAACACAGAA SEQIDNo376 UUCUGUGUUGUACGUGCAGGA SEQIDNo240 AACACAGAAGACCGGGAGCUG SEQIDNo377 CAGCUCCCGGUCUUCUGUGUU SEQIDNo241 AAGACCGGGAGCUGGCCACAG SEQIDNo378 CUGUGGCCAGCUCCCGGUCUU SEQIDNo242 CCUGGAGGAGAUGUGUGUCAA SEQIDNo379 UUGACACACAUCUCCUCCAGG SEQIDNo243 AACUACGUGCACUACUACCCC SEQIDNo380 GGGGUAGUAGUGCACGUAGUU SEQIDNo244 GACGCAGCUGGAGCUCUGCAA SEQIDNo381 UUGCAGAGCUCCAGCUGCGUC SEQIDNo245 AAGAGCGCUGUGGACGCCGGC SEQIDNo382 GCCGGCGUCCACAGCGCUCUU SEQIDNo246 GGACGCCGGCUUCCUGCAGAA SEQIDNo383 UUCUGCAGGAAGCCGGCGUCC SEQIDNo247 GAAGUACUUCCACCUCAUCAA SEQIDNo384 UUGAUGAGGUGGAAGUACUUC SEQIDNo248 AAGUACUUCCACCUCAUCAAC SEQIDNo385 GUUGAUGAGGUGGAAGUACUU SEQIDNo249 CCACCUCAUCAACAGGUUCAA SEQIDNo386 UUGAACCUGUUGAUGAGGUGG SEQIDNo250 CCUCAUCAACAGGUUCAACAA SEQIDNo387 UUGUUGAACCUGUUGAUGAGG SEQIDNo251 AACAGGUUCAACAACGAGGAU SEQIDNo388 AUCCUCGUUGUUGAACCUGUU SEQIDNo252 AACAACGAGGAUGUCUGCACC SEQIDNo389 GGUGCAGACAUCCUCGUUGUU SEQIDNo253 AACGAGGAUGUCUGCACCUGC SEQIDNo390 GCAGGUGCAGACAUCCUCGUU SEQIDNo254 GUUCACCUCUGUUCCCUGGAA SEQIDNo391 UUCCAGGGAACAGAGGUGAAC SEQIDNo255 UGUUCCCUGGAACUCCUUCAA SEQIDNo392 UUGAAGGAGUUCCAGGGAACA SEQIDNo256 AACUCCUUCAACCGCGACGUA SEQIDNo393 UACGUCGCGGUUGAAGGAGUU SEQIDNo257 CUUCAACCGCGACGUACUGAA SEQIDNo394 UUCAGUACGUCGCGGUUGAAG SEQIDNo258 AACCGCGACGUACUGAAGGCC SEQIDNo395 GGCCUUCAGUACGUCGCGGUU SEQIDNo259 AAGGCCCUGUACAGCUUCGCG SEQIDNo396 CGCGAAGCUGUACAGGGCCUU SEQIDNo260 GCCCAUCUCCAUGCACUGCAA SEQIDNo397 UUGCAGUGCAUGGAGAUGGGC SEQIDNo261 CAUCUCCAUGCACUGCAACAA SEQIDNo398 UUGUUGCAGUGCAUGGAGAUG SEQIDNo262 AACAAGUCCUCAGCCGUCCGC SEQIDNo399 GCGGACGGCUGAGGACUUGUU SEQIDNo263 AAGUCCUCAGCCGUCCGCUUC SEQIDNo400 GAAGCGGACGGCUGAGGACUU SEQIDNo264 GCCGUCCGCUUCCAGGGUGAA SEQIDNo401 UUCACCCUGGAAGCGGACGGC SEQIDNo265 CCGCUUCCAGGGUGAAUGGAA SEQIDNo402 UUCCAUUCACCCUGGAAGCGG SEQIDNo266 AAUGGAACCUGCAGCCCCUGC SEQIDNo403 GCAGGGGCUGCAGGUUCCAUU SEQIDNo267 GAACCUGCAGCCCCUGCCCAA SEQIDNo404 UUGGGCAGGGGCUGCAGGUUC SEQIDNo268 AACCUGCAGCCCCUGCCCAAG SEQIDNo405 CUUGGGCAGGGGCUGCAGGUU SEQIDNo269 AAGGUCAUCUCCACACUGGAA SEQIDNo406 UUCCAGUGUGGAGAUGACCUU SEQIDNo270 AAGAGCCCACCCCACAGUGCC SEQIDNo407 GGCACUGUGGGGUGGGCUCUU SEQIDNo271 GCCCCACCAGCCAGGGCCGAA SEQIDNo408 UUCGGCCCUGGCUGGUGGGGC SEQIDNo272 AAGCCCUGCUGGCCCCACCGU SEQIDNo409 ACGGUGGGGCCAGCAGGGCUU SEQIDNo273 UGUCAGCAUUGGUGGGGGCAA SEQIDNo410 UUGCCCCCACCAAUGCUGACA SEQIDNo274 GUCAGCAUUGGUGGGGGCAAA SEQIDNo411 UUUGCCCCCACCAAUGCUGAC
[0171] It was surprisingly found that siRNAs targeted to certain target sequences of the dopamine-beta-hydroxylase gene are particularly effective at inhibiting dopamine-beta-hydroxylase mRNA expression, inhibiting dopamine-beta-hydroxylase expression preventing or reversing progressive optical neuropathy associated with the elevation of intraocular pressure in rats treated by ocular administration of siRNAs targeting certain sequences of the dopamine-beta-hydroxylase gene.
