INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN, ACID LABILE SUBUNIT (IGFALS) AND INSULIN-LIKE GROWTH FACTOR 1 (IGF-1) iRNA COMPOSITIONS AND METHODS OF USE THEREOF

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on Oct. 11, 2019, is named 121301_03904_SL.txt and is 1,164,945 bytes in size.

BACKGROUND OF THE INVENTION

Acromegaly is a progressive and life threatening disease resulting from growth hormone hypersecretion from a benign pituitary tumor, leading to approximately a 10 year reduction in lifespan and a reduced quality of life. Acromegaly is associated with cardiovascular disease including hypertension and cardiac hypertrophy, cerebrovascular disease including stroke, metabolic disease including diabetes, and respiratory disease including sleep apnea. Mortality rates in acromegaly are correlated with growth hormone and IGF-1 levels, with increased growth hormone concentrations being associated with shorter life spans (Holdaway et al., JCEM, 2004). The clinical features most commonly associated with acromegaly are acral enlargement, maxofacial changes, excessive sweating, athralgias, headache, hypogonadal symptoms, visual deficit, fatigue, weight gain, and galactorrhea. Such symptoms may be associated with any of a number of diseases or conditions and, thus, diagnosis of acromegaly often does not occur until several years after the initiation of growth hormone hypersecretion. Definitive diagnosis of acromeagly includes detection of an increased level of insulin-like growth factor-1 (IGF-1) and growth hormone elevation in an oral glucose tolerance test, confirmed by detection of a GH-hypersecreting pituitary tumor, typically by MRI. (The diagnostic criteria for acromegaly are provided in the American Association of Clinical Endocrinologists Medical Guidelines for Clinical Practice for the Diagnosis and Treatment of Acromegaly—2011 Update (Katznelson et al., Endocr. Pract. 17 (Suppl. 4)).

Current treatment options for acromegaly are insufficient for many patients. Surgical removal of the pituitary adenoma by transsphenoidal surgery results in a cure for about 50-60% of patients. Subjects for whom surgical intervention is not possible or does not result in a cure are treated with first-line pharmacological therapy which includes dopamine agonists or sustained-release somatostatin analogs (SSAs). This therapy results in good control for the disease for about 70% of these patients for whom surgery cannot provide a cure. The use of SSAs, however, is limited to subjects expressing a somatostatin receptor on their tumor. Subjects whose disease cannot be controlled by the first-line pharmacological therapy are treated with SOMAVERT® (pegvisomant), a growth hormone receptor antagonist, which is administered by daily subcutaneous injection. Radiotherapy, which suffers from low efficacy and high side effects, is used as a last resort.

The insulin-like growth factor system is also associated with abnormal growth in cancer and metastasis (see, e.g., Samani et al., Endocrine Rev., 2007). The IGF system has become a target for anticancer agents, both as primary and adjunctive therapy.

Currently, treatments for acromegaly and cancer do not fully meet patient needs. Therefore, there is a need for therapies for subjects suffering from acromegaly or cancer.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which affect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an insulin-like growth factor binding protein, acid labile subunit (IGFALS) gene or an insulin-like growth factor-1 (IGF-1) gene. The IGFALS gene or IGF-1 gene may be within a cell, e.g., a cell within a subject, such as a human.

In an aspect, the invention provides a double stranded ribonucleic acid interference (dsRNA) agent for inhibiting expression of insulin-like growth factor binding protein, acid labile subunit (IGFALS), wherein the double stranded dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2.

In certain embodiments, the sense strands and antisense strands comprise sequences selected from any one of the sequences in any one of Tables 3, 5, 6, 8, 12, or 14.

In an aspect, the invention provides a double stranded ribonucleic acid interference (dsRNAi) agent for inhibiting expression of insulin-like growth factor-1 (IGF-1), wherein the double stranded RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 11 or 13 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 12 or 14.

In certain embodiments, the sense strands and antisense strands comprise sequences selected from any one of the sequences in any one of Tables 9, 11, 15, 17, 18, or 20.

In an aspect, the invention provides a double stranded ribonucleic acid interference (dsRNAi) agent for inhibiting expression of insulin-like growth factor binding protein, acid labile subunit (IGFALS), wherein the double stranded RNAi comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 3, 5, 6, 8, 12, or 14.

In an aspect, the invention provides a double stranded ribonucleic acid interference (dsRNAi) agent for inhibiting expression of insulin-like growth factor 1 (IGF-1) wherein the double stranded RNAi comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 9, 11, 15, 17, 18, or 20.

In certain embodiments, the double stranded RNAi comprises at least one modified nucleotide. In some embodiments, substantially all of the nucleotides of the sense strand are modified nucleotides. In some embodiments, substantially all of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In an aspect, the invention provides a double stranded RNAi agent for inhibiting expression of insulin-like growth factor binding protein, acid labile subunit (IGFALS), wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the double stranded RNAi agent comprises a ligand, e.g., the sense strand of the double stranded RNAi agent is conjugated to a ligand, e.g., a ligand is attached at the 3′-terminus of the sense strand.

Accordingly, in certain embodiments, the present invention provides double stranded RNAi agents for inhibiting expression of IGFALS, which comprise a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from nucleotides 11-62, 24-62, 79-117, 79-130, 155-173, 194-216, 194-229, 211-229, 232-293, 254-272, 310-328, 310-349, 324-345, 331-349, 353-371, 353-394, 376-394, 407-425, 439-449, 431-470, 484-515, 497-515, 541-580, 547-568, 596-647, 616-634, 673-691, 694-712, 694-734, 777-799, 781-799, 825-843, 825-855, 869-922, 958-976, 958-988, 1064-1085, 1064-1096, 1067-1085, 1067-1096, 1100-1141, 1111-1129, 1145-1163, 1145-1186, 1159-1186, 1168-1196, 1168-1214, 1193-1214, 1266-1307, 1321-1339, 1342-1373, 1375-1406, 1432-1450, 1454-1472, 1519-1537, 1519-1559, 1534-1555, 1541-1559, 1606-1624, 1606-1637, 1613-1635, 1672-1690, 1672-1712, 1749-1779, 1783-1801, 1805-1823, 1806-1829, 1871-1889, 1871-1919, 1949-1977, 1993-2011, 2013-2042, 2048-2077, 2048-2088, or 2052-2084 of SEQ ID NO: 1, and, in certain embodiments, the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 2 such that the antisense strand is complementary to the at least 15 contiguous nucleotides differing by no more than 3 nucleotides in the sense strand.

Accordingly, in certain embodiments, the present invention provides double stranded RNAi agents for inhibiting expression of insulin-like growth factor 1 (IGF-1), which comprise a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from nucleotides 330-369, 342-369, 432-490, 432-482, 436-462, 534-559, 330-350, 342-362, 348-368, 349-369, 432-452, 435-455, 436-456, 438-458, 440-460, 441-461, 442-462, 449-469, 455-475, 460-480, 461-481, 462-482, 464-484, 470-490, 484-501, 534-554, 536-556, 538-558, 539-559, 542-562, 548-568, 577-597, 582-602, or 640-660 of the nucleotide sequence of SEQ ID NO: 11, and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 12 such that the antisense strand is complementary to the at least 15 contiguous nucleotides differing by no more than 3 nucleotides in the sense strand.

In certain embodiments, the present invention provides double stranded RNAi agents for inhibiting expression of insulin-like growth factor 1 (IGF-1), which comprise a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from nucleotides 6-90, 127-145, 185-238, 247-265, 277-295, 389-417, 430-480, 543-561, 654-690, 750-768, 774-870, 894-930, 1007-1029, 1075-1126, 1144-1162, 1197-1215, 1232-1250, 1293-1311, 1334-1352, 1388-1458, 1463-1490, 1511-1529, 1599-1617, 1643-1661, 1690-1727, 1793-1825, 1843-1861, 2057-2075, 2090-2130, 2192-2228, 2310-2332, 2357-2375, 2521-2539, 2566-2588, 2648-2684, 2793-2811, 2962-2980, 3120-3142, 3208-3233, 3269-3287, 3417-3435, 3449-3467, 3575-3603, 3686-3704, 3721-3739, 3806-3824, 3939-3957, 3982-4018, 4081-4037, 4154-4172, 4271-4289, 4319-4377, 4436-4478, 4484-4502, 4523-4545, 4566-4584, 4610-4660, 4686-4717, 4734-4769, 4780-4798, 4815-4843, 4884-4902, 4911-4929, 5004-5034, 5050-5068, 5171-5256, 5311-5364, 5409-5430, 5551-5588, 5609-5638, 5694-5712, 5715-5758, 5790-5808, 5906-5928, 5934-5952, 6323-6345, 6399-6417, 6461-6497, 6510-6535, 6584-6612, 6629-6647, 6661-6683, 6726-6789, 6796-6824, 6826-6851, 6858-6905, 6910-6927, 7004-7022, 7035-7130, 7144-7162, 7175-7241, and 7252-7270 of the nucleotide sequence of SEQ ID NO: 13, and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 14 such that the antisense strand is complementary to the at least 15 contiguous nucleotides differing by no more than 3 nucleotides in the sense strand.

In certain embodiments, the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from nucleotides 342-369, 432-462, 330-350, 342-362, 348-368, 349-369, 432-452, 435-455, 436-456, 438-458, 440-460, 442-462, 470-490, 481-501, 536-556, or 539-559 of the nucleotide sequence of SEQ ID NO: 11 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 12 such that the antisense strand is complementary to the at least 15 contiguous nucleotides differing by no more than 3 nucleotides in the sense strand. In certain embodiments, the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from nucleotides 340-369, 430-490, 430-482, 434-460, 532-559, 328-350, 340-362, 346-368, 347-369, 430-452, 433-455, 434-456, 436-458, 438-460, 439-461, 440-462, 447-469, 453-475, 458-480, 459-481, 460-482, 461-483, 462-484, 468-490, 479-501, 532-554, 534-556, 536-558, 537-559, 540-562, 546-568, 575-597, 580-602, or 638-660 of the nucleotide sequence of SEQ ID NO: 11, for example nucleotides 342-369, 432-462, 330-350, 342-362, 348-368, 349-369, 432-452, 435-455, 436-456, 438-458, 440-460, 442-462, 470-490, 481-501, 536-556, or 539-559 of the nucleotide sequence of SEQ ID NO: 11, and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 12 such that the antisense strand is complementary to the at least 15 contiguous nucleotides differing by no more than 3 nucleotides in the sense strand.

In certain embodiments, the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from nucleotides 6-90, 127-145, 185-238, 247-265, 277-295, 389-417, 430-480, 543-561, 654-690, 750-768, 774-870, 894-930, 1007-1029, 1075-1126, 1144-1162, 1197-1215, 1232-1250, 1293-1311, 1334-1352, 1388-1458, 1463-1490, 1511-1529, 1599-1617, 1643-1661, 1690-1727, 1793-1825, 1843-1861, 2057-2075, 2090-2130, 2192-2228, 2310-2332, 2357-2375, 2521-2539, 2566-2588, 2648-2684, 2793-2811, 2962-2980, 3120-3142, 3208-3233, 3269-3287, 3417-3435, 3449-3467, 3575-3603, 3686-3704, 3721-3739, 3806-3824, 3939-3957, 3982-4018, 4081-4037, 4154-4172, 4271-4289, 4319-4377, 4436-4478, 4484-4502, 4523-4545, 4566-4584, 4610-4660, 4686-4717, 4734-4769, 4780-4798, 4815-4843, 4884-4902, 4911-4929, 5004-5034, 5050-5068, 5171-5256, 5311-5364, 5409-5430, 5551-5588, 5609-5638, 5694-5712, 5715-5758, 5790-5808, 5906-5928, 5934-5952, 6323-6345, 6399-6417, 6461-6497, 6510-6535, 6584-6612, 6629-6647, 6661-6683, 6726-6789, 6796-6824, 6826-6851, 6858-6905, 6910-6927, 7004-7022, 7035-7130, 7144-7162, 7175-7241, or 7252-7270 of the nucleotide sequence of SEQ ID NO: 13, and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 14 such that the antisense strand is complementary to the at least 15 contiguous nucleotides differing by no more than 3 nucleotides in the sense strand.

In certain embodiments, substantially all of the nucleotides of the sense strand are modified. In certain embodiments, substantially all of the nucleotides of the antisense strand are modified nucleotides. In certain embodiments, substantially all of the nucleotides of both strands are modified. Further, in certain embodiments, the double stranded RNAi agent comprises a ligand, e.g., the double stranded RNAi agent is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In certain embodiments, the present invention also provides double stranded RNAi agents for inhibiting expression of IGFALS, which comprise a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides from nucleotides 11-62, 24-62, 79-117, 79-130, 155-173, 194-216, 194-229, 211-229, 232-293, 254-272, 310-328, 310-349, 324-345, 331-349, 353-371, 353-394, 376-394, 407-425, 439-449, 431-470, 484-515, 497-515, 541-580, 547-568, 596-647, 616-634, 673-691, 694-712, 694-734, 777-799, 781-799, 825-843, 825-855, 869-922, 958-976, 958-988, 1064-1085, 1064-1096, 1067-1085, 1067-1096, 1100-1141, 1111-1129, 1145-1163, 1145-1186, 1159-1186, 1168-1196, 1168-1214, 1193-1214, 1266-1307, 1321-1339, 1342-1373, 1375-1406, 1432-1450, 1454-1472, 1519-1537, 1519-1559, 1534-1555, 1541-1559, 1606-1624, 1606-1637, 1613-1635, 1672-1690, 1672-1712, 1749-1779, 1783-1801, 1805-1823, 1806-1829, 1871-1889, 1871-1919, 1949-1977, 1993-2011, 2013-2042, 2048-2077, 2048-2088, or 2052-2084 of the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 2 such that the antisense strand is complementary to the at least 15 contiguous nucleotides in the sense strand.

In certain embodiments, the present invention provides double stranded ribonucleic acid (RNAi) agent for inhibiting expression of insulin-like growth factor 1 (IGF-1), which comprise a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides selected from the group consisting of nucleotides 330-369, 342-369, 432-490, 432-482, 436-462, 534-559, 330-350, 342-362, 348-368, 349-369, 432-452, 435-455, 436-456, 438-458, 440-460, 441-461, 442-462, 449-469, 455-475, 460-480, 461-481, 462-482, 464-484, 470-490, 484-501, 534-554, 536-556, 538-558, 539-559, 542-562, 548-568, 577-597, 582-602, or 640-660 of the nucleotide sequence of SEQ ID NO: 11 and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 12 such that the antisense strand is complementary to the at least 15 contiguous nucleotides in the sense strand.

In certain embodiments, the agents comprise a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides selected from the group of nucleotides 340-369, 430-490, 430-482, 434-460, 532-559, 328-350, 340-362, 346-368, 347-369, 430-452, 433-455, 434-456, 436-458, 438-460, 439-461, 440-462, 447-469, 453-475, 458-480, 459-481, 460-482, 461-483, 462-484, 468-490, 479-501, 532-554, 534-556, 536-558, 537-559, 540-562, 546-568, 575-597, 580-602, or 638-660 of the nucleotide sequence of SEQ ID NO: 11, for example nucleotides 342-369, 432-462, 330-350, 342-362, 348-368, 349-369, 432-452, 435-455, 436-456, 438-458, 440-460, 442-462, 470-490, 481-501, 536-556, or 539-559 of the nucleotide sequence of SEQ ID NO: 11, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 12 such that the antisense strand is complementary to the at least 15 contiguous nucleotides in the sense strand.

In certain embodiments, the agents comprise a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides selected from the group of nucleotides 6-90, 127-145, 185-238, 247-265, 277-295, 389-417, 430-480, 543-561, 654-690, 750-768, 774-870, 894-930, 1007-1029, 1075-1126, 1144-1162, 1197-1215, 1232-1250, 1293-1311, 1334-1352, 1388-1458, 1463-1490, 1511-1529, 1599-1617, 1643-1661, 1690-1727, 1793-1825, 1843-1861, 2057-2075, 2090-2130, 2192-2228, 2310-2332, 2357-2375, 2521-2539, 2566-2588, 2648-2684, 2793-2811, 2962-2980, 3120-3142, 3208-3233, 3269-3287, 3417-3435, 3449-3467, 3575-3603, 3686-3704, 3721-3739, 3806-3824, 3939-3957, 3982-4018, 4081-4037, 4154-4172, 4271-4289, 4319-4377, 4436-4478, 4484-4502, 4523-4545, 4566-4584, 4610-4660, 4686-4717, 4734-4769, 4780-4798, 4815-4843, 4884-4902, 4911-4929, 5004-5034, 5050-5068, 5171-5256, 5311-5364, 5409-5430, 5551-5588, 5609-5638, 5694-5712, 5715-5758, 5790-5808, 5906-5928, 5934-5952, 6323-6345, 6399-6417, 6461-6497, 6510-6535, 6584-6612, 6629-6647, 6661-6683, 6726-6789, 6796-6824, 6826-6851, 6858-6905, 6910-6927, 7004-7022, 7035-7130, 7144-7162, 7175-7241, or 7252-7270 of the nucleotide sequence of SEQ ID NO: 13, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 14 such that the antisense strand is complementary to the at least 15 contiguous nucleotides in the sense strand.

In certain embodiments, substantially all of the nucleotides of the sense strand are modified nucleotides. In certain embodiments, substantially all of the nucleotides of the antisense strand are modified nucleotides. In certain embodiments, substantially all of the nucleotides of both strands are modified. In preferred embodiments, the double stranded RNAi agent comprises a ligand, e.g., the double stranded RNAi agent is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In certain embodiments, the sense strand and the antisense strand comprise a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 3, 5, 6, 8, 12, or 14 for IGFALS or any one of Tables 9, 11, 15, 17, 18, or 20 for IGF-1.

For example, in certain embodiments, the sense strand and the antisense strand comprise a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense nucleotide sequences selected from the group of the antisense nucleotide sequence of duplexes targeted to IGF selected from the group AD-66722, AD-66748, AD-66746, AD-66747, AD-66733, AD-66752, AD-66739, AD-66738, AD-66725, AD-66740, AD-66750, AD-66729, AD-66745, AD-66749, AD-66720, AD-66724, AD-66726, AD-66766, AD-66761, AD-66755, AD-66751, AD-66719, AD-66727, AD-66744, AD-66760, AD-66753, AD-66721, AD-66716, AD-66743, or AD-66728-AD-77150, AD-77158, AD-74963, AD-77138, AD-75740, AD-74968, AD-74965, AD-75766, AD-75761, AD-75137, AD-74979, AD-74966, AD-75750, AD-77126, AD-74971, AD-74982, AD-77144, AD-77149, AD-75751, AD-75111, AD-77147, AD-74964, AD-74983, AD-75765, AD-74970, AD-75749, AD-77168, AD-77127, AD-75748, AD-75779, AD-75145, AD-74975, AD-77151, AD-75170, AD-75741, AD-75162, AD-74985, AD-75759, AD-75218, AD-74981, AD-75155, AD-74978, AD-77153, AD-75157, AD-75123, AD-75184, AD-77160, AD-75125, AD-75229, AD-77165, AD-75112, AD-75206, AD-75769, AD-75174, AD-75225, AD-75792, AD-75115, AD-74986, AD-77171, AD-75131, AD-77128, AD-75179, AD-75792, AD-77124, AD-75191, AD-75774, AD-75114, AD-74973, AD-77156, AD-75120, AD-75130, AD-74967, AD-75231, AD-74987, AD-77140, AD-74969, AD-75000, AD-75791, AD-75143, AD-77120, AD-77142, AD-75217, AD-75234, AD-75173, AD-75232, AD-75188, AD-75135, AD-75018, AD-77122, AD-75009, AD-75121, AD-75791, AD-77135, AD-75214, AD-74994, AD-75139, AD-75166, AD-75020, AD-77159, AD-75236, AD-77123, AD-77133, AD-74972, AD-75223, AD-75148, AD-75124, AD-75185, AD-75150, AD-74976, AD-74980, AD-75212, AD-75239, AD-75221, AD-75118, AD-75793, AD-75023, AD-75164, AD-74997, AD-74984, AD-75011, AD-75203, AD-77161, AD-75033, AD-75177, AD-75795, AD-77146, AD-75793, AD-75788, AD-75079, AD-75152, AD-77121, AD-75237, AD-75014, AD-75755, AD-75028, AD-75091, AD-75110, AD-75230, AD-75029, AD-75099, AD-77130, AD-75224, AD-75142, AD-75760, AD-75795, AD-77136, AD-75032, AD-75757, AD-75017, AD-75151, AD-75122, AD-75002, AD-75021, AD-75005, AD-75088, AD-75153, AD-75208, AD-74977, AD-75069, AD-75107, AD-74990, AD-75061, AD-75083, AD-75116, AD-75169, AD-75058, AD-74991, AD-75041, AD-77131, AD-75772, AD-77169, AD-75133, AD-75222, AD-75007, AD-75101, AD-77137, AD-75090, AD-77148, AD-75008, AD-77134, AD-74999, AD-75048, AD-75095, AD-74974, AD-75788, AD-75057, AD-75113, AD-77172, AD-75016, AD-75186, AD-75205, AD-75238, or AD-75146; for example duplexes AD-66722, AD-66748, AD-66746, AD-66747, AD-66733, AD-66752, AD-66739, AD-66738, AD-66725, AD-66740, AD-66750, AD-66729, or AD-66745. In certain embodiments, nucleotide sequences selected from the group duplexes targeted to IGF selected from the group AD-66722, AD-66748, AD-66746, AD-66747, AD-66733, AD-66752, AD-66739, AD-66738, AD-66725, AD-66740, AD-66750, AD-66729, and AD-66745. In certain embodiments, the sense strand and the antisense strand comprise a region of complementarity which comprises at least 15 contiguous nucleotides of any one of the sense and antisense nucleotide sequences of the foregoing duplexes.

In certain embodiments, the sense strand and the antisense strand comprise a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences of the duplexes targeted to IGFALS selected from the group AD-62728, AD-62734, AD-68111, AD-68709, AD-68712, AD-68715, AD-68716, AD-68717, AD-68719, AD-68720, AD-68722, AD-68725, AD-68726, AD-68730, AD-68731, AD-73782, AD-73773, AD-73765, AD-73946, AD-73947, AD-73858, AD-73797, AD-73808, AD-73906, AD-73912, AD-73848, AD-73836, AD-73818, AD-73786, AD-73862, AD-73795, AD-73766, AD-73930, AD-73825, AD-73924, AD-73802, AD-73767, AD-73771, AD-73777, AD-73793, AD-73898, AD-73784, AD-73882, AD-73803, AD-73772, AD-73907, AD-73948, AD-73890, AD-73883, AD-73770, AD-73867, AD-73931, AD-73932, AD-73787, AD-73791, AD-73880, AD-73914, AD-73849, AD-73863, AD-73920, AD-73944, AD-73841, AD-73785, AD-73804, AD-73823, AD-73885, AD-73788, AD-73865, AD-73941, AD-73859, AD-73913, AD-73892, AD-73837, AD-73842, AD-73840, AD-73813, AD-73796, AD-73875, AD-73900, AD-73922, AD-73861, AD-73816, AD-73764, AD-73868, AD-73812, AD-73826, AD-73938, AD-73843, AD-73817, AD-73943, AD-73827, AD-73937, AD-73877, AD-73833, AD-73807, AD-73819, AD-73886, AD-73919, AD-73800, AD-76171, AD-76173, AD-76203, AD-76210, AD-76172, AD-76175, AD-76209, AD-76174, AD-76208, AD-76186, AD-76177, AD-76199, AD-76197, or AD-76212.