[0172] In an embodiment, the sense strand of the dopamine-beta-hydroxylase siRNA used in the present disclosure comprises or consists of a sequence selected from the group comprising SEQ ID No 142, SEQ ID No 144, SEQ ID No 145, SEQ ID No 150, SEQ ID No 151, SEQ ID No 153, SEQ ID No 154, SEQ ID No 157, SEQ ID No 158, SEQ ID No 172, SEQ ID No 174, SEQ ID No 175, SEQ ID No 181, SEQ ID No 183, SEQ ID No 184, SEQ ID No 193, SEQ ID No 195, SEQ ID No 196, SEQ ID No 197, SEQ ID No 200, SEQ ID No 201, SEQ ID No 210, SEQ ID No 211, SEQ ID No 231, SEQ ID No 233, SEQ ID No 234, SEQ ID No 235, SEQ ID No 239, SEQ ID No 248, SEQ ID No 250, SEQ ID No 265 and SEQ ID No 269.
[0173] In an embodiment, the siRNA also comprises a corresponding antisense strand comprising SEQ ID No 279, SEQ ID No 280, SEQ ID No 281, SEQ ID No 282, SEQ ID No 287, SEQ ID No 288, SEQ ID No 290, SEQ ID No 291, SEQ ID No 294, SEQ ID No 295, SEQ ID No 309, SEQ ID No 311, SEQ ID No 312, SEQ ID No 318, SEQ ID No 320, SEQ ID No 321, SEQ ID No 330, SEQ ID No 332, SEQ ID No 333, SEQ ID No 334, SEQ ID No 337, SEQ ID No 338, SEQ ID No 347, SEQ ID No 348, SEQ ID No 368, SEQ ID No 370, SEQ ID No 371, SEQ ID No 372, SEQ ID No 376, SEQ ID No 385, SEQ ID No 387, SEQ ID No 402 and SEQ ID No 406.
[0174] In an embodiment, the use of such an siRNA is particularly effective in inhibiting dopamine-beta-hydroxylase mRNA expression, inhibiting dopamine-beta-hydroxylase expression in rats treated by ocular administration of siRNAs targeting certain sequences of the dopamine-beta-hydroxylase gene.
[0175] In an embodiment, the present disclosure relates to a siRNA comprising a sense dopamine-beta-hydroxylase nucleic acid and an anti-sense dopamine-beta-hydroxylase nucleic acid, and the sense dopamine-beta-hydroxylase nucleic acid is substantially identical to a target sequence contained within dopamine-beta-hydroxylase mRNA and the anti-sense dopamine-beta-hydroxylase nucleic acid is complementary to the sense dopamine-beta-hydroxylase nucleic acid. The sense and antisense nucleic acids hybridize to each other to form a double-stranded molecule.
[0176] In an embodiment, the siRNA molecules of the present disclosure inhibit the expression of the dopamine-beta-hydroxylase gene when introduced into a cell expressing said gene.
[0177] In an embodiment, the siRNA molecules of the present disclosure inhibit dopamine-beta-hydroxylase expression and activity in a cell when introduced into a cell expressing dopamine-beta-hydroxylase gene.
[0178] In an embodiment, the siRNA molecules of the present disclosure decrease the expression and activity of dopamine-beta-hydroxylase in rats treated by ocular administration of siRNAs targeting certain sequences of the dopamine-beta-hydroxylase gene.
[0179] In an embodiment, the present disclosure relates to nucleic acid sequences and vectors encoding the siRNA according to the fourth aspect of the present disclosure, as well as to compositions comprising them, useful, for example, in the methods of the present disclosure. Compositions of the present disclosure may additionally comprise transfection enhancing agents. The nucleic acid sequence may be operably linked to an inducible or regulatable promoter. Suitable vectors are discussed above. Preferably the vector is an adeno-associated viral vector.