In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an a basic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, 2′-hydroxly-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, and a nucleotide comprising a 5′-phosphate mimic. In another embodiment, the modified nucleotides comprise a short sequence of 3′-terminal deoxy-thymine nucleotides (dT).

In certain embodiments, the duplex comprises a modified antisense nucleotide sequence targeted to IGFALS provided in Table 5, 8, or 14, or targeted to IGF-1 in Table 11, 17, or 20. In certain embodiments, the duplex comprises a modified sense strand nucleotide sequence targeted to IGFALS provided in Table 5, 8, or 14, or targeted to IGF-1 in Table 11, 17, or 20. In certain embodiments, the duplex comprises the modified sense strand nucleotide sequence and the modified antisense strand nucleotide of any one of the duplexes targeted to IGFALS provided in Table 5, 8, or 14, or targeted to IGF-1 in Table 11, 17, or 20.

In certain embodiments, the region of complementarity between the antisense strand and the target is at least 17 nucleotides in length. For example, the region of complementarity between the antisense strand and the target is 19 to 21 nucleotides in length, for example, the region of complementarity is 21 nucleotides in length. In preferred embodiments, each strand is no more than 30 nucleotides in length.

In some embodiments, at least one strand comprises a 3′ overhang of at least 1 nucleotide, e.g., at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide. In other embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides.

In many embodiments, the double stranded RNAi agent further comprises a ligand. The ligand may be one or more GalNAc attached to the RNAi agent through a monovalent, a bivalent, or a trivalent branched linker. The ligand may be conjugated to the 3′ end of the sense strand of the double stranded RNAi agent. The ligand can be an N-acetylgalactosamine (GalNAc) derivative including, but not limited to

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In various embodiments, the ligand is attached to the 5′ end of the sense strand of the double stranded RNAi agent, the 3′ end of the antisense strand of the double stranded RNAi agent, or the 5′ end of the antisense strand of the double stranded RNAi agent.

In some embodiments, the double stranded RNAi agents of the invention comprise a plurality, e.g., 2, 3, 4, 5, or 6, of GalNAc, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.

In certain embodiments, the dsRNAi is agent conjugated to the ligand as shown in the following schematic:

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and, wherein X is O or S. In one embodiment, the X is O.

In certain embodiments, the ligand is a cholesterol.

In certain embodiments, the region of complementarity comprises any one of the antisense sequences targeted to IGFALS provided in Table 3, 5, 6, 8, 12, or 14 or targeted to IGF-1 in Table 9, 11, 15, 17, 18, or 20. In another embodiment, the region of complementarity consists of any one of the antisense sequences of targeted to IGFALS provided in Table 3, 5, 6, 8, 12, or 14 or targeted to IGF-1 in Table 9, 11, 15, 17, 18, or 20.

In another aspect, the invention provides a double stranded RNAi agent for inhibiting expression of IGFALS or IGF-1, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding IGFALS or IGF-1, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III).

sense: 5′n_p-N_a—(XXX)_i—N_b—YYY—N_b—(ZZZ)_j—N_a-n_q3′

antisense: 3′n_p′-N_a′—(X′X′X′)_k—N_b′—Y′Y′Y′—N_b′—(Z′Z′Z′)_l—N_a′-n_q′5′ (III)

wherein: i, j, k, and l are each independently 0 or 1; p, p′, q, and q′ are each independently 0-6; each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof; each n_p, n_p′, n_q, and n_q′, each of which may or may not be present, independently represents an overhang nucleotide; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides; modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′; and wherein the double stranded RNAi agent comprises a ligand, e.g., the sense strand is conjugated to at least one ligand.

In certain embodiments, i is 0; j is 0; i is 1; j is 1; both i and j are 0; or both i and j are 1. In another embodiment, k is 0; l is 0; k is 1; l is 1; both k and l are 0; or both k and l are 1. In another embodiment, XXX is complementary to X′X′X′, YYY is complementary to Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′. In another embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. In another embodiment, the Y′Y′Y′ motif occurs at the 11, 12 and 13 positions of the antisense strand from the 5′-end. In one embodiment, the Y′ is 2′-O-methyl.

For example, formula (III) can be represented by formula (IIIa):

sense: 5′n_p-N_a—YYY—N_a-n_q3′

antisense: 3′n_p′-N_a′—Y′Y′Y′—N_a′-n_q′5′ (IIIa).

In another embodiment, formula (III) is represented by formula (IIIb):

sense: 5′n_p-N_a—YYY—N_b—ZZZ—N_a-n_q3′

antisense: 3′n_p′-N_a′—Y′Y′Y′—N_b′—Z′Z′Z′—N_a′-n_q′5′ (IIIb)

wherein each N_band N_b′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides.

Alternatively, formula (III) can be represented by formula (IIIc):

sense: 5′n_p-N_a—XXX—N_b—YYY—N_a-n_q3′

antisense: 3′n_p′-N_a′—X′X′X′—N_b′—Y′Y′Y′—N_a′-n_q′5′ (IIIc)

wherein each N_band N_b′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides.

Further, formula (III) can be represented by formula (IIId):

sense: 5′n_p-N_a—XXX—N_b—YYY—N_b—ZZZ—N_a-n_q3′

antisense: 3′n_p′-N_a′—X′X′X′—N_b′—Y′Y′Y′—N_b′—Z′Z′Z′—N_a′-n_q′5′ (IIId)

wherein each N_band N_b′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides and each N_aand N_a′ independently represents an oligonucleotide sequence comprising 2-10 modified nucleotides.

In certain embodiment, the double stranded region is 15-30 nucleotide pairs in length. For example, the double stranded region can be 17-23 nucleotide pairs in length. The double stranded region can be 17-25 nucleotide pairs in length. The double stranded region can be 23-27 nucleotide pairs in length. The double stranded region can be 19-21 nucleotide pairs in length. The double stranded region can be 21-23 nucleotide pairs in length.

In certain embodiments, each strand has 15-30 nucleotides. In other embodiments, each strand has 19-30 nucleotides.

Modifications on the nucleotides are selected from the group including, but not limited to, LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, and combinations thereof. In another embodiment, the modifications on the nucleotides are 2′-O-methyl or 2′-fluoro modifications.

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An exemplary structure of a dsRNAi agent conjugated to the ligand is shown in the following schematic

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In certain embodiments, the ligand can be a cholesterol.

In certain embodiments, the double stranded RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. For example the phosphorothioate or methylphosphonate internucleotide linkage can be at the 3′-terminus of one strand, i.e., the sense strand or the antisense strand; or at the ends of both strands, the sense strand and the antisense strand.

In certain embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5′-terminus of one strand, i.e., the sense strand or the antisense strand; or at the ends of both strands, the sense strand and the antisense strand.

In certain embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the both the 5′- and 3′-terminus of one strand, i.e., the sense strand or the antisense strand; or at the ends of both strands, the sense strand and the antisense strand.

In certain embodiments, the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

In certain embodiments, the Y nucleotides contain a 2′-fluoro modification. In another embodiment, the Y′ nucleotides contain a 2′-O-methyl modification. In another embodiment, p′>0. In some embodiments, p′=2. In some embodiments, q′=0, p=0, q=0, and p′ overhang nucleotides are complementary to the target mRNA. In some embodiments, q′=0, p=0, q=0, and p′ overhang nucleotides are non-complementary to the target mRNA.

In certain embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

In certain embodiments, at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage. In other embodiments, all n_p′ are linked to neighboring nucleotides via phosphorothioate linkages.

In certain embodiments, the dsRNAi agent is selected from the group of any one of the double stranded RNAi agents targeted to IGFALS provided in Table 3, 5, 6, 8, 12, or 14, or targeted to IGF-1 in Table 9, 11, 15, 17, 18, or 20. In certain embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In an aspect, the invention provides a double stranded RNAi agent for inhibiting expression of IGFALS or IGF-1 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding IGFALS or IGF-1, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

sense: 5′n_p-N_a—(XXX)_i—N_b—YYY—N_b—(ZZZ)_j—N_a-n_q3′

antisense: 3′n_p′-N_a′—(X′X′X′)_k-N_b′—Y′Y′Y′—N_b′—(Z′Z′Z′)_l-N_a′-n_q′5′ (III)

wherein i, j, k, and l are each independently 0 or 1; p, p′, q, and q′ are each independently 0-6; each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof; each n_p, n_p′, n_q, and n_q′, each of which may or may not be present independently represents an overhang nucleotide; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y; and wherein the double stranded RNAi agent comprises a ligand, e.g., the double stranded RNAi agent is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

sense: 5′n_p-N_a—(XXX)_i—N_b—YYY—N_b—(ZZZ)_j—N_a-n_q3′

antisense: 3′n_p′-N_a′—(X′X′X′)_k-N_b′—Y′Y′Y′—N_b′—(Z′Z′Z′)_l-N_a′-n_q′5′ (III)

wherein: i, j, k, and l are each independently 0 or 1; each n_p, n_q, and n_q′, each of which may or may not be present, independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6; n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′; and wherein the double stranded RNAi agent comprises a ligand, e.g., the double stranded RNAi agent is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In certain embodiments, the invention provides a d double stranded ribonucleic acid (RNAi) agent for inhibiting expression of IGFALS or IGF-1, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding IGFALS or IGF-1, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

sense: 5′n_p-N_a—(XXX)_i—N_b—YYY—N_b—(ZZZ)_j—N_a-n_q3′

antisense: 3′n_p′-N_a′—(X′X′X′)_k-N_b′—Y′Y′Y′—N_b′—(Z′Z′Z′)_l—N_a′-n_q′5′ (III)

wherein i, j, k, and l are each independently 0 or 1; each n_p, n_q, and n_q′, each of which may or may not be present, independently represents an overhang nucleotide; p, q, and q′ are each independently 0-6; n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y; and wherein the double stranded RNAi agent is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent linker.

In an aspect, the invention provides a double stranded RNAi agent for inhibiting expression of IGFALS or IGF-1, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding IGFALS or IGF-1, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

sense: 5′n_p-(XXX)_i—N_b—YYY N_b—(ZZZ)_j—N_a-n_q3′

antisense: 3′n_p′-N_a′—(X′X′X′)_k-N_b′—Y′Y′Y′—N_b′—(Z′Z′Z′)_l-N_a′-n_q′5′ (III)

wherein i, j, k, and l are each independently 0 or 1; each n_p, n_q, and n_q′, each of which may or may not be present, independently represents an overhang nucleotide; p, q, and q′ are each independently 0-6; n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y; wherein the double stranded RNAi agent comprises a ligand, e.g., the double stranded RNAi agent is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In an aspect, the invention provides a double stranded RNAi agent capable of inhibiting the expression of IGFALS or IGF-1 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding IGFALS of IGF-1, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

sense: 5′n_p-N_a—(XXX)_i—N_b—YYY—N_b—(ZZZ)_j—N_a-n_q3′

antisense: 3′n_p′-N_a′—(X′X′X′)_k-N_b′—Y′Y′Y′—N_b′—(Z′Z′Z′)_l-N_a′-n_q′5′ (III)

wherein i, j, k, and l are each independently 0 or 1; each n_p, n_q, and n_q′, each of which may or may not be present, independently represents an overhang nucleotide; p, q, and q′ are each independently 0-6; n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y; wherein the sense strand comprises at least one phosphorothioate linkage; and wherein the double stranded RNAi agent comprises a ligand, e.g., the double stranded RNAi agent is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

sense: 5′n_p-N_a—YYY—N_a-n_q3′

antisense: 3′n_p′-N_a′-Y′Y′Y′—N_a′-n_q′5′ (IIIa)

wherein each n_p, n_q, and n_q′, each of which may or may not be present, independently represents an overhang nucleotide; p, q, and q′ are each independently 0-6; n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; YYY and Y′Y′Y′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; wherein the sense strand comprises at least one phosphorothioate linkage; wherein the double stranded RNAi agent comprises a ligand, e.g., the double stranded RNAi agent is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In an aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting expression of IGFALS, wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2, wherein substantially all of the nucleotides of the sense strand comprise a modification selected from a 2′-O-methyl modification and a 2′-fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus, wherein substantially all of the nucleotides of the antisense strand comprise a modification selected from a 2′-O-methyl modification and a 2′-fluoro modification, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus and two phosphorothioate internucleotide linkages at the 3′-terminus, and wherein the sense strand is conjugated to one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent linker at the 3′-terminus.

In an aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting expression of insulin-like growth factor 1 (IGF-1), wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:11 or 13 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:12 or 14, wherein substantially all of the nucleotides of the sense strand comprise a modification selected from a 2′-O-methyl modification and a 2′-fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus, wherein substantially all of the nucleotides of the antisense strand comprise a modification selected from a 2′-O-methyl modification and a 2′-fluoro modification, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus and two phosphorothioate internucleotide linkages at the 3′-terminus, and wherein the sense strand is conjugated to one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent linker at the 3′-terminus.

In certain embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides. In certain embodiments, each strand has 19-30 nucleotides.

In certain embodiments, substantially all of the nucleotides of the sense strand are modified.

In certain embodiments, substantially all of the nucleotides of the antisense strand are modified. In certain embodiments, substantially all of the nucleotides of both the sense strand and the antisense strand are modified.

In an aspect, the invention provides a cell containing the dsRNAi agent as described herein.

In an aspect, the invention provides a vector encoding at least one strand of a dsRNAi agent, wherein the RNAi agent comprises a region of complementarity to at least a part of an mRNA encoding IGFALS or IGF-1, wherein the RNAi is 30 base pairs or less in length, and wherein the RNAi agent targets the mRNA for cleavage. In certain embodiments, the region of complementarity is at least 15 nucleotides in length. In certain embodiments, the region of complementarity is 19 to 23 nucleotides in length.

In an aspect, the invention provides a cell comprising a vector as described herein.

In an aspect, the invention provides a pharmaceutical composition for inhibiting expression of an IGFALS or IGF-1 gene, comprising a double stranded RNAi agent of the invention. In one embodiment, the RNAi agent is administered in an unbuffered solution. In certain embodiments, the unbuffered solution is saline or water. In other embodiments, the RNAi agent is administered with a buffer solution. In such embodiments, the buffer solution can comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. For example, the buffer solution can be phosphate buffered saline (PBS).

In an aspect, the invention provides a pharmaceutical composition comprising the double stranded RNAi agent of the invention and a lipid formulation. In certain embodiments, the lipid formulation comprises a LNP. In certain embodiments, the lipid formulation comprises MC3.

In an aspect, the invention provides a method of inhibiting IGFALS or IGF-1 expression in a cell, the method comprising (a) contacting the cell with the double stranded RNAi agent of the invention or a pharmaceutical composition of the invention; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of an IGFALS or IGF-1 gene, thereby inhibiting expression of the IGFALS or IGF-1 gene in the cell. In certain embodiments, the cell is within a subject, for example, a human subject, for example a female human or a male human. In preferred embodiments, IGFALS or IGF-1 expression is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or to below the threshold of detection of the assay method used. Preferably the expression is inhibited by at least 50%. In some embodiments of the methods of the invention, expression of an IGF-1 gene is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the difference between the elevated level associated with the disease and a normal level in an appropriate control subject. Preferably the elevated level is inhibited by at least 50%.

In an aspect, the invention provides a method of treating a subject having a disease or disorder that would benefit from reduction in IGFALS or IGF-1expression, such as an IGF system-associated disease or disorder, the method comprising administering to the subject a therapeutically effective amount of a double stranded RNAi agent of the invention or a pharmaceutical composition of the invention, thereby treating the subject.

In an aspect, the invention provides a method of preventing at least one symptom in a subject having a disease or disorder that would benefit from reduction in IGFALS or IGF-1expression, such as an IGF system-associated disease or disorder, the method comprising administering to the subject a prophylactically effective amount of a double stranded RNAi agent of the invention or a pharmaceutical composition of the invention, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in IGFALS or IGF-1 expression.

In certain embodiments, the administration of the double stranded RNAi to the subject causes a decrease in the IGF-1 signaling pathway. In certain embodiments, the administration of the double stranded RNAi causes a decrease in the level of IGF-1 or IGFALS in the subject, e.g., serum levels of IGF-1 or IGFALS in the subject.

In certain embodiments, the IGF system-associated disease is acromegaly. In certain embodiments, the IGF system-associated disease is gigantism. In another embodiment, the IGF system-associated disease is cancer. In certain embodiments, the cancer is metastatic cancer.

In certain embodiments, the invention further comprises administering an inhibitor of growth hormone to a subject with an IGF system-associated disease.

In certain embodiments, the invention further comprises administering an inhibitor of the IGF pathway signaling to a subject with an IGF system-associated disease.

In certain embodiments, wherein the IGF system-associated disease is acromegaly or gigantism, the subject is further treated for acromegaly or gigantism. In certain embodiments, the treatment for acromegaly or gigantism includes surgery. In certain embodiments, the treatment for acromegaly or gigantism includes radiation. In certain embodiments, the treatment for acromegaly or gigantism includes administration of a therapeutic agent.

In certain embodiments, wherein the IGF system-associated disease is cancer, the subject is further treated for cancer. In certain embodiments, the treatment for cancer includes surgery. In certain embodiments, the treatment for cancer includes radiation. In certain embodiments, the treatment for cancer includes administration of a chemotherapeutic agent.

In various embodiments, the dsRNAi agent is administered at a dose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg. In some embodiments, the dsRNAi agent is administered at a dose of about 10 mg/kg to about 30 mg/kg. In certain embodiments, the dsRNAi agent is administered at a dose selected from 0.5 mg/kg 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, and 30 mg/kg. In certain embodiments, the dsRNAi agent is administered about once per week, once per month, once every other two months, or once a quarter (i.e., once every three months) at a dose of about 0.1 mg/kg to about 5.0 mg/kg.

In certain embodiments, the double stranded RNAi agent is administered to the subject once a week. In certain embodiments, the dsRNAi agent is administered to the subject once a month. In certain embodiments, the dsRNAi agent is administered once per quarter (i.e., every three months). In some embodiment, the dsRNAi agent is administered to the subject subcutaneously.

In various embodiments, the methods of the invention further comprise determining the level of IGF-1 in the subject. In certain embodiments, a decrease in the level of expression or activity of the IGF-1 signaling pathway indicates that the IGF system-associated disease is being treated.

In various embodiments, a surrogate marker of IGF-1 expression is measured. In certain embodiments, a change, preferably a clinically relevant change in the surrogate marker indicating effective treatment of diseases associated with an elevated IGF-level are detected, e.g., decreased serum IGF. In the treatment of acromegaly, a clinically relevant change in one or more signs or symptoms associated with acromegaly as provided below can be used as a surrogate marker for a reduction in IGF-1 expression. In the treatment of cancer, a demonstration of stabilization or reduction of tumor burden using RECIST criteria can be used as a surrogate marker for a reduction of IGF-1 expression or activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing various aspects of the IGF-1 signaling pathways.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an Insulin-like Growth Factor Binding Protein, Acid Labile Subunit (IGFALS) or Insulin-like Growth Factor 1 (IGF-1) gene. The gene may be within a cell, e.g., a cell within a subject, such as a human. The use of these iRNAs enables the targeted degradation of mRNAs of the corresponding gene (IGFALS or IGF-1 gene) in mammals.

The iRNAs of the invention have been designed to target a human IGFALS or a human IGF-1 gene, including portions of the gene that are conserved in the IGFALS or IGF-1 othologs of other mammalian species. Without intending to be limited by theory, it is believed that a combination or sub-combination of the foregoing properties and the specific target sites or the specific modifications in these iRNAs confer to the iRNAs of the invention improved efficacy, stability, potency, durability, and safety.

Accordingly, the present invention also provides methods for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of an IGFALS or IGF-1 gene, e.g., an IGF system-associated disease, such as acromegaly or cancer, such as a cancer in which the tumor expresses IGF-1, using iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an IGFALS or an IGF-1 gene.

Very low dosages of the iRNAs of the invention, in particular, can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of expression of the corresponding target gene (IGFALS or IGF-1gene).

The iRNAs of the invention include an RNA strand (the antisense strand) having a region which is about 30 nucleotides or less in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of an IGFALS or IGF-1 gene.

In certain embodiments, the iRNAs of the invention include an RNA strand (the antisense strand) which can include longer lengths, for example up to 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a part of an mRNA transcript of an IGFALS or an IGF-1 gene.

In some embodiments, the iRNA agents for use in the methods of the invention include an RNA strand (the antisense strand) which can be up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a part of an mRNA transcript of an IGFALS or an IGF-1 gene. In some embodiments, such iRNA agents having longer length antisense strands preferably include a second RNA strand (the sense strand) of 20-60 nucleotides in length wherein the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

Using in vitro and in vivo assays, the present inventors have demonstrated that iRNAs targeting an IGFALS gene or an or IGF-1 gene can mediate RNAi, resulting in significant inhibition of expression of IGFALS or IGF-1, as well as reducing signaling through the IGF-1 pathway which will decrease one or more of the symptoms associated with an IGF system-associated disease, such as acromegaly or cancer. Thus, methods and compositions including these iRNAs are useful for treating a subject having an IGF system-associated disease, such as acromegaly or cancer. The methods and compositions herein are useful for reducing the level of IGFALS or IGF-1 in a subject, e.g., serum or liver IGF-1 in a subject, especially in a subject with acromegaly or a tumor, such as an IGF-1 expressing tumor.

The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of an IGFALS gene or an IGF gene as well as compositions, uses, and methods for treating subjects having diseases and disorders that would benefit from reduction of the expression of an IGFALS gene or an IGF gene.

I. Definitions

In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise. For example, “sense strand or antisense strand” is understood as “sense strand or antisense strand or sense strand and antisense strand.”

The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means±10%. In certain embodiments, about means±5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 18 nucleotides of a 21 nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.

As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range.

As used herein, ranges include both the upper and lower limit.

In the event of a conflict between a sequence and its indicated site on a transcript or other sequence, the nucleotide sequence recited in the specification takes precedence.

Various embodiments of the invention can be combined as determined appropriate by one of skill in the art.

As used herein, “insulin-like growth factor binding protein, acid labile subunit” or “IGFALS” is a serum protein that binds insulin-like growth factors, increasing their half-life and their vascular localization. Production of the encoded protein, predominantly in the liver, which contains twenty leucine-rich repeats, is stimulated by growth hormone. Defects in this gene are a cause of acid-labile subunit deficiency, which manifests itself in delayed and slow puberty. Three transcript variants encoding two different isoforms have been found for this gene. The gene can also be known as ALS or ACLSD. Further information on IGFALS is provided, for example in the NCBI Gene database at www.ncbi.nlm.nih.gov/gene/3483 (which is incorporated herein by reference as of the date of filing this application).