[0180] In an embodiment, the present disclosure relates to a composition comprising the siRNA of the present disclosure and additionally comprise a pharmaceutical agent for preventing or reversing progressive optical neuropathy associated with the elevation of intraocular pressure due to excessive noradrenergic activation, wherein the agent is different from the siRNA.
[0181] In an embodiment, the pharmaceutical agent is selected from the group consisting of an anti-glaucoma agent and most preferably an alpha adrenoceptor agonists, beta adrenoceptor blockers, carbonic anhydrase inhibitors, muscarinic agonists, prostaglandin analogs and rho kinase inhibitors agent and most preferably apraclonidine, brimonidine, betaxolol, levobunolol, metpranolol, timolol, acetazolamide, brinzolamide, dorzolamide, methazolamide, carbachol, pilocarpine, bimatoprost, latanoprost, tafluprost, travaprost, and netarsudil.
[0182] Non-viral delivery siRNA systems involve the creation of nucleic acid transfection reagents. Nucleic acid transfection reagents have two basic properties. First, they must interact in some manner with the nucleic acid cargo. Most often this involves electrostatic forces, which allow the formation of nucleic acid complexes. Formation of a complex ensures that the nucleic acid and transfection reagents are presented simultaneously to the cell membrane. Complexes can be divided into three classes, based on the nature of the delivery reagent: lipoplexes; polyplexes; and lipopolyplexes. Lipoplexes are formed by the interaction of anionic nucleic acids with cationic lipids, polyplexes by interaction with cationic polymers. Lipopolyplex reagents can combine the action of cationic lipids and polymers to deliver nucleic acids. Addition of histone, poly-L-lysine and protamine to some formulations of cationic lipids results in levels of delivery that are higher than either lipid or polymer alone. The combined formulations might also be less toxic. The biocompatible systems most relevant to this purpose are non-viral biodegradable nanocapsules designed especially according to the physical chemistry of nucleic acids. They have an aqueous core surrounded by a biodegradable polymeric envelope, which provides protection and transport of the siRNA into the cytosol and allow the siRNA to function efficiently in vivo.
[0183] In an embodiment, the present disclosure also relates to a cell containing the siRNA according to the present disclosure or the vector of the present disclosure. Preferably the cell is a mammalian cell, more preferably a human cell. It is further preferred that the cell is an isolated cell.
[0184] The following examples further illustrate the present disclosure in detail but are not to be construed to limit the scope thereof.
[0185] siNA molecules described in the present disclosure are tested in one or more of these examples and show to have activity and stability.
Example 1
[0186] Cell culture: SK-N-SH cells expressing dopamine-beta-hydroxylase were maintained in a humidified atmosphere of 5% CO 2 at 37 C. Cells were grown in MEM (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Gibco, UK), 100 U/mL penicillin G, 0.25 g/mL amphotericin B, 100 g/mL streptomycin (Gibco, UK), 25 mM sodium bicarbonate (Merck, Germany) and 25 mM N-2-hydroxyethylpiperazine-N-2-ethanosulfonic acid (HEPES) (Sigma, St. Louis, MO). The cell culture medium was changed every 2 days, and cells reached confluence 3-4 days after initial seeding. For subculturing, cells were dissociated with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Sigma, St. Louis, MO), split 1:15 or 1:20 and subcultured in a 21-cm 2 growth area (Sarstedt, Germany).
Example 2
[0187] Stability of chemically modified siRNAs against dopamine-beta-hydroxylase: siRNA sequences to be used in the study were thaw and incubated at 37 QC during up to 120 min with cell serum-free culture medium added with RNase I (0.25 or 0.50 Units). In contrast to non-modified (natural) siRNAs, chemically modified siRNAs against dopamine-beta-hydroxylase show a significant resistance to degradation in culture medium containing RNAse I (0.50 Units) for up to 120 min (
Example 3
[0188] Dopamine-beta-hydroxylase gene silencing: Total RNA was isolated and purified using the SV Total RNA Isolation System (Promega, USA) according to manufacturer's instructions. RNA quality and concentration were verified in the NanoDrop ND1000 Spectrophotometer (Thermo Scientific, USA), and RNA integrity and genomic DNA contamination were evaluated by agarose gel electrophoresis. Total RNA (1 g) was converted into cDNA using the Maxima Scientific First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific, USA), according to instructions. The following protocol was used: 1.sup.st step, 10 min at 25 C.; 2.sup.nd step, 15 min at 50 C.; 3.sup.rd step, 5 min at 85 C. cDNA was used for qPCR analysis using Maxima SYBR Green qPCR Master Mix (Thermo Scientific, USA) in the StepOnePlus instrument (Applied Biosystems, USA). Primer Assay for dopamine-beta-hydroxylase and for the endogenous control gene GAPDH (Quiagen, Germany) were used. The qPCR reaction was performed in 96-well PCR plates (Sarstedt, Germany) as follows: one cycle of 10 min at 95 C., followed by 40 PCR cycles at 95 C. 15 s and 60 C. 60 s. A melting curve was made immediately after the qPCR, to demonstrate the specificity of the amplification. No template controls were always evaluated for each target gene. Quantification cycle (Cq) values were generated automatically by the StepOnePlus 2.3 Software and the ratio of the target gene was expressed in comparison to the endogenous control gene GAPDH. Real-time PCR efficiencies were found to be between 90% and 110%.