As used herein, “insulin-like growth factor binding protein, acid labile subunit,” used interchangeably with the term “IGFALS,” refers to the naturally occurring gene that encodes an IGF-1 binding protein. The amino acid and complete coding sequences of the reference sequence of the human IGFALS gene may be found in, for example, GenBank Accession No. GI: 225579150 (RefSeq Accession No. NM_004970.2; SEQ ID NO:1; SEQ ID NO:2), GenBank Accession No. GI:225579151 (RefSeq Accession No. NM_001146006.1; SEQ ID NO: 9 and 10). Mammalian orthologs of the human IGFALS gene may be found in, for example, GI:142388344 (RefSeq Accession No. NM_008340.3, mouse; SEQ ID NO:3 and SEQ ID NO:4); GI:71896591 (RefSeq Accession No. NM_053329.2, rat; SEQ ID NO:5 and SEQ ID NO:6); GenBank Accession Nos. GI:544514850 (RefSeq Accession No. XM_005590898.1, cynomolgus monkey; SEQ ID NO:7 and SEQ ID NO:8).

A number of naturally occurring SNPs are known and can be found, for example, in the SNP database at the NCBI at www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=3483 (which is incorporated herein by reference as of the date of filing this application) which lists SNPs in human IGFALS. In preferred embodiments, such naturally occurring variants are included within the scope of the IGFALS gene sequence.

Additional examples of IGFALS mRNA sequences are readily available using publicly available databases, e.g., GenBank, UniProt, and OMIM.

“Insulin-like growth factor 1” or “IGF-1”, also known as MGF, encodes a protein similar to insulin in function and structure and is a member of a family of proteins involved in mediating growth and development. The encoded protein is processed from a precursor, bound by a specific receptor, and secreted. Defects in this gene are a cause of insulin-like growth factor I deficiency. Alternative splicing results in multiple transcript variants encoding different isoforms that may undergo similar processing to generate mature protein. Further information on IGF-1 is provided, for example, in the NCBI Gene database at www.ncbi.nlm.nih.gov/gene/3479 (which is incorporated herein by reference as of the date of filing this application).

As used herein, “insulin-like growth factor 1” is used interchangeably with the term “IGF-1” (and optionally any of the other recognized names listed above) refers to the naturally occurring gene that encodes an insulin-like growth factor 1 protein. The amino acid and complete coding sequences of the reference sequence of the human IGF-1 gene, transcript variant 1, mRNA, may be found in, for example, GenBank Accession No. GI: 930588898 (RefSeq Accession No. NM_001111283.2; SEQ ID NO: 11; SEQ ID NO: 12); human IGF-1 gene, transcript variant 4, mRNA, may be found at GenBank Accession No. GI: 930616505 (RefSeq Accession No. NM_000618.4; SEQ ID NO: 13 and SEQ ID NO:14); and human IGF-1, transcript variant 2, mRNA, may be found at GenBank Accession No. GI: 163659900 (RefSeq Accession No. NM_001111284.1; SEQ ID NO: 15 and 16. Mammalian orthologs of the human IGF-1 gene may be found in, for example, GI: 930155588 (RefSeq Accession No. NM_010512.5, mouseIGF-1; SEQ ID NO:17 and SEQ ID NO:18); GI: 126722710 (RefSeq Accession No. NM_001082478.1, rat; SEQ ID NO:19 and SEQ ID NO:20); GenBank Accession Nos. GI: 544472486 (RefSeq Accession No. XM_005572040.1, cynomolgus monkey; SEQ ID NO:21 and SEQ ID NO:22). Multiple sequence variants for each of the species are known.

A number of naturally occurring SNPs are known and can be found, for example, in the SNP database at the NCBI at www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=3479 (which is incorporated herein by reference as of the date of filing this application) which lists SNPs in human IGF-1. In preferred embodiments, such naturally occurring variants are included within the scope of the IGF-1 gene sequence.

Additional examples of IGF-1 mRNA sequences are readily available using publicly available databases, e.g., GenBank, UniProt, and OMIM.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an IGFALS gene or an IGF-1gene, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an IGFALS gene or an IGF-1gene gene. In one embodiment, the target sequence is within the protein coding region of IGFALS or IGF-1.

The target sequence may be from about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In some embodiments, the target sequence is about 19 to about 30 nucleotides in length. In other embodiments, the target sequence is about 19 to about 25 nucleotides in length. In still other embodiments, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments, the target sequence is about 21 to about 23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety (see, e.g., Table 2). The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

The terms “iRNA,” “RNAi agent,” and “iRNA agent,” “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., inhibits, the expression of the target gene, e.g., an IGFALS gene or an IGF-1 gene, in a cell, e.g., a cell within a subject, such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a single stranded RNAi that interacts with a target RNA sequence, e.g., an IGFALS or IGF-1 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into double-stranded short interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes these dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA (ssRNA) (the antisense strand of an siRNA duplex) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., an IGFALS or IGF-1 gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded RNA that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded RNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

In certain embodiments, an “iRNA” for use in the compositions, uses, and methods of the invention is a double stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., an IGFALS gene or an IGF-1 gene. In some embodiments of the invention, a double stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide or a modified nucleotide. In addition, as used in this specification, an “iRNA” may include ribonucleotides with chemical modifications; an iRNA may include substantial modifications at multiple nucleotides.

As used herein, the term “modified nucleotide” refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, or modified nucleobase, or any combination thereof. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents of the invention include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “iRNA” or “RNAi agent” for the purposes of this specification and claims.

The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23 or more unpaired nucleotides. In some embodiments, the hairpin loop can be 10 or fewer nucleotides. In some embodiments, the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 nucleotides.

Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs. In one embodiment of the RNAi agent, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

In certain embodiments, an iRNA agent of the invention is a dsRNA, each strand of which comprises 19-23 nucleotides, that interacts with a target RNA sequence, e.g., an IGFALS gene or an IGF-1 gene. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

In some embodiments, an iRNA of the invention is a dsRNA of 24-30 nucleotides, or possibly even longer, e.g., 25-35, 27-53, or 27-49 nucleotides, that interacts with a target RNA sequence, e.g., an IGFALS target mRNA sequence or an IGF-1 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of a double strainded iRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end, or both ends of either an antisense or sense strand of a dsRNA. In one embodiment of the dsRNA, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

In certain embodiments, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can include extended lengths longer than 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15 nucleotides in length. In certain embodiments, an extended overhang is on the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′ end of the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′ end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′ end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′ end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

“Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the double stranded RNAi agent, i.e., no nucleotide overhang. A “blunt ended” double stranded RNAi agent is double stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule. The RNAi agents of the invention include RNAi agents with no nucleotide overhang at one end (i.e., agents with one overhang and one blunt end) or with no nucleotide overhangs at either end.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., an IGFALS or IGF-1 mRNA. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., an IGFALS or IGF-1 nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotides of the 5′- or 3′-end of the iRNA. In some embodiments, a double stranded RNAi agent of the invention includes a nucleotide mismatch in the antisense strand. In some embodiments, a double stranded RNAi agent of the invention includes a nucleotide mismatch in the sense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, 3, 2, or 1 nucleotides from the 3′-end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, in the 3′-terminal nucleotide of the iRNA.

The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

As used herein, “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.

As used herein, the term “cleavage region” refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3, or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a double stranded RNAi agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding an IGFALS gene or an IGF-1 gene). For example, a polynucleotide is complementary to at least a part of an IGFALS or IGF-1 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding an IGFALS or IGF-1 gene.

Accordingly, in some embodiments, the sense strand polynucleotides and the antisense polynucleotides disclosed herein are fully complementary to the target IGFALS or IGF-1sequence.

In one embodiment, the antisense polynucleotides disclosed herein are fully complementary to the target IGFALS sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target IGFALS sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs:1, 3, 5, 7, or 9, or a fragment of any one of SEQ ID NOs:1, 3, 5, 7, or 9, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target IGFALS sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 3, 5, 6, 8, 12, or 14, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 3, 5, 6, 8, 12, or 14, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target IGFALS sequence and comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 3, 5, 6, 8, 12, or 14, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 3, 5, 6, 8, 12, or 14, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In one embodiment, the antisense polynucleotides disclosed herein are fully complementary to the target IGF-1 sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target IGF-1 sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs:11, 13, 15, 17, 19, or 21, or a fragment of any one of SEQ ID NOs:11, 13, 15, 17, 19, or 21, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target IGF-1 sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 9, 11, 15, 17, 18, or 20, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 9, 11, 15, 17, 18, or 20, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target IGF-1 sequence and comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 9, 11, 15, 17, 18, or 20, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 9, 11, 15, 17, 18, or 20, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In an aspect of the invention, an agent for use in the methods and compositions of the invention is a single-stranded antisense oligonucleotide molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence within the target mRNA. The single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense oligonucleotide molecule may be about 14 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense oligonucleotide molecule may comprise a sequence that is at least 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.

The phrase “contacting a cell with an iRNA,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an iRNA includes contacting a cell in vitro with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly. Thus, for example, the iRNA may be put into physical contact with the cell by the individual performing the method, or alternatively, the iRNA may be put into a situation that will permit or cause it to subsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating the cell with the iRNA. Contacting a cell in vivo may be done, for example, by injecting the iRNA into or near the tissue where the cell is located, or by injecting the iRNA into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the iRNA may contain or be coupled to a ligand, e.g., GalNAc3, that directs the iRNA to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an iRNA and subsequently transplanted into a subject.

In certain embodiments, contacting a cell with an iRNA includes “introducing” or “delivering the iRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusion or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be done by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and US Publication No. 2005/0281781, the entire contents of which are hereby incorporated herein by reference. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or are known in the art.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA is transcribed. LNPs are described in, for example, U.S. Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose) that expresses the target gene, either endogenously or heterologously. It is understood that the sequence of the PHD gene must be sufficiently complementary to the antisense strand of the iRNA agent for the agent to be used in the indicated species. In certain embodiments, the subject is a human, such as a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in an IGFALS gene or an IGF-1 gene expression or replication; a human at risk for a disease, disorder or condition that would benefit from reduction in IGFALS or IGF-1 gene expression; a human having a disease, disorder or condition that would benefit from reduction in IGFALS or IGF-1 gene expression; or human being treated for a disease, disorder or condition that would benefit from reduction in IGFALS or IGF-1 gene expression, as described herein. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human.

As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more symptoms associated with IGFALS or IGF-1 gene expression or IGFALS or IGF-1 protein production, e.g., acromegaly, cancer. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.

The term “lower” or “reduce” in the context of the level of IGFALS or IGF-1 gene expression or IGFALS or IGF-1 protein production in a subject, or a disease marker or symptom refers to a statistically significant decrease in such level. The decrease can be, for example, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the level of detection for the detection method. In certain embodiments, the decrease is down to a level accepted as within the range of normal for an individual without such disorder which can also be referred to as a normalization of a level. For example, lowering cholesterol to 180 mg/dl or lower would be considered to be within the range of normal for a subject. A subject having a cholesterol level of 230 mg/dl with a cholesterol level decreased to 210 mg/dl would have a cholesterol level that was decreased by 40% (230−210/230−180=20/50=40% reduction). In certain embodiments, the reduction is the normalization of the level of a sign or symptom of a disease, a reduction in the difference between the subject level of a sign of the disease and the normal level of the sign for the disease (e.g., the upper level of normal when the level must be reduced to reach a normal level, and the lower level of normal when the level must be increased to reach a normal level). In certain embodiments, the methods include a clinically relevant inhibition of expression of IGFALS or IGF-1, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of IGFALS or IGF-1.

As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder or condition thereof, that would benefit from a reduction in expression of an IGFALS gene or an IGF-1gene or production of an IGFALS or an IGF-1protein, refers to a reduction in the likelihood that a subject will develop a symptom associated with such a disease, disorder, or condition, e.g., a symptom of IGFALS or IGF-1 gene expression, such as the presence of elevated levels of proteins in the IGF signaling pathway, e.g., acromegaly or cancer. The failure to develop a disease, disorder or condition, or the reduction in the development of a symptom or comorbidity associated with such a disease, disorder or condition (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms or disease progression (e.g., delayed cancer progression as determined using RECIST criteria) by days, weeks, months or years is considered effective prevention. Prevention may require the administration of more than one dose.

As used herein, the term “IGF system-associated disease,” used interchangeable with the terms “insulin-like growth factor binding protein, acid labile subunit-associated disease,” “IGFALS-associated disease,” “IGF-associated disease,” or “IGF-1-associated disease” is a disease or disorder that is caused by, or associated with IGFALS or IGF gene expression or IGFALS or IGF protein production. The term “IGF system-associated disease” includes a disease, disorder or condition that would benefit from a decrease in IGFALS or IGF-1 gene expression, replication, or protein activity. Non-limiting examples of IGF system-associated diseases include, for example, acromegaly, gigantism, and cancer, especially metastatic cancer.

In certain embodiments, an IGF system-associated disease-associated disease is acromegaly.

“Therapeutically effective amount,” as used herein, is intended to include the amount of an iRNA that, when administered to a patient for treating a subject having acromealgy, cancer, or IGF system-associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease or its related comorbidities). The “therapeutically effective amount” may vary depending on the iRNA, how it is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, stage of pathological processes mediated by IGFALS or IGF-1 gene expression, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated. Treatment may require the administration of more than one dose.

“Prophylactically effective amount,” as used herein, is intended to include the amount of an iRNA that, when administered to a subject who does not yet experience or display symptoms of acromealgy, cancer, or other IGF system-associated disease-associated diseases, but who may be predisposed to an IGF system-associated disease-associated disease, is sufficient to prevent or delay the development or progression of the disease or one or more symptoms of the disease for a clinically significant period of time. The “prophylactically effective amount” may vary depending on the iRNA, how it is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effective amount” also includes an amount of an iRNA that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. iRNAs employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs, or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes). A “sample derived from a subject” can refer to blood drawn from the subject, or plasma derived therefrom. In certain embodiments when detecting a level of IGF-1, a “sample” preferably refers to a tissue or body fluid from a subject in which IGF-1 is detectable prior to administration of an agent of the invention, e.g., a liver biopsy from a subject with a acromegaly, a tumor. In certain subjects, e.g., healthy subjects, the level of IGF-1 may not be detectable in a number of body fluids, cell types, and tissues.

I. iRNAs of the Invention

The present invention provides iRNAs which inhibit the expression of an IGFALS gene or an IGF-1 gene. In preferred embodiments, the iRNA includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of an IGFALS gene or an IGF-1 gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human having an IGF system-associated disease-associated disease, e.g., acromeagly. The dsRNAi agent includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of an IGFALS gene or an IGF-1 gene. The region of complementarity is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the IGFALS gene or the IGF-1 gene, the iRNA inhibits the expression of the IGFALS gene or the IGF-1 gene (e.g., a human, a primate, a non-primate, or a bird IGFALS gene or IGF-1 gene) by at least 20%, preferably at least 30%, as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, western blotting or flowcytometric techniques. In preferred embodiments, inhibition of expression is determined by the qPCR method provided in the examples. For in vitro assessment of activity, percent inhibition is determined using the methods provided in Example 2 at a single dose at a 10 nM duplex final concentration. For in vivo studies, the level after treatment can be compared to, for example, an appropriate historical control or a pooled population sample control to determine the level of reduction, e.g., when a baseline value is no available for the subject.

A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of an IGFALS gene or IGF-1 gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

Generally, the duplex structure is about 15 to 30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention. Similarly, the region of complementarity to the target sequence is about 15 to 30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

In some embodiments, the dsRNA is about 15 to 23 nucleotides in length, or about 25 to 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well-known in the art that dsRNAs longer than about 21-23 nucleotides in length may serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 9 to about 36 base pairs, e.g., 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target IGFALS or IGF-1 gene expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can have superior inhibitory properties relative to their blunt-ended counterparts. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end, or both ends of an antisense or sense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

Double stranded RNAi compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Similarly, single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.

In an aspect, a dsRNA of the invention for inhibiting the expression of an IGFALS gene includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence. The sense strand is selected from the group of sequences provided in any one of Tables 3, 5, 6, 8, 12, and 14, and the corresponding antisense strand of the sense strand is selected from the group of sequences in any one of Tables 3, 5, 6, 8, 12, and 14. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of an IGFALS gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in any one of Table 3, 5, 6, 8, 12, and 14, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Table 3, 5, 6, 8, 12, and 14. In certain embodiments, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In other embodiments, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

In an aspect, a dsRNA of the invention for inhibiting the expression of an IGF-1 gene includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence. The sense strand is selected from the group of sequences provided in any one of Tables 9, 11, 15, 17, 18, and 20, and the corresponding antisense strand of the sense strand is selected from the group of sequences in any one of Tables 9, 11, 15, 17, 18, and 20. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of an IGF-1 gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in any one of Table 9, 11, 15, 17, 18, and 20, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Table 9, 11, 15, 17, 18, and 20. In certain embodiments, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In other embodiments, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

It will be understood that, although the sequences in Tables 3, 6, 9, 12, 15, and 18 are not described as modified or conjugated sequences, the RNA of the iRNA of the invention e.g., a dsRNA of the invention, may comprise any one of the sequences set forth in any one of Tables 3, 6, 9, 12, 15, and 18, or the sequences of any one of Tables 5, 8, 11, 14, 17, and 20 that are modified, or the sequences of any one of Tables 5, 8, 11, 14, 17, and 20 that are conjugated to a ligand. In other words, the invention encompasses dsRNAs of any one of Tables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, and 20 which are un-modified, un-conjugated, modified, or conjugated, as described herein.

The skilled person is well aware that dsRNAs having a duplex structure of between about 20 and 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in any one of Tables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, and 20, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes having one of the sequences of Tables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, and 20 minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences of Tables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, and 20, and differing in their ability to inhibit the expression of an IGFALS gene or an IGF-1 gene by not more than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated to be within the scope of the present invention.

In addition, the RNAs provided in Tables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, and 20 identify a site(s) in an IGFALS transcript or IGF-1 transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least about 15 contiguous nucleotides from one of the sequences provided in Tables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, and 20 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in an IGFALS gene or an IGF-1 gene.

While a target sequence is generally about 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art or provided herein) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, in Tables 3 and 5 represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., in Tables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, and 20, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch is not located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, for a 23 nucleotide iRNA agent the strand which is complementary to a region of an IGFALS gene or an IGF-1 gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of an IGFALS gene or IGF-1 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of an IGFALS gene or an IGF-1 gene is important, especially if the particular region of complementarity in an IGFALS gene or an IGF-1 gene is known to have polymorphic sequence variation within the population.

II. Modified iRNAs of the Invention

In certain embodiments, the RNA of the iRNA of the invention e.g., a dsRNA, is unmodified, and does not comprise, e.g., chemical modifications or conjugations known in the art and described herein. In other embodiments, the RNA of an iRNA of the invention, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of an iRNA or substantially all of the nucleotides of an iRNA are modified, i.e., not more than 5, 4, 3, 2, or 1 unmodified nucleotides are present in a strand of the iRNA.

The nucleic acids featured in the invention can be synthesized or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified iRNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

Representative US Patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH₂component parts.

Representative US Patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

Suitable RNA mimetics are contemplated for use in iRNAs provided herein, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound in which an RNA mimetic that has been shown to have excellent hybridization properties is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative US patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the invention are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂-4 wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′-position: OH; F; O-, S-, or N-alkyl; 0, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C₁to C₁₀alkyl or C₂to C₁₀alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_nO]_mCH₃, O(CH₂)._nOCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)_nON[(CH₂)_nCH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁to C₁₀lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂. Further exemplary modifications include: 5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides, 5′-Me-2′-deoxynucleotides, (both R and S isomers in these three families); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F) Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative US patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as deoxy-thymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative US Patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

In some embodiments, the iRNA of the invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomer with bonds between C1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated by reference).

The RNA of an iRNA can also be modified to include one or more bicyclic sugar moities. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. Thus, in some embodiments an agent of the invention may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′ bridge. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the invention include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the invention include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH₂—O—N(CH₃)-2′ (see, e.g., US Patent Publication No. 2004/0171570); 4′-CH₂—N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference.

Additional representative US Patents and US Patent Publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or more constrained ethyl nucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge. In one embodiment, a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.”

An iRNA of the invention may also include one or more “conformationally restricted nucleotides” (“CRN”). CRN are nucleotide analogs with a linker connecting the C2′ and C4′ carbons of ribose or the C3 and -C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, US Patent Publication No. 2013/0190383; and PCT publication WO 2013/036868, the entire contents of each of which are hereby incorporated herein by reference.

Representative US publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference. Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.

Other modifications of the nucleotides of an iRNA of the invention include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic on the antisense strand of an iRNA. Suitable phosphate mimics are disclosed in, for example US Patent Publication No. 2012/0157511, the entire contents of which are incorporated herein by reference.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double stranded RNAi agents of the invention include agents with chemical modifications as disclosed, for example, in WO2013/075035, the entire contents of each of which are incorporated herein by reference. WO2013/075035 provides motifs of three identical modifications on three consecutive nucleotides into a sense strand or antisense strand of a dsRNAi agent, particularly at or near the cleavage site. In some embodiments, the sense strand and antisense strand of the dsRNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense or antisense strand. The dsRNAi agent may be optionally conjugated with a GalNAc derivative ligand, for instance on the sense strand.

More specifically, when the sense strand and antisense strand of the double stranded RNAi agent are completely modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of a dsRNAi agent, the gene silencing activity of the dsRNAi agent was observed.

Accordingly, the invention provides double stranded RNAi agents capable of inhibiting the expression of a target gene (i.e., IGFALS or IGF-1 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be, independently, 12-30 nucleotides in length. For example, each strand may independently be 14-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex double stranded RNA (“dsRNA”), also referred to herein as “dsRNAi agent.” The duplex region of a dsRNAi agent may be 12-30 nucleotide pairs in length. For example, the duplex region can be 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In certain embodiments, the sense and antisense strands may be even longer. For example, in certain embodiments, the sense strand and the antisense strand are independently 25-35 nucleotides in length. In certain embodiments, each the sense and the antisense strand are independently 27-53 nucleotides in length, e.g., 27-49, 31-49, 33-49, 35-49, 37-49, and 39-49 nucleotides in length. In certain embodiments, the dsRNAi agent may contain one or more overhang regions or capping groups at the 3′-end, 5′-end, or both ends of one or both strands. The overhang can be, independently, 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. In certain embodiments, at least one strand of the dsRNAi agent comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the dsRNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the dsRNAi agent comprise an overhang of at least 1 nucleotide.

In certain embodiments, the overhang regions can include extended overhang regions as provided above. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In certain embodiments, the nucleotides in the overhang region of the dsRNAi agent can each independently be a modified or unmodified nucleotide including, but no limited to 2′-sugar modified, such as, 2′-F, 2′-O-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand, or both strands of the dsRNAi agent may be phosphorylated. hi some embodiments, the overhang region(s) contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In some embodiments, the overhang is present at the 3′-end of the sense strand, antisense strand, or both strands. In some embodiments, this 3′-overhang is present in the antisense strand. In some embodiments, this 3′-overhang is present in the sense strand.

The dsRNAi agent may contain only a single overhang, which can strengthen the interference activity of the RNAi, without affecting its overall stability. For example, the single-stranded overhang may be located at the 3′-end of the sense strand or, alternatively, at the 3′-end of the antisense strand. The RNAi may also have a blunt end, located at the 5′-end of the antisense strand (or the 3′-end of the sense strand) or vice versa. Generally, the antisense strand of the dsRNAi agent has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. While not wishing to be bound by theory, the asymmetric blunt end at the 5′-end of the antisense strand and 3′-end overhang of the antisense strand favor the guide strand loading into RISC process.

In certain embodiments, the dsRNAi agent is a double ended bluntmer of 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 7, 8, 9 from the 5′ end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′ end.

In other embodiments, the dsRNAi agent is a double ended bluntmer of 20 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 8, 9, 10 from the 5′ end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′ end.

In yet other embodiments, the dsRNAi agent is a double ended bluntmer of 21 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′ end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′ end.

In certain embodiments, the dsRNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′ end; the antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′ end, wherein one end of the RNAi agent is blunt, while the other end comprises a 2 nucleotide overhang. Preferably, the 2 nucleotide overhang is at the 3′-end of the antisense strand.

When the 2 nucleotide overhang is at the 3′-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand. In certain embodiments, every nucleotide in the sense strand and the antisense strand of the dsRNAi agent, including the nucleotides that are part of the motifs are modified nucleotides. In certain embodiments each residue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g., in an alternating motif. Optionally, the dsRNAi agent further comprises a ligand (preferably GalNAc₃).

In certain embodiments, the dsRNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3 ‘ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3’ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

In certain embodiments, the dsRNAi agent comprises sense and antisense strands, wherein the dsRNAi agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at position 11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand and the 5′ end of the second strand form a blunt end and the second strand is 1-4 nucleotides longer at its 3′ end than the first strand, wherein the duplex region region which is at least 25 nucleotides in length, and the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotide of the second strand length to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, and wherein Dicer cleavage of the dsRNAi agent preferentially results in an siRNA comprising the 3′-end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the dsRNAi agent further comprises a ligand.

In certain embodiments, the sense strand of the dsRNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.

In certain embodiments, the antisense strand of the dsRNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand.

For a dsRNAi agent having a duplex region of 17-23 nucleotides in length, the cleavage site of the antisense strand is typically around the 10, 11, and 12 positions from the 5′-end. Thus the motifs of three identical modifications may occur at the 9, 10, 11 positions; the 10, 11, 12 positions; the 11, 12, 13 positions; the 12, 13, 14 positions; or the 13, 14, 15 positions of the antisense strand, the count starting from the first nucleotide from the 5′-end of the antisense strand, or, the count starting from the first paired nucleotide within the duplex region from the 5′-end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the dsRNAi agent from the 5′-end.

The sense strand of the dsRNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

In some embodiments, the sense strand of the dsRNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand and the other motifs may be a wing modification. The term “wing modification” herein refers to a motif occurring at another portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other then the chemistries of the motifs are distinct from each other, and when the motifs are separated by one or more nucleotide than the chemistries can be the same or different. Two or more wing modifications may be present. For instance, when two wing modifications are present, each wing modification may occur at one end relative to the first motif which is at or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the dsRNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that may be present on the sense strand.

In some embodiments, the wing modification on the sense strand or antisense strand of the dsRNAi agent typically does not include the first one or two terminal nucleotides at the 3′-end, 5′-end, or both ends of the strand.

In other embodiments, the wing modification on the sense strand or antisense strand of the dsRNAi agent typically does not include the first one or two paired nucleotides within the duplex region at the 3′-end, 5′-end, or both ends of the strand.

When the sense strand and the antisense strand of the dsRNAi agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex region, and have an overlap of one, two, or three nucleotides.

When the sense strand and the antisense strand of the dsRNAi agent each contain at least two wing modifications, the sense strand and the antisense strand can be so aligned that two modifications each from one strand fall on one end of the duplex region, having an overlap of one, two, or three nucleotides; two modifications each from one strand fall on the other end of the duplex region, having an overlap of one, two or three nucleotides; two modifications one strand fall on each side of the lead motif, having an overlap of one, two or three nucleotides in the duplex region.

In some embodiments, every nucleotide in the sense strand and antisense strand of the dsRNAi agent, including the nucleotides that are part of the motifs, may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2′-hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking 0 of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′- or 5′-terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of a dsRNAi agent or may only occur in a single strand region of a dsRNAi agent. For example, a phosphorothioate modification at a non-linking O position may only occur at one or both ends, may only occur in a terminal region, e.g., at a position on a terminal nucleotide, or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at the ends. The 5′-end or ends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′- or 3′-overhang, or in both. For example, it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′- or 5′-overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications.

Overhangs need not be homologous with the target sequence.

In some embodiments, each residue of the sense strand and antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, 2′-hydroxyl, or 2′-fluoro. The strands can contain more than one modification. In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro.

At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-O-methyl or 2′-fluoro modifications, or others.

In certain embodiments, the N_aor N_bcomprise modifications of an alternating pattern. The term “alternating motif” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.

The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.

In some embodiments, the dsRNAi agent of the invention comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′ to 3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 5′ to 3′ of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′ to 3′ of the strand and the alternating motif in the antisenese strand may start with “BBAABBAA” from 5′ to 3′ of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

In some embodiments, the dsRNAi agent comprises the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the sense strand initially has a shift relative to the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the antisense strand initially, i.e., the 2′-O-methyl modified nucleotide on the sense strand base pairs with a 2′-F modified nucleotide on the antisense strand and vice versa. The 1 position of the sense strand may start with the 2′-F modification, and the 1 position of the antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modifications on three consecutive nucleotides to the sense strand or antisense strand interrupts the initial modification pattern present in the sense strand or antisense strand. This interruption of the modification pattern of the sense or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides to the sense or antisense strand may enhance the gene silencing activity against the target gene.

In some embodiments, when the motif of three identical modifications on three consecutive nucleotides is introduced to any of the strands, the modification of the nucleotide next to the motif is a different modification than the modification of the motif. For example, the portion of the sequence containing the motif is “ . . . N_aYYYN_b. . . ,” where “Y” represents the modification of the motif of three identical modifications on three consecutive nucleotide, and “N_a” and “N_b” represent a modification to the nucleotide next to the motif “YYY” that is different than the modification of Y, and where N_aand N_bcan be the same or different modifications. Alternatively, N_aor N_bmay be present or absent when there is a wing modification present.

The iRNA may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand, antisense strand, or both strands in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand. In one embodiment, a double-stranded RNAi agent comprises 6-8phosphorothioate internucleotide linkages. In some embodiments, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-end and two phosphorothioate internucleotide linkages at the 3′-end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5′-end or the 3′-end.

In some embodiments, the dsRNAi agent comprises a phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region may contain two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within the duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. These terminal three nucleotides may be at the 3′-end of the antisense strand, the 3′-end of the sense strand, the 5′-end of the antisense strand, or the 5′ end of the antisense strand.

In some embodiments, the 2-nucleotide overhang is at the 3′-end of the antisense strand, and there are two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. Optionally, the dsRNAi agent may additionally have two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand.

In one embodiment, the dsRNAi agent comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mistmatch may occur in the overhang region or the duplex region. The base pair may be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

In certain embodiments, the dsRNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand independently selected from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.

In certain embodiments, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.

In other embodiments, the nucleotide at the 3′-end of the sense strand is deoxy-thymine (dT) or the nucleotide at the 3′-end of the antisense strand is deoxy-thymine (dT). For example, there is a short sequence of deoxy-thymine nucleotides, for example, two dT nucleotides on the 3′-end of the sense, antisense strand, or both strands.

In certain embodiments, the sense strand sequence may be represented by formula (I):

5′n_p-N_a—(XXX)_i—N_b—YYY—N_b—(ZZZ)_j—N_a-n_q3′ (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_aindependently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_bindependently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_pand n_qindependently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY, and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably YYY is all 2′-F modified nucleotides.

In some embodiments, the N_aor N_bcomprises modifications of alternating pattern.

In some embodiments, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the dsRNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13) of the sense strand, the count starting from the first nucleotide, from the 5′-end; or optionally, the count starting at the first paired nucleotide within the duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas:

5′n_p-N_a—YYY—N_b—ZZZ—N_a-n_q3′ (Ib);

5′n_p-N_a—XXX—N_b—YYY—N_a-n_q3′ (Ic); or

5′n_p-N_a—XXX—N_b—YYY—N_b—ZZZ—N_a-n_q3′ (Id).

When the sense strand is represented by formula (Ib), N_brepresents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_aindependently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. When the sense strand is represented as formula (Ic), N_brepresents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_acan independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Id), each N_bindependently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Preferably, N_bis 0, 1, 2, 3, 4, 5, or 6 Each N_acan independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula:

5′n_p-N_a—YYY—N_a-n_q3′ (Ia).

When the sense strand is represented by formula (Ia), each N_aindependently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may be represented by formula (II):

5′n_q′-N_a′-(Z′Z′Z′)_k—N_b′—Y′Y′Y′—N_b′-(X′X′X′)_l-N′_a-n_p′3′ (II)

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_a′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_b′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_p′ and n_q′ independently represent an overhang nucleotide;

wherein N_b′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In some embodiments, the N_a′ or N_b′ comprises modifications of alternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the dsRNAi agent has a duplex region of 17-23 nucleotides in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the first nucleotide, from the 5′-end; or optionally, the count starting at the first paired nucleotide within the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, 13.

In certain embodiments, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In certain embodiments, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1. The antisense strand can therefore be represented by the following formulas:

5′n_q′-N_a′-Z′Z′Z′—N_b′—Y′Y′Y′—N_a′-n_p′3′ (IIb);

5′n_q′-N_a′-Y′Y′Y′—N_b′-X′X′X′-n_p′3′ (IIc); or

5′n_q′-N_a′-Z′Z′Z′—N_b′—Y′Y′Y′—N_b′-X′X′X′—N_a′-n_p′3′ (IId).

When the antisense strand is represented by formula (IIb), N_b′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IIc), N_b′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IId), each N_b′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N_bis 0, 1, 2, 3, 4, 5, or 6.

In other embodiments, k is 0 and l is 0 and the antisense strand may be represented by the formula:

5′n_p′-N_a′—Y′Y′Y′—N_a′-n_q′3′ (Ia).

When the antisense strand is represented as formula (IIa), each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′, and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.

In some embodiments, the sense strand of the dsRNAi agent may contain YYY motif occurring at 9, 10, and 11 positions of the strand when the duplex region is 21 nt, the count starting from the first nucleotide from the 5′-end, or optionally, the count starting at the first paired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.

In some embodiments the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the first nucleotide from the 5′-end, or optionally, the count starting at the first paired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with a antisense strand being represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively.

Accordingly, the dsRNAi agents for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the iRNA duplex represented by formula (III):

sense: 5′n_p-N_a—(XXX)_i—N_b—YYY—N_b—(ZZZ)_j—N_a-n_q3′

antisense: 3′n_p′-N_a′—(X′X′X′)_k-N_b′—Y′Y′Y′—N_b′—(Z′Z′Z′)_l-N_a′-n_q′5′ (III)

wherein:

j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein each n_p′, n_p, n_q′, and n_q, each of which may or may not be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forming an iRNA duplex include the formulas below:

5′n_p-N_a—YYY—N_a-n_q3′

3′n_p′-N_a′-Y′Y′Y′—N_a′n_q′5′ (IIIa)

5′n_p-N_a—YYY—N_b—ZZZ—N_a-n_q3′

3′n_p′-N_a′-Y′Y′Y′—N_b′-Z′Z′Z′—N_a′n_q′5′ (IIIb)

5′n_p-N_a—XXX—N_b—YYY—N_a-n_q3′

3′n_p′-N_a′-X′X′X′—N_b′—Y′Y′Y′—N_a′-n_q′5′ (IIIc)

5′n_p-N_a—XXX—N_b—YYY—N_b—ZZZ—N_a-n_q3′

3′n_p′-N_a′-X′X′X′—N_b′—Y′Y′Y′—N_b′-Z′Z′Z′—N_a-n_q′5′ (IIId)

When the dsRNAi agent is represented by formula (IIIa), each N_aindependently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented by formula (IIIb), each N_bindependently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5, or 1-4 modified nucleotides. Each N_aindependently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented as formula (IIIc), each N_b, N_b′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_aindependently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented as formula (IIId), each N_b, N_b′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_a, N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_a, N_a′, N_b, and N_b′ independently comprises modifications of alternating pattern.

Each of X, Y, and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same or different from each other.

When the dsRNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides may form a base pair with one of the Y′ nucleotides. Alternatively, at least two of the Y nucleotides form base pairs with the corresponding Y′ nucleotides; or all three of the Y nucleotides all form base pairs with the corresponding Y′ nucleotides.

When the dsRNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may form a base pair with one of the Z′ nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding T nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding T nucleotides.

When the dsRNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides may form a base pair with one of the X′ nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X′ nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X′ nucleotides.

In certain embodiments, the modification on the Y nucleotide is different than the modification on the Y′ nucleotide, the modification on the Z nucleotide is different than the modification on the Z′ nucleotide, and/or the modification on the X nucleotide is different than the modification on the X′ nucleotide.

In certain embodiments, when the dsRNAi agent is represented by formula (IIId), the N_amodifications are 2′-O-methyl or 2′-fluoro modifications. In other embodiments, when the RNAi agent is represented by formula (IIId), the N_amodifications are 2′-O-methyl or 2′-fluoro modifications and n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In yet other embodiments, when the RNAi agent is represented by formula (IIId), the N_amodifications are 2′-O-methyl or 2′-fluoro modifications, n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker (described below). In other embodiments, when the RNAi agent is represented by formula (IIId), the N_amodifications are 2′-O-methyl or 2′-fluoro modifications, n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In some embodiments, when the dsRNAi agent is represented by formula (IIIa), the N_amodifications are 2′-O-methyl or 2′-fluoro modifications, n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In some embodiments, the dsRNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In some embodiments, the dsRNAi agent is a multimer containing three, four, five, six, or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In one embodiment, two dsRNAi agents represented by at least one of formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, and one or both of the 3′ ends, and are optionally conjugated to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.

Various publications describe multimeric iRNAs that can be used in the methods of the invention. Such publications include U.S. Pat. No. 7,858,769, WO2007/091269, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520 the entire contents of each of which are hereby incorporated herein by reference.

As described in more detail below, the iRNA that contains conjugations of one or more carbohydrate moieties to an iRNA can optimize one or more properties of the iRNA. In many cases, the carbohydrate moiety will be attached to a modified subunit of the iRNA. For example, the ribose sugar of one or more ribonucleotide subunits of a iRNA can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

The iRNA may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is a serinol backbone or diethanolamine backbone.

In certain embodiments, the iRNA for use in the methods of the invention for inhibiting the expression of an IGFALS gene is an agent selected from the agents listed in any one of Tables 3, 5, 6, 8, 12, and 14. These agents may further comprise a ligand. These agents may further comprise a ligand.

In certain embodiments, the iRNA for use in the methods of the invention for inhibiting the expression of an IGF-1 gene is an agent selected from the agents listed in any one of Tables 9, 11, 15, 17, 18, and 20. These agents may further comprise a ligand.

III. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involves chemically linking to the iRNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the iRNA e.g., into a cell. For example, the ligand can be attached to the sense strand, antisense strand or both strands, at the 3′-end, 5′-end or both ends. For instance, the ligand may be conjugated to the sense strand. In preferred embodiments, the ligand is conjugated to the 3′-end of the sense strand. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060). In certain embodiments, the modification can include a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In certain embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands do not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, monovalent or multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucoseamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic. In certain embodiments, ligands include monovalent or multivalent galactose. In certain embodiments, ligands include cholesterol.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated iRNAs of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

In the ligand-conjugated iRNAs and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

A. Lipid Conjugates

In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In certain embodiments, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In other embodiments, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of, or in addition to, the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:24). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:25) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:26) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:27) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, an iRNA further comprises a carbohydrate. The carbohydrate conjugated iRNA is advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri-, and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In certain embodiments, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide.

In other embodiments, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group:

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In certain embodiments, the ligand is an N-acetylgalactosamine (GalNAc) or GalNAc derivative. In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker.

In one embodiment, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent. In another embodiment, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.

In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex.

The GalNAc or GalNAc derivative may be conjugated to the 3′ end of the sense strand of the double stranded RNAi agent, the 5′ end of the sense strand of the double stranded RNAi agent, the 3′ end of the antisense strand of the double stranded RNAi agent, or the 5′ end of the antisense strand of the double stranded RNAi agent. In certain embodiments, the monosaccharide is an N-acetylgalactosamine, such as

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Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

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when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell permeation peptide.

Additional carbohydrate conjugates suitable for use in the present invention include those described in PCT Publication Nos. WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic, or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms. A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential, or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

i. Redox Cleavable Linking Groups

In certain embodiments, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In other embodiments, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S-. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—, —S—P(O)(H)—S—, and —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

iii. Acid Cleavable Linking Groups

In other embodiments, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

iv. Ester-Based Linking Groups

In other embodiments, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

v. Peptide-Based Cleaving Groups

In yet other embodiments, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH-). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

In some embodiments, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,

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when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention, a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In certain embodiments, a dsRNA of the invention is conjugated to a monovalent, a bivalent or a trivalent branched linker selected from the group of structures shown in any of formula (XXXII)-(XXXV):

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wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;

P^2A, P^2B, P^3A, P^3B, P^4A, P^4B, P^5A, P^5B, P^5C, T^2A, T^2B, T^3A, T^3B, T^4A, T^4B, T^4A, T^5B, T^5Care each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH, or CH₂O;

Q^2A, Q^2B, Q^3A, Q^3B, Q^4A, Q^4B, Q^5A, Q^5B, Q^5Care independently for each occurrence absent, alkylene, substituted alkylene wherin one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^N), C(R′)═C(R″), C≡C, or C(O);

R^2A, R^2B, R^3A, R^3B, R^4A, R^4B, R^5A, R^5B, R^5Care each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^a)C(O), —C(O)—CH(R^a)—NH—, CO, CH═N-O,

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or heterocyclyl;

L^2A, L^2B, L^3A, L^3B, L^4A, L^4B, L^5A, L^5B, and L^5Crepresent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R^ais H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):

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- wherein L^5A, L^5Band L^5Crepresent a monosaccharide, such as GalNAc derivative.

Examples of suitable monovalent, bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.

Representative US Patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAi agents, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

IV. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a disease, disorder, or condition associated with IGFALS gene or IGF-1 gene expression) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when a dsRNAi agent is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J, et al (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J., et al (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O, et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H, et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R, et al (2003) J. Mol. Biol 327:761-766; Verma, U N, et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N, et al (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S, et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y, et al (2005) Cancer Gene Ther. 12:321-328; Pal, A, et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E, et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A, et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

A. Vector encoded iRNAs of the Invention

iRNA targeting an IGFALS gene or an IGF-1 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A, et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are known in the art.

V. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the iRNA are useful for treating a disease or disorder associated with the expression or activity of an IGFALS gene or an IGF-1 gene. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery.

The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of an IGFALS gene or an IGF-1 gene. In general, a suitable dose of an iRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. Typically, a suitable dose of an iRNA of the invention will be in the range of about 0.1 mg/kg to about 5.0 mg/kg, preferably about 0.3 mg/kg and about 3.0 mg/kg. A repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as every other day or once a year. In certain embodiments, the iRNA is administered about once per month to about once per quarter (i.e., about once every three months).

After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration weekly or biweekly for three months, administration can be repeated once per month, for six months, or a year; or longer.

The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

In other embodiments, a single dose of the pharmaceutical compositions can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals. In some embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered once per week. In other embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered bi-monthly.

The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as known in the art. For example, a mouse model of acromegaly was developed by Kovacs et al. (1997, Endocrinology) the entire contents of which are incorporated herein by reference. Bovine growth hormone transgenic mice also exhibit features of acromegaly (Palmiter et al., Science (1983), Olsson et al., Am J Phys Endo Metab (2003), Berryman et al, GH and IGF Res (2004), Izzard et al., GH and IGF Res (2009), Blutke et al., Mol and Cell Endo (2014)). Multiple animal models of cancer are known in the art.

The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular administration.

The iRNA can be delivered in a manner to target a particular tissue (e.g., liver cells).

Pharmaceutical compositions and formulations for topical or transdermal administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C_1-20alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof). Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate RNA interference. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types.

A liposome containing an iRNA agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNA agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the iRNA agent and condense around the iRNA agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of iRNA agent.

If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Feigner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging iRNA agent preparations into liposomes.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleic acids rather than complex with it. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of two or more of phospholipid, phosphatidylcholine, and cholesterol.

Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185 and 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Feigner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_M1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_M1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_m′or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

In some embodiments, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver iRNA agents to macrophages.

Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated iRNAs in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size, and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of iRNA agent (see, e.g., Feigner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer iRNA agent into the skin. In some implementations, liposomes are used for delivering iRNA agent to epidermal cells and also to enhance the penetration of iRNA agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with iRNA agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include iRNAs can be delivered, for example, subcutaneously by infection in order to deliver iRNAs to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described in WO/2008/042973.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in “Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided as micellar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of iRNA, an alkali metal C₈to C₂₂alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which contains the RNAi and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the RNAi, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

Phenol or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAi agents of the invention may be fully encapsulated in a lipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipid particle. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). LNPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; US Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.C1), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.C1), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

In some embodiments, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles.

In some embodiments, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid can be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or a PEG-distearyloxypropyl (Ci₈). The conjugated lipid that prevents aggregation of particles can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see US20090023673, which is incorporated herein by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

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LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are described in Table 1.

TABLE 1

Exemplary lipid formulations

cationic lipid/non-cationic lipid/

cholesterol/PEG-lipid conjugate

Ionizable/Cationic Lipid
Lipid:siRNA ratio

SNALP-1
1,2-Dilinolenyloxy-N,N-
DLinDMA/DPPC/Cholesterol/PEG-cDMA

dimethylaminopropane (DLinDMA)
(57.1/7.1/34.4/1.4)

lipid:siRNA ~7:1

2-XTC
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DPPC/Cholesterol/PEG-cDMA

[1,3]-dioxolane (XTC)
57.1/7.1/34.4/1.4

lipid:siRNA ~7:1

LNP05
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG

[1,3]-dioxolane (XTC)
57.5/7.5/31.5/3.5

lipid:siRNA ~6:1

LNP06
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG

[1,3]-dioxolane (XTC)
57.5/7.5/31.5/3.5

lipid:siRNA ~11:1

LNP07
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG

[1,3]-dioxolane (XTC)
60/7.5/31/1.5,

lipid:siRNA ~6:1

LNP08
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG

[1,3]-dioxolane (XTC)
60/7.5/31/1.5,

lipid:siRNA ~11:1

LNP09
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG

[1,3]-dioxolane (XTC)
50/10/38.5/1.5

Lipid:siRNA 10:1

LNP10
(3aR,5s,6aS)-N,N-dimethyl-2,2-
ALN100/DSPC/Cholesterol/PEG-DMG

di((9Z,12Z)-octadeca-9,12-
50/10/38.5/1.5

dienyl)tetrahydro-3aH-
Lipid:siRNA 10:1

cyclopenta[d][1,3]dioxol-5-amine

(ALN100)

LNP11
(6Z,9Z,28Z,31Z)-heptatriaconta-
MC-3/DSPC/Cholesterol/PEG-DMG

6,9,28,31-tetraen-19-yl 4-
50/10/38.5/1.5

(dimethylamino)butanoate (MC3)
Lipid:siRNA 10:1

LNP12
1,1′-(2-(4-(2-((2-(bis(2-
Tech G1/DSPC/Cholesterol/PEG-DMG

hydroxydodecyl)amino)ethyl)(2-
50/10/38.5/1.5

hydroxydodecyl)amino)ethyl)piperazin-
Lipid:siRNA 10:1

1-yl)ethylazanediyl)didodecan-2-ol

(Tech G1)

LNP13
XTC
XTC/DSPC/Chol/PEG-DMG

50/10/38.5/1.5

Lipid:siRNA: 33:1

LNP14
MC3
MC3/DSPC/Chol/PEG-DMG

40/15/40/5

Lipid:siRNA: 11:1

LNP15
MC3
MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG

50/10/35/4.5/0.5

Lipid:siRNA: 11:1

LNP16
MC3
MC3/DSPC/Chol/PEG-DMG

50/10/38.5/1.5

Lipid:siRNA: 7:1

LNP17
MC3
MC3/DSPC/Chol/PEG-DSG

50/10/38.5/1.5

Lipid:siRNA: 10:1

LNP18
MC3
MC3/DSPC/Chol/PEG-DMG

50/10/38.5/1.5

Lipid:siRNA: 12:1

LNP19
MC3
MC3/DSPC/Chol/PEG-DMG

50/10/35/5

Lipid:siRNA: 8:1

LNP20
MC3
MC3/DSPC/Chol/PEG-DPG

50/10/38.5/1.5

Lipid:siRNA: 10:1

LNP21
C12-200
C12-200/DSPC/Chol/PEG-DSG

50/10/38.5/1.5

Lipid:siRNA: 7:1

LNP22
XTC
XTC/DSPC/Chol/PEG-DSG

50/10/38.5/1.5

Lipid:siRNA: 10:1

DSPC: distearoylphosphatidylcholine

DPPC: dipalmitoylphosphatidylcholine

PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000)

PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000)

PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)

SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.