Example 4
[0189] Dopamine-beta-hydroxylase expression: Cells were rinsed twice with cold phosphate-buffered saline (PBS) and incubated with 100 L RIPA lysis buffer (154 mM NaCl, 65.2 mM TRIZMA base, 1 mM EDTA, 1% NP-40 (IGEPAL), 6 mM sodium deoxycholate) containing protease inhibitors: 1 mM PMSF, 1 g/mL leupeptine and 1 g/mL aprotinin; and phosphatase inhibitors: 1 mM Na 3 VO 4 and 1 mM NaF. Cells were scraped and briefly sonicated. Equal amounts of total protein (30 g) were separated on a % SDS-polyacrylamide gel and electrotransfered to a nitrocellulose membrane in Tris-Glycine transfer buffer containing 20% methanol. The transblot sheets were blocked in 5% non-fat dry milk in Tris-buffered saline (TBS) for 60 min and then incubated overnight, at 4 C., with the antibodies against dopamine-beta-hydroxylase and GAPDH, diluted in 2.5% non-fat dry milk in TBS-Tween 20 (0.1% vol/vol). The immunoblots were subsequently washed and incubated with fluorescently-labelled secondary antibodies (1:20,000; AlexaFluor 680, Molecular Probes) for 60 min at room temperature (RT) and protected from light. Membranes were washed and imaged by scanning at both 700 nm and 800 nm with an Odyssey Infrared Imaging System (LI-COR Biosciences).
Example 5
[0190] Dopamine-beta-hydroxylase activity: Cells were rinsed twice with cold phosphate-buffered saline (PBS) and pre-incubated for 15 minutes in Hanks media at 37 C. Hanks media had the following composition (in mM): NaCl 140, KCl 5, MgSO.sub.4-7H.sub.2O 0.8, K2HPO.sub.4 0.33, KH.sub.2PO.sub.4 0.44, MgCl.sub.2 0.6H.sub.2O 1.0, CaCl.sub.2 0.025, Tris-HCl 9.75, pH 7.4. The reaction was initiated by adding 3 M L-dihydroxyphenylalanine plus ascorbic acid (at 1 mM; co-factor) to the Hanks media in the absence and the presence of 1 M nepicastat, for 360 minutes. During the pre-incubation and the incubation, cells were continuously shacked and maintained at 37 C. in a water bath. The reaction was stopped through the rapid removal of the incubation solution through aspiration with a Pasteur pipette, followed by a quick wash with Hanks media. Subsequently, cells were added with 0.2 M perchloric acid and stored at 4 C. for 24 hours. Thereafter, 900 l of perchloric acid in which the cells were kept was used for the quantification of noradrenaline by means of high-pressure liquid chromatography with electrochemical detection (HPLC-EC).
Example 6
[0191] In vivo dopamine-beta-hydroxylase inhibition and experimental glaucoma studies: Wistar rats were delivered with 4-6 weeks of age and used for the treatment with dopamine-beta-hydroxylase inhibitors or siRNAs against dopamine-beta-hydroxylase at least one week of quarantine. All animal interventions were conducted according to the European Directive 86/609, and the guidelines Guide for the Care and Use of Laboratory Animals, 7th edition, 1996, Institute for Laboratory Animal Research (I LAR), Washington, DC.
Example 7
[0192] The induction of high intra ocular pressure (experimental glaucoma) can be obtained according with the procedures previously described by Ishikawa et al. (Ishikawa, Yoshitomi, Zorumski & Izumi, 2015), namely the topical application of hydroxyanmphetamine or by means of elevated hydrostatic pressure in an in vitro model with retinal organotypic cultures (Madeira et al., 2015).
[0193] While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present disclosure, including methods, as well as the best mode thereof, of making and using this disclosure, the following examples are provided to further enable those skilled in the art to practice this disclosure and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the disclosure, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present disclosure will be apparent to those skilled in the art in view of the present disclosure.
[0194] All documents mentioned in this specification, including reference to sequence database identifiers, are incorporated herein by reference in their entirety. Unless otherwise specified, when reference to sequence database identifiers is made, the version number is 1.
[0195] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the disclosure and apply equally to all aspects and embodiments which are described. The disclosure is further described in the following non-limiting examples.
[0196] Additional aspects of the invention will be apparent to those skilled in the art, or may be learned from the practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
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