XTC comprising formulations are described, e.g., in International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which is hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in US Patent Publication No. 2010/0324120, filed Jun. 10, 2010, the entire contents of which are hereby incorporated by reference.

ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.

C12-200 comprising formulations are described in WO2010/129709, which is hereby incorporated by reference.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions, or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids or esters or salts thereof, bile acids or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG), and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses, and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular, or intrahepatic administration can include sterile aqueous solutions which can also contain buffers, diluents, and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Formulations include those that target the liver when treating hepatic disorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol, or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The iRNAs of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution either in the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic, and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin, and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate, and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral, and parenteral routes, and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins, and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the iRNAs are formulated as microemulsions. A microemulsion can be defined as a system of water, oil, and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij® 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex® 300, Captex® 355, Capmul® MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.

Microemulsions of the present invention can also contain additional components and additives such as sorbitan monostearate (Grill® 3), Labrasol®, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

iii. Microparticles

An iRNA of the invention may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Such compounds are well known in the art.

v. Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

vii. Other Components

The compositions of the present invention can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol, or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA and (b) one or more agents which function by a non-iRNA mechanism and which are useful in treating an IGFALS or IGF-1-associated disorder.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by IGFALS or IGF-1 expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

VI. Methods of the Invention

The present invention also provides methods of inhibiting expression of an IGFALS gene or IGF-1 gene in a cell. The methods include contacting a cell with an RNAi agent, e.g., double stranded RNAi agent, in an amount effective to inhibit expression of IGFALS or IGF-1 in the cell, thereby inhibiting expression of IGFALS or IGF-1 in the cell.

Contacting of a cell with an iRNA, e.g., a double stranded RNAi agent, may be done in vitro or in vivo. Contacting a cell in vivo with the iRNA includes contacting a cell or group of cells within a subject, e.g., a human subject, with the iRNA. Combinations of in vitro and in vivo methods of contacting a cell are also possible. Contacting a cell may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In preferred embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc₃ligand, or any other ligand that directs the RNAi agent to a site of interest.

The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating”, “suppressing”, and other similar terms, and includes any level of inhibition.

The phrase “inhibiting expression of an IGFALS or “inhibiting expression of an IGF-1” is intended to refer to inhibition of expression of any IGFALS gene or IGF-1 gene (such as, e.g., a mouse IGFALS gene or IGF-1 gene, a rat IGFALS gene or IGF-1 gene, a monkey IGFALS gene or IGF-1 gene, or a human IGFALS gene or IGF-1 gene) as well as variants or mutants of an IGFALS gene or IGF-1 gene. Thus, the IGFALS gene or IGF-1 gene may be a wild-type IGFALS gene or IGF-1 gene, a mutant IGFALS gene or IGF-1 gene (such as a mutant IGFALS gene or IGF-1 gene), or a transgenic IGFALS gene or IGF-1 gene in the context of a genetically manipulated cell, group of cells, or organism.

“Inhibiting expression of an IGFALS gene” or “inhibiting expression of an IGF-1 gene” includes any level of inhibition of an IGFALS gene or an IGF-1 gene, e.g., at least partial suppression of the expression of an IGFALS gene or an IGF-1 gene. The expression of the IGFALS gene or an IGF-1 gene may be assessed based on the level, or the change in the level, of any variable associated with IGFALS gene or an IGF-1 gene expression, e.g., IGFALS mRNA or IGF-1 mRNA level or an IGFALS protein level or an IGF-1 protein level. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject.

Inhibition may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with IGFALS or IGF-1 expression compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

In some embodiments of the methods of the invention, expression of an IGFALS or IGF-1 gene is inhibited by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay. In some embodiments, the inhibition of expression of an IGFALS gene or an IGF-1 gene results in normalization of the level of IGF-1 such that the difference between the level before treatment and a normal control level is reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

Inhibition of the expression of an IGFALS gene or an IGF-1 gene may be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which an IGFALS gene or an IGF-1 gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with an iRNA of the invention, or by administering an iRNA of the invention to a subject in which the cells are or were present) such that the expression of an IGFALS gene or an IGF-1 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s) not treated with an iRNA or not treated with an iRNA targeted to the gene of interest). In preferred embodiments, the inhibition is assessed by the method provided in Example 2 and expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells, using the following formula:

$\frac{(mRNA in control cells) - (mRNA in treated cells)}{(mRNA in control cells)} \cdot 100 %$

In other embodiments, inhibition of the expression of an IGFALS gene or an IGF-1 gene may be assessed in terms of a reduction of a parameter that is functionally linked to IGFALS or IGF-1 gene expression, e.g., IGFALS or IGF-1 protein expression or IGF signaling pathways. IGFALS or IGF-1 gene silencing may be determined in any cell expressing IGFALS or IGF-1, either endogenous or heterologous from an expression construct, and by any assay known in the art.

Inhibition of the expression of an IGFALS or IGF-1 protein may be manifested by a reduction in the level of the IGFALS or IGF-1 protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above, for the assessment of mRNA suppression, the inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess the inhibition of the expression of an IGFALS or IGF-1 gene includes a cell or group of cells that has not yet been contacted with an RNAi agent of the invention. For example, the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent.

In certain embodiments, inhibition of expression of an IGF-1 gene may be manifested in a reduction in the difference between a normal level of IGF-1 mRNA or protein and an abnormal level of IGF-1 mRNA or protein in a subject or in a specific tissue in the subject, e.g., mRNA in the liver of the subject or IGF-1 protein in subject serum. That is, inhibition may be manifested in a normalization of expression as compared to an appropriate control.

The level of IGFALS mRNA or IGF-1 mRNA that is expressed by a cell or group of cells, or the level of circulating IGFALS mRNA or IGF-1 mRNA, may be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of expression of IGFALS or IGF-1 in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the IGFALS gene or IGF-1 gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy™ RNA preparation kits (Qiagen®) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis. Circulating IGFALS or IGF-1 mRNA may be detected using methods the described in PCT Publication WO2012/177906, the entire contents of which are hereby incorporated herein by reference.

In some embodiments, the level of expression of IGFALS or IGF-1 is determined using a nucleic acid probe. The term “probe”, as used herein, refers to any molecule that is capable of selectively binding to a specific IGFALS or IGF-1. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or northern analyses, polymerase chain reaction (PCR) analyses and probe arrays.

One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to IGFALS mRNA or IGF-1 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of IGFALS mRNA or IGF-1 mRNA.

An alternative method for determining the level of expression of IGFALS or IGF-1 in a sample involves the process of nucleic acid amplification and/or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, the level of expression of IGFALS or IGF-1 is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™ System).

The expression levels of IGFALS or IGF-1 mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of IGFALS or IGF-1 expression level may also comprise using nucleic acid probes in solution.

In preferred embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of these methods is described and exemplified in the Examples presented herein.

The level of IGFALS or IGF-1 protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like.

In some embodiments, the efficacy of the methods of the invention in the treatment of an IGF system-associated disease is assessed by a decrease in IGFALS mRNA or IGF-1 mRNA level (by liver biopsy) or IGFALS or IGF-1 protein level, typically determined in serum.

In some embodiments, the efficacy of the methods of the invention in the treatment of acromegaly can be monitored by evaluating a subject for normalization of at least one sign or symptom of acromegaly previously displayed in the subject including, elevated IGF-1 level, sleep apnea, joint pain, symptomatic carpal tunnel syndrome, hypertension, biventricular cardiac hypertrophy, cardiac arrhythmia, fatigue, and weakness. These symptoms may be assessed in vitro or in vivo using any method known in the art. Although the nadir GH suppression ofter administration of glucose can be considered the “gold standard” test for acromegaly (Katznelson et al., 2011, Endocrine Practice), suppression may not be observed after treatment with the RNAi agents provided herein due to their proposed mechanism of action. Moreover, subjects may have accomplished clinically relevant beneficial outcomes with lowering of IGF-1 without reaching normal GH levels.

It is understood that normal IGF-1 levels are dependent both on the age and gender of the subject, with younger subjects having lower IGF-1 levels than older subjects. Therefore, when comparing IGF-1 levels to determine the lowering or normalizing of the level, an appropriate control must be selected. Appropriate controls include, for example, an IGF-1 level prior to treatment (when available) or an age and gender matched control. In certain embodiments, IGF-1 levels are monitored or tested on multiple occasions to confirm a change in IGF-1 level in a subject. In preferred embodiments, the IGF-1 level is decreased sufficiently to provide a clinically beneficial outcome for the subject.

In some embodiments, the efficacy of the method of the invention in treatment of cancer can be monitored by evaluating a subject for maintenance or preferably reduction of tumor burden of the primary tumor or metastatic tumor(s) or the prevention of metastasis. Methods for detection and monitoring of tumor burden are known in the art, e.g., RECIST criteria as provided in Eisenhauer et al., 2009, New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur. J. Cancer. 45:228-247.

In some embodiments of the methods of the invention, the iRNA is administered to a subject such that the iRNA is delivered to a specific site within the subject. The inhibition of expression of IGFALS or IGF-1 may be assessed using measurements of the level or change in the level of IGFALS or IGF-1 mRNA or IGFALS or IGF-1 protein in a sample derived from fluid or tissue from the specific site within the subject.

As used herein, the terms detecting or determining a level of an anlyte are understood to mean performing the steps to determine if a material, e.g., protein, RNA, is present. As used herein, methods of detecting or determining include detection or determination of an anlyte level that is below the level of detection for the method used.

VII. Methods of Treating or Preventing IGF System-Associated Diseases

The present invention also provides methods of using an iRNA of the invention or a composition containing an iRNA of the invention to reduce or inhibit IGFALS or IGF-1 expression in a cell. The methods include contacting the cell with a dsRNA of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of an IGFALS gene or an IGF-1 gene, thereby inhibiting expression of the IGFALS gene or an IGF-1 gene in the cell. Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of IGFALS or IGF-1 may be determined by determining the mRNA expression level of IGFALS or IGF-1, e.g., in a liver sample, using methods routine to one of ordinary skill in the art, e.g., northern blotting, qRT-PCR; by determining the protein level of IGFALS or IGF-1 using methods routine to one of ordinary skill in the art, such as western blotting, immunological techniques. A reduction in the expression of IGFALS or IGF-1 may also be assessed indirectly by measuring a decrease in biological activity of IGFALS or IGF-1 or measuring the level of IGF-1 in a subject sample (e.g., a serum sample).

In the methods of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may be any cell that expresses an IGFALS or IGF-1 gene, typically a liver cell. A cell suitable for use in the methods of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell, e.g., a human liver cell.

IGFALS expression or IGF-1expression is inhibited in the cell by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to a level below the level of detection of the assay. IGFALS expression or IGF-1 expression is inhibited in the cell such that the difference between the level of expression in a subject with an IGF system-associated disease and the normal level of expression is reduce by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

The in vivo methods of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the IGFALS gene or IGF-1 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection.

In some embodiments, the administration is via a depot injection. A depot injection may release the iRNA in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of IGFALS or IGF-1, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods for inhibiting the expression of an IGFALS or IGF-1 gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets an IGFALS or an IGF-1 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the IGFALS gene or the IGF-1 gene, thereby inhibiting expression of the IGFALS gene or the IGF-1 gene in the cell. Reduction in gene expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, described herein. In one embodiment, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in the IGFALS gene or the IGF-1 gene or protein expression.

The present invention further provides methods of treatment of a subject in need thereof. The treatment methods of the invention include administering an iRNA of the invention to a subject, e.g., a subject that would benefit from a reduction or inhibition of IGFALS or IGF-1 expression, in a therapeutically effective amount of an iRNA targeting an IGFALS gene or an IGF-1 gene or a pharmaceutical composition comprising an iRNA targeting an IGFALS gene or an IGF-1 gene.

An iRNA of the invention may be administered as a “free iRNA.” A free iRNA is administered in the absence of a pharmaceutical composition. The naked iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.

Alternatively, an iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction or inhibition of IGFALS gene or an IGF-1 gene expression are those having a disorder of elevated growth hormone, e.g., acromegaly, or a disorder of elevated insulin signaling, e.g., cancer. In another embodiment, a subject having a disorder of elevated growth hormone has one or more signs or symptoms associated with acromegaly or elevated growth hormone including, but not limited to, elevated IGF-1 level, somatic enlargement (soft tissue and bony overgrowth), excessive sweating, jaw overgrowth, sleep apnea, osteoarthropathy, joint pain, symptomatic carpal tunnel syndrome, hypertension, biventricular cardiac hypertrophy, cardiac arrhythmia, fatigue, weakness, diabetes mellitus, menstrual irregularities in women and sexual dysfunction in men, headache, and visual field loss (attributable to optic chiasmal compression) and diplopia (due to cranial nerve palsy); in conjunction with an elevated growth hormone level. Treatment of a subject that would benefit from a reduction or inhibition of IGFALS or IGF-1 gene expression and normalization of growth hormone levels includes therapeutic treatment (e.g., of a subject is suffering from acromegaly) and prophylactic treatment (e.g., of a subject does not meet the diagnostic criteria of acromegaly or may have elevated or fluctuating growth hormone, or IGFALS, or IGF-1 levels, or a subject may be at risk of developing acromegaly). Treatment of a subject that would benefit from a reduction or inhibition of IGFALS gene expression or IGF-1 gene expression can also include treatment of cancer.

The invention further provides methods for the use of an iRNA or a pharmaceutical composition thereof, e.g., for treating a subject that would benefit from reduction or inhibition of IGFALS or IGF-1 expression, e.g., a subject having a disorder of elevated growth hormone, in combination with other pharmaceuticals or other therapeutic methods, e.g., with known pharmaceuticals or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, in certain embodiments, an iRNA targeting IGFALS or IGF-1 is administered in combination with an agent useful in treating a disorder of elevated growth hormone as described elsewhere herein.

The invention provides methods for the treatment of cancer, e.g., IGF-1 dependent cancer, IGF-1 receptor positive cancer, or metastatic or potentially metastatic cancer. In certain embodiments, the iRNAs of the invention are used in conjunction with various standards of treatment of cancer, e.g., chemotherapeutic agents, surgery, radiation; and combinations thereof.

The iRNA and additional therapeutic agents may be administered at the same time or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times or by another method known in the art or described herein.

In one embodiment, the method includes administering a composition featured herein such that expression of the target IGFALS gene or IGF-1 gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, expression of the target IGFALS gene or IGF-1 gene is decreased for an extended duration, e.g., at least about two, three, four days or more, e.g., about one week, two weeks, three weeks, or four weeks or longer.

Preferably, the iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target IGFALS gene or IGF-1 gene. Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described herein.

Administration of the iRNA according to the methods of the invention may result in a reduction of the severity, signs, symptoms, or markers of such diseases or disorders in a patient with a disorder of elevated growth hormone, elevated IGFALS, elevated IGF-1, or an IGF-1 responsive tumor. By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay used.

Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker, or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of a disorder of IGF signaling may be assessed, for example, by periodic monitoring of IGF-1 or IGFALS levels, e.g., serum IGF-1 or IGFALS levels. For subjects suffering from acromegaly a decrease in one or more signs or symptoms including, but not limited to sleep apnea, joint pain, symptomatic carpal tunnel syndrome, hypertension, biventricular cardiac hypertrophy, cardiac arrhythmia, fatigue, and weakness can be an indication of treatment of acromegaly. Similarly a delay or lessening of the severity of the co-morbidities associated with acromegaly such as hypertension, hypertrophy, stroke, diabetes, and sleep apnea can demonstrate efficacy of treatment.

Efficacy of treatment of cancer can be demonstrated by stabilization or a decrease in tumor burden as demonstrated by a stabilization or decrease in tumor burden of the primary tumor, metastatic tumors, or the delay or prevention of tumor metastasis. Diagnostic and monitoring methods are known in the art and are also provided herein.

Comparisons of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA targeting IGFALS or IGF-1, or pharmaceutical composition thereof, “effective against” an IGF system-associated disorder indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating IGF system-associated disorders.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale. Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using an iRNA or iRNA formulation as described herein.

Subjects can be administered a therapeutic amount of iRNA, such as about 0.01 mg/kg to about 200 mg/kg.

The iRNA can be administered by intravenous infusion over a period of time, on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. Administration of the iRNA can reduce IGFALS or IGF-1 levels, e.g., in a cell, tissue, blood, urine, or other compartment of the patient by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the level of detection of the assay method used. It is noted that a reduction in IGFALS will not likely result in a decrease in growth hormone levels in a subject with acromegaly. Administration of the iRNA can reduce the difference in the subject IGF-1 levels and a normal IGF-1 level, e.g., in a cell, tissue, blood, urine, or other compartment of the patient by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Alternatively, the iRNA can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired daily dose of iRNA to a subject. The injections may be repeated over a period of time. The administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as every other day or to once a year. In certain embodiments, the iRNA is administered about once per month to about once per quarter (i.e., about once every three months).

IX. Diagnostic Criteria and Treatment for Acromegaly

Diagnostic criteria for acromegaly are set forth in the American Association of Clinical Endocrinologists Medical Guidelines for Clinical Practice for the Diagnosis and Treatment of Acromegaly—2011 Update (Katznelson et al., Endocr. Pract. 17(Suppl. 4), incorporated herein by reference. Further details and citations can be found therein.

Acromegaly is a clinical syndrome that, depending on its stage of progression, may not manifest with clear diagnostic features. Diagnosis should be considered in patients with 2 or more of the following comorbidities: new-onset diabetes, diffuse arthralgias, new-onset or difficult-to-control hypertension, cardiac disease including biventricular hypertrophy and diastolic or systolic dysfunction, fatigue, headaches, carpal tunnel syndrome, sleep apnea syndrome, diaphoresis, loss of vision, colon polyps, and progressive jaw malocclusion. A serum IGF-I level, if accompanied by a large number of results from age- and sex-matched normal subjects, is a good tool to assess integrated GH secretion and is excellent for diagnosis, monitoring, and especially screening. A random IGF-I value (a marker of integrated GH secretion) should be measured for diagnosis and for monitoring after a therapeutic intervention. Serum GH assays are not standardized and should not be used interchangably. The nadir GH suppression after administration of glucose has been considered the “gold standard” test for acromegaly, however, a conflict exists regarding the threshold for diagnosis. The panel recommends that GH measurements be performed at baseline, then every 30 minutes for a total of 120 minutes after administration of glucose. The inability to suppress serum GH to less than 1 ng/mL after glucose administration is typically considered the diagnostic criterion for acromegaly, however, in a consensus guideline in 2000, the diagnosis of acromegaly was excluded if the patient had a random GH measurement less than 0.4 ng/mL and a normal IGF-I value. Although a nadir GH concentration of less than 1 ng/mL after administration of glucose is the standard recommendation for a normal response, the 2011 panel suggests consideration of a lower nadir GH cut point at 0.4 ng/mL after glucose administration because of the enhanced assay sensitivity and more frequent finding of modest GH hypersecretion. A diagnosis of acromegaly will be made by one of skill in the art considering the totality of the evidence for the patient under consideration.

Once a biochemical diagnosis of acromegaly has been made, a magnetic resonance imaging (MRI) scan of the pituitary gland should be performed because a pituitary GH-secreting adenoma is the most common cause of acromegaly. Visual field testing should be performed if there is optic chiasmal compression noted on the MRI or if the patient has complaints of reduced peripheral vision. Further biochemical testing should include a serum prolactin level (to evaluate for hyperprolactinemia) and assessment of anterior and posterior pituitary function (for potential hypopituitarism).

The goals of therapy for acromegaly are to (1) control biochemical indices of activity, (2) control tumor size and prevent local mass effects, (3) reduce signs and symptoms of disease, (4) prevent or improve medical comorbidities, and (5) prevent early mortality. The primary mode of therapy is surgery, which is recommended for all patients with microadenomas and for all patients who have macroadenomas with associated mass effects. In patients with macroadenomas without mass effects, and with low likelihood of surgical cure, a role for surgical de-bulking of macroadenomas to improve the response to subsequent medical therapy has been advocated, as well as primary medical therapy alone. Medical therapy is generally used in the adjuvant setting. Irradiation, either conventional fractionated RT or stereotactic radiosurgery, is largely relegated to an adjuvant role. Availability of specific therapeutic options and cost of these interventions are taken into account with decisions regarding therapy.

The goal of surgical interventions is to decrease tumor volume, thereby decreasing production of excess growth hormone and decompress the mass effect of macroadenomas on any normal remaining pituitary gland tissues, optic nerve, or surrounding critical structures. Surgical interventions can be curative for many subjects. Surgically resected tissue should be analyzed to understand the tumor biology to potentially provide guidance for treatment. Biochemical analyses are also performed post-operatively to assess the surgical outcome.

Medical therapy is used in conjunction with surgery. Studies have provided conflicting results regarding the benefits of treatment with medical interventions prior to surgery to change the nature of the tumor. The iRNAs provided herein can be used at any time in conjunction with surgical intervention (i.e., before or after surgery).

Adjunctive medical therapy is used in patients who cannot achieve a complete cure by surgical intervention. Medical therapies fall into three categories: dopamine agonists, somatostatin analogs (SSAs), and a GH receptor antagonist. Each of the medical interventions presents different risks and benefits, including substantial costs of some of the therapies.

Dopamine agonists include cabergoline and bromocriptine. The agents are a good first line therapy, especially in patients with mild biochemical activity, as they are relatively inexpensive and orally administered. However, side effects include gastrointestinal upset, orthostatic hypotension, headache, and nasal congestion.

Somatostatin analogs (SSAs) include octreotide (Sandostatin®) LAR (long-acting release, administered as an intramuscular injection) and lanreotide (Somatuline®) Autogel (administered as a deep subcutaneous depot injection). SSAs are less convenient for use than dopamine agonists as they must be administered by injection (50 mcg three times daily Sandostatin® Injection subcutaneously for 2 weeks followed by Sandostatin® LAR 20 mg intragluteally every 4 weeks for 3 months; or 60, 90, or 120 mg of Somatuline® every 28 days by deep subcutaneous injection). SSAs are effective in normalizing IGF-I and GH levels in approximately 55% of patients. The clinical and biochemical responses to SSAs are inversely related to tumor size and degree of GH hypersecretion. Octreotide LAR and lanreotide Autogel have similar efficacy profiles. In patients with an inadequate response to SSAs, the addition of cabergoline or pegvisomant (Somavert®) may be effective for further lowering one or both of GH and IGF-1 levels. Potential side effects of SSAs, include gastrointestinal upset, malabsorption, constipation, gallbladder disease, hair loss, and bradycardia.

Pegvisomant, a GH receptor antagonist, is administered by daily subcutaneous injection. Side effects of pegvisomant, include flulike illness, allergic reactions, and increase in liver enzymes. Patients treated with pegvisomant must undergo routine liver enzyme tests. Because endogenous GH levels increase with pegvisomant administration and pegvisomant may be cross-measured in GH assays, serum GH levels are not specific and should not be monitored in patients receiving pegvisomant. Instead, serum IGF-1 levels are monitored.

Combinations of various medical therapies may be useful in the treatment of some acromegaly patients.

Radiation therapy is used as an adjunctive treatment is patients who do not respond sufficiently to surgical or medical interventions.

Similar treatment strategies are used in children with gigantism, a type of acromegaly, which refers to excess GH secretion that occurs during childhood when the growth plates are open, leading to accelerated vertical growth.

Some of the comorbidities of acromegaly resolve upon decreasing the level of GH or decreasing the responsiveness of the subject to GH. However, others are not. Unlike soft tissue changes, bone enlargement is not reversible. Surgical interventions (e.g., carpal tunnel release, joint replacement surgery), physical therapy, and analgesic medications can be used to treat conditions associated with bone or soft tissue overgrowth. Respiratory disorders including sleep apnea and higher susceptibility to respiratory infections can be treated with standard interventions and preventive strategies (e.g., influenza and pneumococcal vaccinations). Cardiovascular disease, hypertension, and stroke can be managed using standard monitoring (e.g., blood pressure, cholesterol, and lipid level monitoring) and medical treatment. Subjects should be monitored for the development of type 2 diabetes and neoplasia, particularly colon polyps and neoplasia. Subjects should also be monitored for psychological complications related to the physical changes and deformities that can occur with the disease. As used herein, treatment can include, but does not require, resolution of the co-morbidities of acromegaly. Treatment can include, but does not require, prevention or reduction of the development of one or more of the comorbidities associated with acromegaly. As used herein, treatment for acromegaly can further include, but does not require, treatment of one or more of the comorbidities associated with acromegaly.

IX. Response Evaluation Criteria and Treatment of Cancer

Methods for detection of tumors and assessment of tumor burden are well known in the art. For example, the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines were revised in 2008 and are fully set forth in Eisenhauer et al., 2009, New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur. J. Cancer. 45:228-247. These guidelines can be used to determine if a subject has tumor regression or no tumor progression as demonstrated by a complete response (CR) or partial response (PR), or stable disease (SD), respectively, as provided therein for at least a sufficient time that the CR, PR, or SD is detected meets the threshold of treatment or effective treatment as provided herein. A subject with only progressive disease (PD) after administration of an iRNA provided herein is not considered to have a favorable response to or be effectively treated by the iRNA. The development of PD after a period of CR, PR, or PD is understood as having been effectively treated by the iRNA provided herein.

It is understood that the iRNA agents provided herein can be used in conjunction with other interventions for the treatment of cancer, e.g., surgery, chemotherapy, or radiation.

This invention is further illustrated by the following examples which should not be construed as limiting. The entire contents of all references, patents and published patent applications cited throughout this application, as well as the FIGURE and Sequence Listing, are hereby incorporated herein by reference.

EXAMPLES
Example 1. iRNA Synthesis
Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

IGFALS Transcripts and siRNA Design

A set of siRNAs targeting the human IGFALS, “insulin-like growth factor binding protein, acid labile subunit”, (human: NCBI refseqID NM_004970; NCBI GeneID: 3483), as well as toxicology-species IGFALS orthologs (cynomolgus monkey: XM_005590898; mouse: NM_008340; rat, NM_053329) were designed using custom R and Python scripts. The human NM_004970 REFSEQ mRNA has a length of 2168 bases.

The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 19mer siRNA from position 50 through position 2160 (the coding region and 3′ UTR) was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct siRNA designs targeting a large number of vertebrate genes. Subsets of the IGFALS siRNAs were designed with perfect or near-perfect matches between human, cynomolgus and rhesus monkey. A further subset was designed with perfect or near-perfect matches to mouse and rat IGFALS orthologs. For each strand of the siRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the siRNA and all potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed region, defined here as positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the siRNA, defined here as positions 10-11 of the antisense oligonucleotide. The relative weight of the mismatches was 2.8; 1.2:1 for seed mismatches, cleavage site, and other positions up through antisense position 19. Mismatches in the first position were ignored. A specificity score was calculated for each strand by summing the value of each weighted mismatch. Preference was given to siRNAs whose antisense score in human and cynomolgus monkey was >=2.0 and predicted efficacy was >=50% knockdown of the IGFALS transcript.

A detailed list of the unmodified IGFALS sense and antisense strand sequences is shown in Tables 3, 6, and 12.

A detailed list of the modified IGFALS sense and antisense strand sequences is shown in Tables 5, 8, and 14.

IGF-1 Transcripts and siRNA Design

A set of siRNAs targeting the human insulin like growth factor 1, “IGF1” (human: e.g., NCBI refseqID NM_000618; NCBI GeneID: 3479), as well as toxicology-species IGF1 orthologs (cynomolgus monkey: e.g., XM_005572039; mouse: e.g., NM_010512; rat, e.g., NM_178866) were designed using custom R and Python scripts. The human NM_00618 REFSEQ mRNA has a length of 7366 bases.

The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 19mer siRNA from position 265 through position 7366 (the coding region and 3′ UTR) was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct siRNA designs targeting a large number of vertebrate genes. Subsets of the IGF1 siRNAs were designed with perfect or near-perfect matches between human and cynomolgus monkey. A further subset was designed with perfect or near-perfect matches to human, cynomolgus monkey and mouse IGF1 orthologs. A further subset was designed with perfect or near-perfect matches to mouse and rat IGF1 orthologs. For each strand of the siRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the siRNA and all potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed region, defined here as positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the siRNA, defined here as positions 10-11 of the antisense oligonucleotide. The relative weight of the mismatches was 2.8; 1.2:1 for seed mismatches, cleavage site, and other positions up through antisense position 19. Mismatches in the first position were ignored. A specificity score was calculated for each strand by summing the value of each weighted mismatch. Preference was given to siRNAs whose antisense score in human and cynomolgus monkey was >, 3.0 and predicted efficacy was >=70% knockdown of the human IGF1 transcripts.

A detailed list of the unmodified IGF-1 sense and antisense strand sequences is shown in Tables 9, 15, and 18.

A detailed list of the modified IGF-1 sense and antisense strand sequences is shown in Tables 11, 17, and 20.

siRNA Synthesis siRNA sequences were synthesized at 1 μmol scale on a Mermade 192 synthesizer (BioAutomation) using the solid support mediated phosphoramidite chemistry. The solid support was controlled pore glass (500 A) loaded with custom GalNAc ligand or universal solid support (AM biochemical). Ancillary synthesis reagents, 2′-F and 2′-O-Methyl RNA and deoxy phosphoramidites were obtained from Thermo-Fisher (Milwaukee, Wis.) and Hongene (China). 2′F 2′-O-Methyl, GNA (glycol nucleic acids), 5′phosphate and other modifications are introduced using the corresponding phosphoramidites. Synthesis of 3′ GalNAc conjugated single strands was performed on a GalNAc modified CPG support. Custom CPG universal solid support was used for the synthesis of antisense single strands. Coupling time for all phosphoramidites (100 mM in acetonitrile) is 5 min employing 5-Ethylthio-1H-tetrazole (ETT) as activator (0.6 M in acetonitrile). Phosphorothioate linkages were generated using a 50 mM solution of 3-((Dimethylamino-methylidene) amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, Mass., USA)) in anhydrous acetonitrile/pyridine (1:1 v/v). Oxidation time was 3 minutes. All sequences were synthesized with final removal of the DMT group (“DMT off”).

Upon completion of the solid phase synthesis, oligoribonucleotides were cleaved from the solid support and deprotected in sealed 96 deep well plates using 200 μL Aqueous Methylamine reagents at 60° C. for 20 minutes. For sequences containing 2′ ribo residues (2′-OH) that are protected with a tert-butyl dimethyl silyl (TBDMS) group, a second step deprotection is performed using TEA.3HF (triethylamine trihydro fluoride) reagent. To the methylamine deprotection solution, 200 uL of dimethyl sulfoxide (DMSO) and 300 ul TEA.3HF reagent were added and the solution was incubated for additional 20 min at 60° C. At the end of cleavage and deprotection step, the synthesis plate was allowed to come to room temperature and is precipitated by addition of 1 mL of acetontile: ethanol mixture (9:1). The plates are cooled at −80 C for 2 hours, superanatant was decanted carefully with the aid of a multi channel pipette. The oligonucleotide pellet was re-suspended in 20 mM NaOAc buffer and is desalted using a 5 mL HiTrap size exclusion column (GE Healthcare) on an AKTA Purifier System equipped with an A905 autosampler and a Frac 950 fraction collector. Desalted samples are collected in 96-well plates. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV (260 nm) for quantification and a selected set of samples by IEX chromatography to determine purity.

Annealing of single strands was performed on a Tecan liquid handling robot. Equimolar mixture of sense and antisense single strands were combined and annealed in 96 well plates. After combining the complementary single strands, the 96-well plate was sealed tightly and heated in an oven at 100° C. for 10 minutes and allowed to come slowly to room temperature over a period 2-3 hours. The concentration of each duplex was normalized to 10 μM in 1×PBS.

Example 2—In Vitro Screening
Cell Culture and Transfections

Hep3B (ATCC) were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 384-well plate and incubated at room temperature for 15 minutes. Firty μl of DMEM containing ˜5×10³cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM and in some cases 1 nM final duplex concentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit

RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs (Invitrogen, cat #61012). Briefly, 50 μl of Lysis/Binding Buffer and 25 μl of lysis buffer containing 3 μl of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed 2 times with 1500 Wash Buffer A and once with Wash Buffer B. Beads were then washed with 1500 Elution Buffer, re-captured and supernatant removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25× dNTPs, 1 μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H₂O per reaction was added to RNA isolated above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 hours 37° C. Plates were then incubated at 81° C. for 8 minutes.

Real time PCR

Two μl of cDNA were added to a master mix containing 0.5 μl of GAPDH TaqMan Probe (Hs99999905 ml), 0.5 μl IGFALS probe (HS00744047 S1) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche). Each duplex was tested at least two times and data were normalized to naïve cells or cells transfected with a non-targeting control siRNA.

To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells.

TABLE 2

Abbreviations of nucleotide monomers used in nucleic

acid sequence representation. It will be understood

that these monomers, when present in an oligonucleotide,

are mutually linked by 5′-3′-phosphodiester bonds.

Abbrevi-

ation
Nucleotide(s)

A
Adenosine-3′-phosphate

Af
2′-fluoroadenosine-3′-phosphate

Afs
2′-fluoroadenosine-3′-phosphorothioate

As
adenosine-3′-phosphorothioate

C
cytidine-3′-phosphate

Cf
2′-fluorocytidine-3′-phosphate

Cfs
2′-fluorocytidine-3′-phosphorothioate

Cs
cytidine-3′-phosphorothioate

G
guanosine-3′-phosphate

Gf
2′-fluoroguanosine-3′-phosphate

Gfs
2′-fluoroguanosine-3′-phosphorothioate

Gs
guanosine-3′-phosphorothioate

T
5′-methyluridine-3′-phosphate

Tf
2′-fluoro-5-methyluridine-3′-phosphate

Tfs
2′-fluoro-5-methyluridine-3′-phosphorothioate

Ts
5-methyluridine-3′-phosphorothioate

U
Uridine-3′-phosphate

Uf
2′-fluorouridine-3′-phosphate

Ufs
2′-fluorouridine-3′-phosphorothioate

Us
uridine-3′-phosphorothioate

N
any nucleotide (G, A, C, T or U)

a
2′-O-methyladenosine-3′-phosphate

as
2′-O-methyladenosine-3′- phosphorothioate

c
2′-O-methylcytidine-3′-phosphate

cs
2′-O-methylcytidine-3′- phosphorothioate

g
2′-O-methylguanosine-3′-phosphate

gs
2′-O-methylguanosine-3′- phosphorothioate

t
2′-O-methyl-5-methyluridine-3′-phosphate

ts
2′-O-methyl-5-methyluridine-3′-phosphorothioate

u
2′-O-methyluridine-3′-phosphate

us
2′-O-methyluridine-3′-phosphorothioate

s
phosphorothioate linkage

L96
N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol

Hyp-(GalNAc-alkyl)3

dT
2′-deoxythymidine-3′-phosphate

dC
2′-deoxycytidine-3′-phosphate

Y44
inverted abasic DNA (2-hydroxymethyl-tetrahydrofurane-5-

phosphate)

(Tgn)
Thymidine-glycol nucleic acid (GNA) S-Isomer

P
Phosphate

VP
Vinyl-phosphate

(Aam)
2′-O-(N-methylacetamide)adenosine-3′-phosphate

TABLE 3

Unmodified Sense and Antisense Strand Sequences of IGFALS dsRNAs

SEQ

SEQ

Duplex
Sense

Position in
ID
Antisense

Position in
ID

name
name
Sense Sequence
NM_004970.2
NO
name
Antisense Sequence
NM_004970.2
NO

AD-62728
A-125826
ACAGAUGAGCUCAGCGUCUUU
196-216
28
A-125827
AAAGACGCUGAGCUCAUCUGUGU
194-216
85

AD-62741
A-125832
AGCUCAGCGUCUUUUGCAGUU
203-223
29
A-125833
AACUGCAAAAGACGCUGAGCUCA
201-223
86

AD-68729
A-138233
CUGUGGCUGGACGGCAACAAA
318-337 C21A
30
A-138234
UUUGUUGCCGUCCAGCCACAGGG
316-337 C21A
87

AD-68720
A-138215
GGACGGCAACAACCUCUCGUA
326-345 C21A
31
A-138216
UACGAGAGGUUGUUGCCGUCCAG
324-345 C21A
88

AD-68717
A-138209
AACCGUCUGAGCAGGCUGGAA
549-568 G21A
32
A-138210
UUCCAGCCUGCUCAGACGGUUGU
547-568 G21A
89

AD-62737
A-125830
GGACCUCAACCUGGGUUGGAA
558-578
33
A-125831
UUCCAACCCAGGUUGAGGUCCCA
556-578
90

AD-62713
A-125820
UGGCAACAAACUGACUUACCU
645-665
34
A-125821
AGGUAAGUCAGUUUGUUGCCAGC
643-665
91

AD-62742
A-125786
GGUGCUGGCGGGCAACAGGCU
678-698
35
A-125787
AGCCUGUUGCCCGCCAGCACCAG
676-698
92

AD-62719
A-125838
GCUGGACCUGAGCAGGAACGA
747-767 C21A
36
A-125839
UCGUUCCUGCUCAGGUCCAGCUC
745-767 C21A
93

AD-62724
A-125840
CUGGACCUGAGCAGGAACGCA
748-768 G21A
37
A-125841
UGCGUUCCUGCUCAGGUCCAGCU
746-768 G21A
94

AD-68728
A-138231
GGCCAUCAAGGCAAACGUGUU
776-795
38
A-138232
AACACGUUUGCCUUGAUGGCCCG
774-795
95

AD-62717
A-125806
GCAACCUCAUCGCUGCCGUGA
833-853 G21A
39
A-125807
UCACGGCAGCGAUGAGGUUGCGG
831-853 G21A
96

AD-62731
A-125796
GCUGCCGUGGCCCCGGGCGCA
844-864 C21A
40
A-125797
UGCGCCCGGGGCCACGGCAGCGA
842-864 C21A
97

AD-62726
A-125794
UGGCCCCGGGCGCCUUCCUGA
851-871 G21A
41
A-125795
UCAGGAAGGCGCCCGGGGCCACG
849-871 G21A
98

AD-68727
A-138229
GGGCCUGAAGGCGCUGCGAUA
872-891 G21A
42
A-138230
UAUCGCAGCGCCUUCAGGCCCAG
870-891 G21A
99

AD-62715
A-125774
GCGUGGCUGGCCUCCUGGAGA
911-931 G21A
43
A-125775
UCUCCAGGAGGCCAGCCACGCGG
909-931 G21A
100

AD-68710
A-138195
GGCCUCCUGGAGGACACGUUA
921-940 C21A
44
A-138196
UAACGUGUCCUCCAGGAGGCCAG
919-940 C21A
101

AD-62743
A-125802
UGGGCCACAACCGCAUCCGGA
1043-1063 C21A
45
A-125803
UCCGGAUGCGGUUGUGGCCCAGC
1041-1063 C21A
102

AD-62711
A-125788
CCACAACCGCAUCCGGCAGCU
1047-1067
46
A-125789
AGCUGCCGGAUGCGGUUGUGGCC
1045-1067
103

AD-68709
A-138193
AGCUGGCUGAGCGCAGCUUUA
1066-1085 G21A
47
A-138194
UAAAGCUGCGCUCAGCCAGCUGC
1064-1085 G21A
104

AD-68724
A-138223
CUCACGCUAGACCACAACCAA
1110-1129 G21A
48
A-138224
UUGGUUGUGGUCUAGCGUGAGCA
1108-1129 G21A
105

AD-62734
A-125782
CCUCACCAACGUGGCGGUCAU
1161-1181
49
A-125783
AUGACCGCCACGUUGGUGAGGCC
1159-1181
106

AD-68111
A-135415
CCUCACCAACGUGGCGGUCAU
1161-1181
50
A-135416
AUGACCGCCACGUUGGUGAGGCC
1159-1181
107

AD-68719
A-138213
CACCAACGUGGCGGUCAUGAA
1166-1185
51
A-138214
UUCAUGACCGCCACGUUGGUGAG
1164-1185
108

AD-68712
A-138199
ACCAACGUGGCGGUCAUGAAA
1167-1186 C21A
52
A-138200
UUUCAUGACCGCCACGUUGGUGA
1165-1186 C21A
109

AD-62730
A-125780
UCAUGAACCUCUCUGGGAACU
1178-1198
53
A-125781
AGUUCCCAGAGAGGUUCAUGACC
1176-1198
110

AD-68711
A-138197
ACCUCUCUGGGAACUGUCUCA
1186-1205 C21A
54
A-138198
UGAGACAGUUCCCAGAGAGGUUC
1184-1205 C21A
111

AD-68713
A-138201
CUCUCUGGGAACUGUCUCCGA
1188-1207 G21A
55
A-138202
UCGGAGACAGUUCCCAGAGAGGU
1186-1207 G21A
112

AD-62732
A-125812
GAACUGUCUCCGGAACCUUCA
1194-1214 C21A
56
A-125813
UGAAGGUUCCGGAGACAGUUCCC
1192-1214 C21A
113

AD-68715
A-138205
GGAACUGUCUCCGGAACCUUA
1195-1214 C21A
57
A-138206
UAAGGUUCCGGAGACAGUUCCCA
1193-1214 C21A
114

AD-62738
A-125784
CUGUCUCCGGAACCUUCCGGA
1197-1217
58
A-125785
UCCGGAAGGUUCCGGAGACAGUU
1195-1217
115

AD-62736
A-125814
GUCUCCGGAACCUUCCGGAGA
1199-1219 C21A
59
A-125815
UCUCCGGAAGGUUCCGGAGACAG
1197-1219 C21A
116

AD-62712
A-125804
CGGAACCUUCCGGAGCAGGUA
1204-1224 G21A
60
A-125805
UACCUGCUCCGGAAGGUUCCGGA
1202-1224 G21A
117

AD-68723
A-138221
AACCUUCCGGAGCAGGUGUUA
1209-1228 C21A
61
A-138222
UAACACCUGCUCCGGAAGGUUCC
1207-1228 C21A
118

AD-62739
A-125800
CUUCCGGAGCAGGUGUUCCGA
1210-1230 G21A
62
A-125801
UCGGAACACCUGCUCCGGAAGGU
1208-1230 G21A
119

AD-62723
A-125824
CAUCUCCAGCAUCGAAGAACA
1302-1322
63
A-125825
UGUUCUUCGAUGCUGGAGAUGCU
1300-1322
120

AD-62745
A-125834
CUCCAGCAUCGAAGAACAGAA
1305-1325 G21A
64
A-125835
UUCUGUUCUUCGAUGCUGGAGAU
1303-1325 G21A
121

AD-62733
A-125828
CAGCAUCGAAGAACAGAGCCU
1308-1328
65
A-125829
AGGCUCUGUUCUUCGAUGCUGGA
1306-1328
122

AD-68718
A-138211
UUCCUCAAGGACAACGGCCUA
1329-1348 C21A
66
A-138212
UAGGCCGUUGUCCUUGAGGAAGA
1327-1348 C21A
123

AD-62744
A-125818
AAGGACAACGGCCUCGUGGGA
1333-1353 C21A
67
A-125819
UCCCACGAGGCCGUUGUCCUUGA
1331-1353 C21A
124

AD-68721
A-138217
GCUGCUGGAGCUCGACCUGAA
1388-1407 C21A
68
A-138218
UUCAGGUCGAGCUCCAGCAGCUC
1386-1407 C21A
125

AD-62727
A-125810
UGACCUCCAACCAGCUCACGA
1403-1423 C21A
69
A-125811
UCGUGAGCUGGUUGGAGGUCAGG
1401-1423 C21A
126

AD-62740
A-125816
CAACCAGCUCACGCACCUGCA
1410-1430 C21A
70
A-125817
UGCAGGUGCGUGAGCUGGUUGGA
1408-1430 C21A
127

AD-62716
A-125790
UGGAGUACCUGCUGCUCUCCA
1460-1480 C21A
71
A-125791
UGGAGAGCAGCAGGUACUCCAGC
1458-1480 C21A
128

AD-62725
A-125778
UGCAGCGGGCCUUCUGGCUGA
1523-1543 G21A
72
A-125779
UCAGCCAGAAGGCCCGCUGCAGG
1521-1543 G21A
129

AD-62714
A-125836
GCAGCGGGCCUUCUGGCUGGA
1524-1544
73
A-125837
UCCAGCCAGAAGGCCCGCUGCAG
1522-1544
130

AD-68716
A-138207
UUCUGGCUGGACGUCUCGCAA
1536-1555 C21A
74
A-138208
UUGCGAGACGUCCAGCCAGAAGG
1534-1555 C21A
131

AD-62721
A-125792
GGCUGGACGUCUCGCACAACA
1538-1558 C21A
75
A-125793
UGUUGUGCGAGACGUCCAGCCAG
1536-1558 C21A
132

AD-62718
A-125822
UCAGGAAUAACUCCUUGCAGA
1577-1597
76
A-125823
UCUGCAAGGAGUUAUUCCUGAGG
1575-1597
133

AD-68722
A-138219
UCAGCCUCAGGAACAACUCAA
1615-1634 C21A
77
A-138220
UUGAGUUGUUCCUGAGGCUGAGG
1613-1634 C21A
134

AD-68725
A-138225
CAGCCUCAGGAACAACUCACU
1616-1635
78
A-138226
AGUGAGUUGUUCCUGAGGCUGAG
1614-1635
135

AD-68714
A-138203
AGCCUCAGGAACAACUCACUA
1617-1636 G21A
79
A-138204
UAGUGAGUUGUUCCUGAGGCUGA
1615-1636 G21A
136

AD-62722
A-125808
UCCAGGCCAUCUGUGAGGGGA
1766-1786 G21A
80
A-125809
UCCCCUCACAGAUGGCCUGGACG
1764-1786 G21A
137

AD-62735
A-125798
GGGGACAGGUCCUCAGUGUCA
1956-1976 C21A
81
A-125799
UGACACUGAGGACCUGUCCCCAG
1954-1976 C21A
138

AD-68731
A-138237
UGUCAUCAAUUAAAGGCAAAA
2054-2073 G21A
82
A-138238
UUUUGCCUUUAAUUGAUGACAGC
2052-2073 G21A
139

AD-68730
A-138235
UCAAUUAAAGGCAAAGGCAAU
2059-2078
83
A-138236
AUUGCCUUUGCCUUUAAUUGAUG
2057-2078
140

AD-68726
A-138227
AAAGGCAAAGGCAAUCGAAUA
2065-2084 C21A
84
A-138228
UAUUCGAUUGCCUUUGCCUUUAA
2063-2084 C21A
141

TABLE 4

IGFALS Single Dose Screen in Hep3B Cells

Hep3B

10 nM
10 nM
1 nM
1 nM
0.1 nM
0.1 nM

DuplexID
Avg
SD
Avg
SD
Avg
SD

AD-68729
53.9
16.0
57.5
3.8
97.4
7.0

AD-68720
48.4
4.1
73.2
27.9
116.3
9.3

AD-68717
22.6
5.6
55.4
8.3
101.8
26.9

AD-62742
126.5
20.7
ND

124.7
13.1

AD-62719
106.8
25.1
ND

66.5
4.7

AD-62724
87.8
8.4
ND

75.1
5.7

AD-68728
56.3
7.6
78.6
4.4
110.6
26.0

AD-62717
98.6
1.7
ND

102.9
56.2

AD-62731
105.7
3.4
ND

51.1
18.3

AD-62726
70.7
37.7
ND

93.5
8.9

AD-68727
68.9
14.6
94.8
12.8
124.7
23.3

AD-62715
118.5
41.8
ND

128.4
17.9

AD-68710
91.0
34.2
91.7
14.2
90.5
1.1

AD-62743
81.6
18.1
ND

123.9
20.9

AD-62711
107.0
11.3
ND

92.0
11.5

AD-68709
42.4
2.2
38.1
1.4
76.1
1.4

AD-68724
53.5
16.7
40.9
7.9
73.8
1.2

AD-62734
43.1
29.6
ND

115.2
0.3

AD-68111
29.5
14.8
37.5
0.7
92.1
3.9

AD-68719
45.4
18.9
59.6
5.7
108.7
25.0

AD-68712
40.8
1.9
58.0
4.7
97.4
13.1

AD-62730
98.4
14.2
ND

119.5
17.2

AD-68711
97.3
23.0
86.6
5.7
110.7
14.8

AD-68713
79.6
10.0
93.6
23.9
103.3
4.2

AD-62732
89.0
13.3
ND

66.0
14.3

AD-68715
48.7
13.1
71.9
6.8
78.7
7.2

AD-62738
133.2
34.5
ND

123.4
13.0

AD-62736
113.6
22.9
ND

84.9
13.9

AD-62712
68.5
22.6
ND

96.9
25.6

AD-68723
83.3
14.5
84.4
13.8
71.8
25.4

AD-62739
99.4
13.8
ND

105.6
24.4

AD-68718
83.1
6.8
78.2
0.1
119.8
36.8

AD-62744
98.8
10.4
ND

139.1
24.7

AD-68721
61.8
8.0
81.3
4.2
99.2
14.2

AD-62727
91.6
25.9
ND

86.6
46.5

AD-62740
138.9
16.0
ND

117.3
55.5

AD-62716
81.5
0.2
ND

127.8
20.9

AD-62725
109.1
6.1
ND

103.8
17.0

AD-62714
73.9
8.9
ND

64.0
16.0

AD-68716
18.2
0.3
28.6
17.6
40.2
10.8

AD-62721
62.0
7.7
ND

91.7
6.1

AD-68722
19.2
0.5
51.1
0.7
53.4
22.2

AD-68725
20.3
7.5
23.0
6.2
67.6
15.6

AD-68714
58.9
2.8
73.5
19.0
92.6
8.9

AD-62722
120.7
69.9
ND

115.0
25.4

AD-62735
60.2
29.8
ND

100.2
22.6

AD-68731
33.5
27.2
11.8
0.9
26.6
7.4

AD-68730
14.5
0.9
24.5
7.5
44.4
8.3

AD-68726
17.0
10.0
28.6
11.8
64.5
3.7

AD-62728
46.3
20.5
ND

49.5
10.2

AD-62741
116.8
48.6
ND

143.6
35.9

AD-62737
94.7
8.6
ND

75.2
55.1

AD-62713
83.0
20.3
ND

89.3
31.9

AD-62723
103.5
32.2
ND

66.5
22.2

AD-62745
66.7
4.4
ND

85.7
61.2

AD-62733
107.1
1.3
ND

35.9
3.8

AD-62718
129.6
42.7
ND

87.7
39.2

AD-1955
102.5
25.0

Mock
103.0
18.8

Naïve
118.0
23.5

Data are expressed as percent message remaining relative to AD-1955 non-targeting control.

TABLE 5

IGFALS Modified Sequences

Start

Sense

Position

Duplex
Oligo

SEQ ID
Antisense

SEQ
in SEQ

Name
Name
Sense Sequence
NO
Oligo Name
Antisense Sequence
ID NO
ID NO: 1

AD-68729
A-138233
csusguggCfuGfGfAfcggcaacaaaL96
142
A-138234
usUfsuguUfgCfCfguccAfgCfcacagsgsg
199
316

AD-68720
A-138215
gsgsacggCfaAfCfAfaccucucguaL96
143
A-138216
usAfscgaGfaGfGfuuguUfgCfcguccsasg
200
324

AD-68717
A-138209
asasccguCfuGfAfGfcaggcuggaaL96
144
A-138210
usUfsccaGfcCfUfgcucAfgAfcgguusgsu
201
547

AD-62742
A-125786
GfsgsUfgCfuGfgCfGfGfgCfaAfcAfgGfcUfL96
145
A-125787
asGfscCfuGfuUfgCfccgCfcAfgCfaCfcsasg
202
676

AD-62719
A-125838
GfscsUfgGfaCfcUfGfAfgCfaGfgAfaCfgAfL96
146
A-125839
usCfsgUfuCfcUfgCfucaGfgUfcCfaGfcsusc
203
745

AD-62724
A-125840
CfsusGfgAfcCfuGfAfGfcAfgGfaAfcGfcAfL96
147
A-125841
usGfscGfuUfcCfuGfcucAfgGfuCfcAfgscsu
204
746

AD-68728
A-138231
gsgsccauCfaAfGfGfcaaacguguuL96
148
A-138232
asAfscacGfuUfUfgccuUfgAfuggccscsg
205
774

AD-62717
A-125806
GfscsAfaCfcUfcAfUfCfgCfuGfcCfgUfgAfL96
149
A-125807
usCfsaCfgGfcAfgCfgauGfaGfgUfuGfcsgsg
206
831

AD-62731
A-125796
GfscsUfgCfcGfuGfGfCfcCfcGfgGfcGfcAfL96
150
A-125797
usGfscGfcCfcGfgGfgccAfcGfgCfaGfcsgsa
207
842

AD-62726
A-125794
UfsgsGfcCfcCfgGfGfCfgCfcUfuCfcUfgAfL96
151
A-125795
usCfsaGfgAfaGfgCfgccCfgGfgGfcCfascsg
208
849

AD-68727
A-138229
gsgsgccuGfaAfGfGfcgcugcgauaL96
152
A-138230
usAfsucgCfaGfCfgccuUfcAfggcccsasg
209
870

AD-62715
A-125774
GfscsGfuGfgCfuGfGfCfcUfcCfuGfgAfgAfL96
153
A-125775
usCfsuCfcAfgGfaGfgccAfgCfcAfcGfcsgsg
210
909

AD-68710
A-138195
gsgsccucCfuGfGfAfggacacguuaL96
154
A-138196
usAfsacgUfgUfCfcuccAfgGfaggccsasg
211
919

AD-62743
A-125802
UfsgsGfgCfcAfcAfAfCfcGfcAfuCfcGfgAfL96
155
A-125803
usCfscGfgAfuGfcGfguuGfuGfgCfcCfasgsc
212
1041

AD-62711
A-125788
CfscsAfcAfaCfcGfCfAfuCfcGfgCfaGfcUfL96
156
A-125789
asGfscUfgCfcGfgAfugcGfgUfuGfuGfgscsc
213
1045

AD-68709
A-138193
asgscuggCfuGfAfGfcgcagcuuuaL96
157
A-138194
usAfsaagCfuGfCfgcucAfgCfcagcusgsc
214
1064

AD-68724
A-138223
csuscacgCfuAfGfAfccacaaccaaL96
158
A-138224
usUfsgguUfgUfGfgucuAfgCfgugagscsa
215
1108

AD-62734
A-125782
CfscsUfcAfcCfaAfCfGfuGfgCfgGfuCfaUfL96
159
A-125783
asUfsgAfcCfgCfcAfcguUfgGfuGfaGfgscsc
216
1159

AD-68111
A-135415
cscsucacCfaAfCfGfuggcggucauL96
160
A-135416
asUfsgacCfgCfCfacguUfgGfugaggscsc
217
1159

AD-68719
A-138213
csasccaaCfgUfGfGfcggucaugaaL96
161
A-138214
usUfscauGfaCfCfgccaCfgUfuggugsasg
218
1164

AD-68712
A-138199
ascscaacGfuGfGfCfggucaugaaaL96
162
A-138200
usUfsucaUfgAfCfcgccAfcGfuuggusgsa
219
1165

AD-62730
A-125780
UfscsAfuGfaAfcCfUfCfuCfuGfgGfaAfcUfL96
163
A-125781
asGfsuUfcCfcAfgAfgagGfuUfcAfuGfascsc
220
1176

AD-68711
A-138197
ascscucuCfuGfGfGfaacugucucaL96
164
A-138198
usGfsagaCfaGfUfucccAfgAfgaggususc
221
1184

AD-68713
A-138201
csuscucuGfgGfAfAfcugucuccgaL96
165
A-138202
usCfsggaGfaCfAfguucCfcAfgagagsgsu
222
1186

AD-62732
A-125812
GfsasAfcUfgUfcUfCfCfgGfaAfcCfuUfcAfL96
166
A-125813
usGfsaAfgGfuUfcCfggaGfaCfaGfuUfcscsc
223
1192

AD-68715
A-138205
gsgsaacuGfuCfUfCfcggaaccuuaL96
167
A-138206
usAfsaggUfuCfCfggagAfcAfguuccscsa
224
1193

AD-62738
A-125784
CfsusGfuCfuCfcGfGfAfaCfcUfuCfcGfgAfL96
168
A-125785
usCfscGfgAfaGfgUfuccGfgAfgAfcAfgsusu
225
1195

AD-62736
A-125814
GfsusCfuCfcGfgAfAfCfcUfuCfcGfgAfgAfL96
169
A-125815
usCfsuCfcGfgAfaGfguuCfcGfgAfgAfcsasg
226
1197

AD-62712
A-125804
CfsgsGfaAfcCfuUfCfCfgGfaGfcAfgGfuAfL96
170
A-125805
usAfscCfuGfcUfcCfggaAfgGfuUfcCfgsgsa
227
1202

AD-68723
A-138221
asasccuuCfcGfGfAfgcagguguuaL96
171
A-138222
usAfsacaCfcUfGfcuccGfgAfagguuscsc
228
1207

AD-62739
A-125800
CfsusUfcCfgGfaGfCfAfgGfuGfuUfcCfgAfL96
172
A-125801
usCfsgGfaAfcAfcCfugcUfcCfgGfaAfgsgsu
229
1208

AD-68718
A-138211
ususccucAfaGfGfAfcaacggccuaL96
173
A-138212
usAfsggcCfgUfUfguccUfuGfaggaasgsa
230
1327

AD-62744
A-125818
AfsasGfgAfcAfaCfGfGfcCfuCfgUfgGfgAfL96
174
A-125819
usCfscCfaCfgAfgGfccgUfuGfuCfcUfusgsa
231
1331

AD-68721
A-138217
gscsugcuGfgAfGfCfucgaccugaaL96
175
A-138218
usUfscagGfuCfGfagcuCfcAfgcagcsusc
232
1386

AD-62727
A-125810
UfsgsAfcCfuCfcAfAfCfcAfgCfuCfaCfgAfL96
176
A-125811
usCfsgUfgAfgCfuGfguuGfgAfgGfuCfasgsg
233
1401

AD-62740
A-125816
CfsasAfcCfaGfcUfCfAfcGfcAfcCfuGfcAfL96
177
A-125817
usGfscAfgGfuGfcGfugaGfcUfgGfuUfgsgsa
234
1408

AD-62716
A-125790
UfsgsGfaGfuAfcCfUfGfcUfgCfuCfuCfcAfL96
178
A-125791
usGfsgAfgAfgCfaGfcagGfuAfcUfcCfasgsc
235
1458

AD-62725
A-125778
UfsgsCfaGfcGfgGfCfCfuUfcUfgGfcUfgAfL96
179
A-125779
usCfsaGfcCfaGfaAfggcCfcGfcUfgCfasgsg
236
1521

AD-62714
A-125836
GfscsAfgCfgGfgCfCfUfuCfuGfgCfuGfgAfL96
180
A-125837
usCfscAfgCfcAfgAfaggCfcCfgCfuGfcsasg
237
1522

AD-68716
A-138207
ususcuggCfuGfGfAfcgucucgcaaT96
181
A-138208
usUfsgcgAfgAfCfguccAfgCfcagaasgsg
238
1534

AD-62721
A-125792
GfsgsCfuGfgAfcGfUfCfuCfgCfaCfaAfcAfL96
182
A-125793
usGfsuUfgUfgCfgAfgacGfuCfcAfgCfcsasg
239
1536

AD-68722
A-138219
uscsagccUfcAfGfGfaacaacucaaL96
183
A-138220
usUfsgagUfuGfUfuccuGfaGfgcugasgsg
240
1613

AD-68725
A-138225
csasgccuCfaGfGfAfacaacucacuL96
184
A-138226
asGfsugaGfuUfGfuuccUfgAfggcugsasg
241
1614

AD-68714
A-138203
asgsccucAfgGfAfAfcaacucacuaL96
185
A-138204
usAfsgugAfgUfUfguucCfuGfaggcusgsa
242
1615

AD-62722
A-125808
UfscsCfaGfgCfcAfUfCfuGfuGfaGfgGfgAfL96
186
A-125809
usCfscCfcUfcAfcAfgauGfgCfcUfgGfascsg
243
1764

AD-62735
A-125798
GfsgsGfgAfcAfgGfUfCfcUfcAfgUfgUfcAfL96
187
A-125799
usGfsaCfaCfuGfaGfgacCfuGfuCfcCfcsasg
244
1954

AD-68731
A-138237
usgsucauCfaAfUfUfaaaggcaaaaL96
188
A-138238
usUfsuugCfcUfUfuaauUfgAfugacasgsc
245
2052

AD-68730
A-138235
uscsaauuAfaAfGfGfcaaaggcaauL96
189
A-138236
asUfsugcCfuUfUfgccuUfuAfauugasusg
246
2057

AD-68726
A-138227
asasaggcAfaAfGfGfcaaucgaauaL96
190
A-138228
usAfsuucGfaUfUfgccuUfuGfccuuusasa
247
2063

AD-62728
A-125826
AfscsAfgAfuGfaGfCfUfcAfgCfgUfcUfuUfL96
191
A-125827
asAfsaGfaCfgCfuGfagcUfcAfuCfuGfusgsu
248
774

AD-62741
A-125832
AfsgsCfuCfaGfcGfUfCfuUfuUfgCfaGfuUfL96
192
A-125833
asAfscUfgCfaAfaAfgacGfcUfgAfgCfuscsa
249
201

AD-62737
A-125830
GfsgsAfcCfuCfaAfCfCfuGfgGfuUfgGfaAfL96
193
A-125831
usUfscCfaAfcCfcAfgguUfgAfgGfuCfcscsa
250
556

AD-62713
A-125820
UfsgsGfcAfaCfaAfAfCfuGfaCfuUfaCfcUfL96
194
A-125821
asGfsgUfaAfgUfcAfguuUfgUfuGfcCfasgsc
251
643

AD-62723
A-125824
CfsasUfcUfcCfaGfCfAfuCfgAfaGfaAfcAfL96
195
A-125825
usGfsuUfcUfuCfgAfugcUfgGfaGfaUfgscsu
252
1300

AD-62745
A-125834
CfsusCfcAfgCfaUfCfGfaAfgAfaCfaGfaAfL96
196
A-125835
usUfscUfgUfuCfuUfcgaUfgCfuGfgAfgsasu
253
1301

AD-62733
A-125828
CfsasGfcAfuCfgAfAfGfaAfcAfgAfgCfcUfL96
197
A-125829
asGfsgCfuCfuGfuUfcuuCfgAfuGfcUfgsgsa
254
1306

AD-62718
A-125822
UfscsAfgGfaAfuAfAfCfuCfcUfuGfcAfgAfL96
198
A-125823
usCfsuGfcAfaGfgAfguuAfuUfcCfuGfasgsg
255
1575

Example 3—Knockdown of IGFALs Expression with an IGFALS siRNA Decreases Expression of IGF-1

A series of siRNAs targeting mouse IGFALS were designed and tested for the ability to knockdown expression of IGFALs mRNA in 6-8 week old C57Bl/6 female mice (n=3 per group). A single 10 mg/kg dose of AD-62713, AD-62724, AD-62745, or AD-62728; or PBS control, was administered subcutaneously on day 1. On day 7, the mice were sacrificed to assess knockdown of IGFLALS mRNA in liver and IGFALS and IGF-1 protein in serum.

AD-62728 was found to be most effective in decreasing expression of IGFALS mRNA and protein. Specifically, at day 7, IFGALS mRNA expression in the liver was found to be about 15% of the PBS control. At day 7 after treatment with AD-62713 and AD-62745, IGFALS mRNA expression in the liver was found to be about 65% of the PBS control for both duplexes.

A decrease in serum IGFALS protein levels was found to correspond to the decrease in IGFALS mRNA in the liver. Specifically, AD-62728 decreased the serum IGFALS protein level to about 3.9 μg/ml, as compared to about 6.4 μg/ml in the PBS control. AD-62713 and AD-62745 decreased the serum IGFALS level to about 5.2 μg/ml and 4.6 μg/ml, respectively.

A decrease in serum IGF-1 was also observed in response to treatment with the duplexes. Specifically, AD-62727 decreased the serum IGF-1 protein level to about 13 ng/ml as compared to about 34 ng/ml in the PBS control. AD-62713 and AD-62745 decreased serum IGF-1 levels to about 20 ng/ml and 27 ng/ml, respectively.

Further, in a multidose study, AD-62728 was demonstrated to be effective in knockdown of expression of IGFALS mRNA in liver in an expected dose response manner Specifically, C57Bl/6 female mice, 6-8 weeks of age (n=3 per group) were administered either four doses of AD-62728 at 1 mg/kg or 3 mg/kg once weekly, or two doses at 3 mg/kg or 10 mg/kg every other week; or a PBS control. IGFALS mRNA knockdown was observed in the expected dose response manner.

Example 4—In Vitro Screening
Bioinformatics

A set of double stranded RNAi agents targeting human IGFALS (human NCBI refseq ID: NM_004970; NCBI GeneID: 3483, SEQ ID NO: 1) were designed using custom R and Python scripts. The human IGFALS REFSEQ mRNA has a length of 2168 bases.

The rationale and method for the set of agent designs is as follows: the predicted efficacy for every potential 19mer siRNA from position 10 through position 2168 was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct siRNA designs targeting a large number of vertebrate genes. The custom Python script built the set of agents by systematically selecting a siRNA every 11 bases along the target mRNA starting at position 10. At each of the positions, the neighboring agent (one position to the 5′ end of the mRNA, one position to the 3′ end of the mRNA) was swapped into the design set if the predicted efficacy was better than the efficacy at the exact every-11th siRNA. Low complexity agents, i.e., those with Shannon Entropy measures below 1.35 were excluded from the set.

In Vitro Dual-Glo® Screening
Cell Culture and Transfections

Cos7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂in DMEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. Dual-Glo® Luciferase constructs were generated in the psiCHECK2 plasmid and contained approximately 2.0 kb (human) IGFALS sequences (SEQ ID NO: 23). Dual-luciferase plasmids were co-transfected with double stranded agents into 3000 cells using Lipofectamine RNAiMax (Invitrogen, Carlsbad Calif. cat #13778-150). For each well of a 384 well plate, 0.1 μl of Lipofectamine was added to 3 ng of plasmid vector and agent in 15 μl of Opti-MEM and allowed to complex at room temperature for 15 minutes. The mixture was then added to the cells resuspended in 35 ul of fresh complete media. Cells were incubated for 48 hours before luciferase was measured. Single dose experiments were performed at 10 nM final duplex concentration.

Dual-Glo® Luciferase Assay

Forty-eight hours after the siRNAs were transfected, Firefly (transfection control) and Renilla (fused to IGFALS target sequence in 3′ UTR, SEQ ID NO: 23) luciferase were measured. First, media was removed from cells. Then Firefly luciferase activity was measured by adding 20 μl of Dual-Glo® Luciferase Reagent mixed with 20 μl of complete media to each well. The mixture was incubated at room temperature for 30 minutes before luminescense (500 nm) was measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 20 ul of room temperature of Dual-Glo® Stop & Glo® Reagent to each well and the plates were incubated for 20 minutes before luminescence was again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent quenched the firefly luciferase signal and sustained luminescence for the Renilla luciferase reaction. Double stranded RNAi agent activity was determined by normalizing the Renilla (IGFALS) signal to the Firefly (control) signal within each well. The magnitude of agent activity was then assessed relative to cells that were transfected with the same vector but were not treated with agent or were treated with a non-targeting double stranded RNAi agent. All transfections were done in quadruplicates.

TABLE 6

Unmodified Sense and Antisense Strand Sequences of IGFALS dsRNAs

Antisense

Range in

Duplex
Sense Oligo

SEQ

Oligo

SEQ ID
SEQ ID

Name
Name
Sense oligo sequence
ID NO
Range
Name
Antisense oligo sequence
NO
NO: 1

AD-73764
A-147667
AGGGCAGGGGUGGCCGGCA
256
11-29
A-147668
UGCCGGCCACCCCUGCCCU
441
11-29

AD-73765
A-147669
CCGGCACAGCAGACGUACA
257
24-42
A-147670
UGUACGUCUGCUGUGCCGG
442
24-42

AD-73766
A-147671
AGACGUACCCUCCCUCGCU
258
34-52
A-147672
AGCGAGGGAGGGUACGUCU
443
34-52

AD-73767
A-147673
UCCCUCGCUGCCUGCCUGA
259
44-62
A-147674
UCAGGCAGGCAGCGAGGGA
444
44-62

AD-73768
A-147675
UGCCUGCAGCCUGCCCUGA
260
56-74
A-147676
UCAGGGCAGGCUGCAGGCA
445
56-74

AD-73769
A-147677
UGCCCUGCAUGCAGGAUGA
261
67-85
A-147678
UCAUCCUGCAUGCAGGGCA
446
67-85

AD-73770
A-147679
AGGAUGGCCCUGAGGAAAG
262
79-97
A-147680
CUUUCCUCAGGGCCAUCCU
447
79-97

AD-73771
A-147681
CUGAGGAAAGGAGGCCUGA
263
88-106
A-147682
UCAGGCCUCCUUUCCUCAG
448
88-106

AD-73772
A-147683
AGGCCUGGCCCUGGCGCUA
264
99-117
A-147684
UAGCGCCAGGGCCAGGCCU
449
99-117

AD-73773
A-147685
GCGCUGCUGCUGCUGUCCU
265
112-130
A-147686
AGGACAGCAGCAGCAGCGC
450
112-130

AD-73774
A-147687
UGCUGUCCUGGGUGGCACU
266
122-140
A-147688
AGUGCCACCCAGGACAGCA
451
122-140

AD-73775
A-147689
UGGCACUGGGCCCCCGCAA
267
134-152
A-147690
UUGCGGGGGCCCAGUGCCA
452
134-152

AD-73776
A-147691
GCCCCCGCAGCCUGGAGGA
268
143-161
A-147692
UCCUCCAGGCUGCGGGGGC
453
143-161

AD-73777
A-147693
UGGAGGGAGCAGACCCCGA
269
155-173
A-147694
UCGGGGUCUGCUCCCUCCA
454
155-173

AD-73778
A-147695
AGACCCCGGAACGCCGGGA
270
165-183
A-147696
UCCCGGCGUUCCGGGGUCU
455
165-183

AD-73779
A-147697
CGCCGGGGGAAGCCGAGGA
271
176-194
A-147698
UCCUCGGCUUCCCCCGGCG
456
176-194

AD-73780
A-147699
CGAGGGCCCAGCGUGCCCA
272
189-207
A-147700
UGGGCACGCUGGGCCCUCG
457
189-207

AD-73781
A-147701
AGCGUGCCCGGCCGCCUGU
273
198-216
A-147702
ACAGGCGGCCGGGCACGCU
458
198-216

AD-73782
A-147703
GCCUGUGUCUGCAGCUACA
274
211-229
A-147704
UGUAGCUGCAGACACAGGC
459
211-229

AD-73783
A-147705
UGCAGCUACGAUGACGACA
275
220-238
A-147706
UGUCGUCAUCGUAGCUGCA
460
220-238

AD-73784
A-147707
GACGACGCGGAUGAGCUCA
276
232-250
A-147708
UGAGCUCAUCCGCGUCGUC
461
232-250

AD-73785
A-147709
AUGAGCUCAGCGUCUUCUA
277
242-260
A-147710
UAGAAGACGCUGAGCUCAU
462
242-260

AD-73786
A-147711
UCUUCUGCAGCUCCAGGAA
278
254-272
A-147712
UUCCUGGAGCUGCAGAAGA
463
254-272

AD-73787
A-147713
UCCAGGAACCUCACGCGCA
279
265-283
A-147714
UGCGCGUGAGGUUCCUGGA
464
265-283

AD-73788
A-147715
UCACGCGCCUGCCUGAUGA
280
275-293
A-147716
UCAUCAGGCAGGCGCGUGA
465
275-293

AD-73789
A-147717
UGAUGGAGUCCCGGGCGGA
281
288-306
A-147718
UCCGCCCGGGACUCCAUCA
466
288-306

AD-73790
A-147719
CGGGCGGCACCCAAGCCCU
282
299-317
A-147720
AGGGCUUGGGUGCCGCCCG
467
299-317

AD-73791
A-147721
CAAGCCCUGUGGCUGGACA
283
310-328
A-147722
UGUCCAGCCACAGGGCUUG
468
310-328

AD-73792
A-147723
UGGCUGGACGGCAACAACA
284
319-337
A-147724
UGUUGUUGCCGUCCAGCCA
469
319-337

AD-73793
A-147725
AACAACCUCUCGUCCGUCA
285
331-349
A-147726
UGACGGACGAGAGGUUGUU
470
331-349

AD-73794
A-147727
UCCGUCCCCCCGGCAGCCU
286
343-361
A-147728
AGGCUGCCGGGGGGACGGA
471
343-361

AD-73795
A-147729
CGGCAGCCUUCCAGAACCU
287
353-371
A-147730
AGGUUCUGGAAGGCUGCCG
472
353-371

AD-73796
A-147731
CAGAACCUCUCCAGCCUGA
288
364-382
A-147732
UCAGGCUGGAGAGGUUCUG
473
364-382

AD-73797
A-147733
AGCCUGGGCUUCCUCAACA
289
376-394
A-147734
UGUUGAGGAAGCCCAGGCU
474
376-394

AD-73798
A-147735
UUCCUCAACCUGCAGGGCA
290
385-403
A-147736
UGCCCUGCAGGUUGAGGAA
475
385-403

AD-73799
A-147737
CAGGGCGGCCAGCUGGGCA
291
397-415
A-147738
UGCCCAGCUGGCCGCCCUG
476
397-415

AD-73800
A-147739
AGCUGGGCAGCCUGGAGCA
292
407-425
A-147740
UGCUCCAGGCUGCCCAGCU
477
407-425

AD-73801
A-147741
CUGGAGCCACAGGCGCUGA
293
418-436
A-147742
UCAGCGCCUGUGGCUCCAG
478
418-436

AD-73802
A-147743
CGCUGCUGGGCCUAGAGAA
294
431-449
A-147744
UUCUCUAGGCCCAGCAGCG
479
431-449

AD-73803
A-147745
CUAGAGAACCUGUGCCACA
295
442-460
A-147746
UGUGGCACAGGUUCUCUAG
480
442-460

AD-73804
A-147747
UGUGCCACCUGCACCUGGA
296
452-470
A-147748
UCCAGGUGCAGGUGGCACA
481
452-470

AD-73805
A-147749
ACCUGGAGCGGAACCAGCU
297
464-482
A-147750
AGCUGGUUCCGCUCCAGGU
482
464-482

AD-73806
A-147751
AACCAGCUGCGCAGCCUGA
298
475-493
A-147752
UCAGGCUGCGCAGCUGGUU
483
475-493

AD-73807
A-147753
CGCAGCCUGGCACUCGGCA
299
484-502
A-147754
UGCCGAGUGCCAGGCUGCG
484
484-502

AD-73808
A-147755
UCGGCACGUUUGCACACAA
300
497-515
A-147756
UUGUGUGCAAACGUGCCGA
485
497-515

AD-73809
A-147757
UUGCACACACGCCCGCGCU
301
506-524
A-147758
AGCGCGGGCGUGUGUGCAA
486
506-524

AD-73810
A-147759
CCCGCGCUGGCCUCGCUCA
302
517-535
A-147760
UGAGCGAGGCCAGCGCGGG
487
517-535

AD-73811
A-147761
UCGCUCGGCCUCAGCAACA
303
529-547
A-147762
UGUUGCUGAGGCCGAGCGA
488
529-547

AD-73812
A-147763
AGCAACAACCGUCUGAGCA
304
541-559
A-147764
UGCUCAGACGGUUGUUGCU
489
541-559

AD-73813
A-147767
CUGGAGGACGGGCUCUUCA
305
562-580
A-147768
UGAAGAGCCCGUCCUCCAG
490
562-580

AD-73814
A-147769
CUCUUCGAGGGCCUCGGCA
306
574-592
A-147770
UGCCGAGGCCCUCGAAGAG
491
574-592

AD-73815
A-147771
GGCCUCGGCAGCCUCUGGA
307
583-601
A-147772
UCCAGAGGCUGCCGAGGCC
492
583-601

AD-73816
A-147773
UCUGGGACCUCAACCUCGA
308
596-614
A-147774
UCGAGGUUGAGGUCCCAGA
493
596-614

AD-73817
A-147775
AACCUCGGCUGGAAUAGCA
309
607-625
A-147776
UGCUAUUCCAGCCGAGGUU
494
607-625

AD-73818
A-147777
UGGAAUAGCCUGGCGGUGA
310
616-634
A-147778
UCACCGCCAGGCUAUUCCA
495
616-634

AD-73819
A-147779
CGGUGCUCCCCGAUGCGGA
311
629-647
A-147780
UCCGCAUCGGGGAGCACCG
496
629-647

AD-73820
A-147781
GAUGCGGCGUUCCGCGGCA
312
640-658
A-147782
UGCCGCGGAACGCCGCAUC
497
640-658

AD-73821
A-147783
UUCCGCGGCCUGGGCAGCA
313
649-667
A-147784
UGCUGCCCAGGCCGCGGAA
498
649-667

AD-73822
A-147785
GCAGCCUGCGCGAGCUGGU
314
662-680
A-147786
ACCAGCUCGCGCAGGCUGC
499
662-680

AD-73823
A-147787
GAGCUGGUGCUGGCGGGCA
315
673-691
A-147788
UGCCCGCCAGCACCAGCUC
500
673-691

AD-73824
A-147789
CUGGCGGGCAACAGGCUGA
316
682-700
A-147790
UCAGCCUGUUGCCCGCCAG
501
682-700

AD-73825
A-147791
AGGCUGGCCUACCUGCAGA
317
694-712
A-147792
UCUGCAGGUAGGCCAGCCU
502
694-712

AD-73826
A-147793
ACCUGCAGCCCGCGCUCUU
318
704-722
A-147794
AAGAGCGCGGGCUGCAGGU
503
704-722

AD-73827
A-147795
CGCUCUUCAGCGGCCUGGA
319
716-734
A-147796
UCCAGGCCGCUGAAGAGCG
504
716-734

AD-73828
A-147797
CGGCCUGGCCGAGCUCCGA
320
726-744
A-147798
UCGGAGCUCGGCCAGGCCG
505
726-744

AD-73829
A-147799
AGCUCCGGGAGCUGGACCU
321
737-755
A-147800
AGGUCCAGCUCCCGGAGCU
506
737-755

AD-73830
A-147801
CUGGACCUGAGCAGGAACA
322
748-766
A-147802
UGUUCCUGCUCAGGUCCAG
507
748-766

AD-73831
A-147803
AGGAACGCGCUGCGGGCCA
323
760-778
A-147804
UGGCCCGCAGCGCGUUCCU
508
760-778

AD-73832
A-147805
CGGGCCAUCAAGGCAAACA
324
772-790
A-147806
UGUUUGCCUUGAUGGCCCG
509
772-790

AD-73833
A-147807
AAGGCAAACGUGUUCGUGA
325
781-799
A-147808
UCACGAACACGUUUGCCUU
510
781-799

AD-73834
A-147809
UUCGUGCAGCUGCCCCGGA
326
793-811
A-147810
UCCGGGGCAGCUGCACGAA
511
793-811

AD-73835
A-147813
AGAAACUCUACCUGGACCA
327
815-833
A-147814
UGGUCCAGGUAGAGUUUCU
512
815-833

AD-73836
A-147815
CCUGGACCGCAACCUCAUA
328
825-843
A-147816
UAUGAGGUUGCGGUCCAGG
513
825-843

AD-73837
A-147817
CCUCAUCGCUGCCGUGGCA
329
837-855
A-147818
UGCCACGGCAGCGAUGAGG
514
837-855

AD-73838
A-147819
CGUGGCCCCGGGCGCCUUA
330
849-867
A-147820
UAAGGCGCCCGGGGCCACG
515
849-867

AD-73839
A-147821
GGCGCCUUCCUGGGCCUGA
331
859-877
A-147822
UCAGGCCCAGGAAGGCGCC
516
859-877

AD-73840
A-147823
UGGGCCUGAAGGCGCUGCA
332
869-887
A-147824
UGCAGCGCCUUCAGGCCCA
517
869-887

AD-73841
A-147825
CGCUGCGAUGGCUGGACCU
333
881-899
A-147826
AGGUCCAGCCAUCGCAGCG
518
881-899

AD-73842
A-147827
UGGACCUGUCCCACAACCA
334
893-911
A-147828
UGGUUGUGGGACAGGUCCA
519
893-911

AD-73843
A-147829
CACAACCGCGUGGCUGGCA
335
904-922
A-147830
UGCCAGCCACGCGGUUGUG
520
904-922

AD-73844
A-147831
UGGCUGGCCUCCUGGAGGA
336
914-932
A-147832
UCCUCCAGGAGGCCAGCCA
521
914-932

AD-73845
A-147833
CCUGGAGGACACGUUCCCA
337
924-942
A-147834
UGGGAACGUGUCCUCCAGG
522
924-942

AD-73846
A-147835
UUCCCCGGUCUGCUGGGCA
338
937-955
A-147836
UGCCCAGCAGACCGGGGAA
523
937-955

AD-73847
A-147837
UGCUGGGCCUGCGUGUGCU
339
947-965
A-147838
AGCACACGCAGGCCCAGCA
524
947-965

AD-73848
A-147839
CGUGUGCUGCGGCUGUCCA
340
958-976
A-147840
UGGACAGCCGCAGCACACG
525
958-976

AD-73849
A-147841
CUGUCCCACAACGCCAUCA
341
970-988
A-147842
UGAUGGCGUUGUGGGACAG
526
970-988

AD-73850
A-147843
AACGCCAUCGCCAGCCUGA
342
979-997
A-147844
UCAGGCUGGCGAUGGCGUU
527
979-997

AD-73851
A-147845
AGCCUGCGGCCCCGCACCU
343
991-1009
A-147846
AGGUGCGGGGCCGCAGGCU
528
991-1009

AD-73852
A-147847
CGCACCUUCAAGGACCUGA
344
1003-1021
A-147848
UCAGGUCCUUGAAGGUGCG
529
1003-1021

AD-73853
A-147849
AAGGACCUGCACUUCCUGA
345
1012-1030
A-147850
UCAGGAAGUGCAGGUCCUU
530
1012-1030

AD-73854
A-147851
UUCCUGGAGGAGCUGCAGA
346
1024-1042
A-147852
UCUGCAGCUCCUCCAGGAA
531
1024-1042

AD-73855
A-147853
CUGCAGCUGGGCCACAACA
347
1036-1054
A-147854
UGUUGUGGCCCAGCUGCAG
532
1036-1054

AD-73856
A-147855
CCACAACCGCAUCCGGCAA
348
1047-1065
A-147856
UUGCCGGAUGCGGUUGUGG
533
1047-1065

AD-73857
A-147857
UCCGGCAGCUGGCUGAGCA
349
1058-1076
A-147858
UGCUCAGCCAGCUGCCGGA
534
1058-1076

AD-73858
A-147859
UGGCUGAGCGCAGCUUUGA
350
1067-1085
A-147860
UCAAAGCUGCGCUCAGCCA
535
1067-1085

AD-73859
A-147861
AGCUUUGAGGGCCUGGGGA
351
1078-1096
A-147862
UCCCCAGGCCCUCAAAGCU
536
1078-1096

AD-73860
A-147863
UGGGGCAGCUUGAGGUGCU
352
1091-1109
A-147864
AGCACCUCAAGCUGCCCCA
537
1091-1109

AD-73861
A-147865
UUGAGGUGCUCACGCUAGA
353
1100-1118
A-147866
UCUAGCGUGAGCACCUCAA
538
1100-1118

AD-73862
A-147867
ACGCUAGACCACAACCAGA
354
1111-1129
A-147868
UCUGGUUGUGGUCUAGCGU
539
1111-1129

AD-73863
A-147869
AACCAGCUCCAGGAGGUCA
355
1123-1141
A-147870
UGACCUCCUGGAGCUGGUU
540
1123-1141

AD-73864
A-147871
AGGAGGUCAAGGCGGGCGA
356
1133-1151
A-147872
UCGCCCGCCUUGACCUCCU
541
1133-1151

AD-73865
A-147873
CGGGCGCUUUCCUCGGCCU
357
1145-1163
A-147874
AGGCCGAGGAAAGCGCCCG
542
1145-1163

AD-73866
A-147875
CUCGGCCUCACCAACGUGA
358
1156-1174
A-147876
UCACGUUGGUGAGGCCGAG
543
1156-1174

AD-73867
A-147877
AACGUGGCGGUCAUGAACA
359
1168-1186
A-147878
UGUUCAUGACCGCCACGUU
544
1168-1186

AD-73868
A-147879
UCAUGAACCUCUCUGGGAA
360
1178-1196
A-147880
UUCCCAGAGAGGUUCAUGA
545
1178-1196

AD-73869
A-147881
UCUGGGAACUGUCUCCGGA
361
1189-1207
A-147882
UCCGGAGACAGUUCCCAGA
546
1189-1207

AD-73870
A-147883
UCUCCGGAACCUUCCGGAA
362
1200-1218
A-147884
UUCCGGAAGGUUCCGGAGA
547
1200-1218

AD-73871
A-147885
UUCCGGAGCAGGUGUUCCA
363
1211-1229
A-147886
UGGAACACCUGCUCCGGAA
548
1211-1229

AD-73872
A-147887
GGUGUUCCGGGGCCUGGGA
364
1221-1239
A-147888
UCCCAGGCCCCGGAACACC
549
1221-1239

AD-73873
A-147889
CUGGGCAAGCUGCACAGCA
365
1234-1252
A-147890
UGCUGUGCAGCUUGCCCAG
550
1234-1252

AD-73874
A-147891
UGCACAGCCUGCACCUGGA
366
1244-1262
A-147892
UCCAGGUGCAGGCUGUGCA
551
1244-1262

AD-73875
A-147895
CAGCUGCCUGGGACGCAUA
367
1266-1284
A-147896
UAUGCGUCCCAGGCAGCUG
552
1266-1284

AD-73876
A-147897
GACGCAUCCGCCCGCACAA
368
1277-1295
A-147898
UUGUGCGGGCGGAUGCGUC
553
1277-1295

AD-73877
A-147899
CGCACACCUUCACCGGCCU
369
1289-1307
A-147900
AGGCCGGUGAAGGUGUGCG
554
1289-1307

AD-73878
A-147901
UCACCGGCCUCUCGGGGCU
370
1298-1316
A-147902
AGCCCCGAGAGGCCGGUGA
555
1298-1316

AD-73879
A-147903
UCGGGGCUCCGCCGACUCU
371
1309-1327
A-147904
AGAGUCGGCGGAGCCCCGA
556
1309-1327

AD-73880
A-147905
CGACUCUUCCUCAAGGACA
372
1321-1339
A-147906
UGUCCUUGAGGAAGAGUCG
557
1321-1339

AD-73881
A-147907
CAAGGACAACGGCCUCGUA
373
1332-1350
A-147908
UACGAGGCCGUUGUCCUUG
558
1332-1350

AD-73882
A-147909
GGCCUCGUGGGCAUUGAGA
374
1342-1360
A-147910
UCUCAAUGCCCACGAGGCC
559
1342-1360

AD-73883
A-147911
UUGAGGAGCAGAGCCUGUA
375
1355-1373
A-147912
UACAGGCUCUGCUCCUCAA
560
1355-1373

AD-73884
A-147913
AGAGCCUGUGGGGGCUGGA
376
1364-1382
A-147914
UCCAGCCCCCACAGGCUCU
561
1364-1382

AD-73885
A-147915
GGGCUGGCGGAGCUGCUGA
377
1375-1393
A-147916
UCAGCAGCUCCGCCAGCCC
562
1375-1393

AD-73886
A-147917
UGCUGGAGCUCGACCUGAA
378
1388-1406
A-147918
UUCAGGUCGAGCUCCAGCA
563
1388-1406

AD-73887
A-147919
GACCUGACCUCCAACCAGA
379
1399-1417
A-147920
UCUGGUUGGAGGUCAGGUC
564
1399-1417

AD-73888
A-147921
UCCAACCAGCUCACGCACA
380
1408-1426
A-147922
UGUGCGUGAGCUGGUUGGA
565
1408-1426

AD-73889
A-147923
ACGCACCUGCCCCACCGCA
381
1420-1438
A-147924
UGCGGUGGGGCAGGUGCGU
566
1420-1438

AD-73890
A-147925
CACCGCCUCUUCCAGGGCA
382
1432-1450
A-147926
UGCCCUGGAAGAGGCGGUG
567
1432-1450

AD-73891
A-147927
UCCAGGGCCUGGGCAAGCU
383
1442-1460
A-147928
AGCUUGCCCAGGCCCUGGA
568
1442-1460

AD-73892
A-147929
GCAAGCUGGAGUACCUGCU
384
1454-1472
A-147930
AGCAGGUACUCCAGCUUGC
569
1454-1472

AD-73893
A-147931
UACCUGCUGCUCUCCCGCA
385
1465-1483
A-147932
UGCGGGAGAGCAGCAGGUA
570
1465-1483

AD-73894
A-147933
CUCUCCCGCAACCGCCUGA
386
1474-1492
A-147934
UCAGGCGGUUGCGGGAGAG
571
1474-1492

AD-73895
A-147935
CCGCCUGGCAGAGCUGCCA
387
1485-1503
A-147936
UGGCAGCUCUGCCAGGCGG
572
1485-1503

AD-73896
A-147937
AGCUGCCGGCGGACGCCCU
388
1496-1514
A-147938
AGGGCGUCCGCCGGCAGCU
573
1496-1514

AD-73897
A-147939
GACGCCCUGGGCCCCCUGA
389
1507-1525
A-147940
UCAGGGGGCCCAGGGCGUC
574
1507-1525

AD-73898
A-147941
CCCCUGCAGCGGGCCUUCU
390
1519-1537
A-147942
AGAAGGCCCGCUGCAGGGG
575
1519-1537

AD-73899
A-147943
GGGCCUUCUGGCUGGACGU
391
1529-1547
A-147944
ACGUCCAGCCAGAAGGCCC
576
1529-1547

AD-73900
A-147945
UGGACGUCUCGCACAACCA
392
1541-1559
A-147946
UGGUUGUGCGAGACGUCCA
577
1541-1559

AD-73901
A-147947
ACAACCGCCUGGAGGCAUU
393
1553-1571
A-147948
AAUGCCUCCAGGCGGUUGU
578
1553-1571

AD-73902
A-147949
GAGGCAUUGCCCAACAGCA
394
1564-1582
A-147950
UGCUGUUGGGCAAUGCCUC
579
1564-1582

AD-73903
A-147951
CAACAGCCUCUUGGCACCA
395
1575-1593
A-147952
UGGUGCCAAGAGGCUGUUG
580
1575-1593

AD-73904
A-147953
UUGGCACCACUGGGGCGGA
396
1585-1603
A-147954
UCCGCCCCAGUGGUGCCAA
581
1585-1603

AD-73905
A-147955
UGGGGCGGCUGCGCUACCU
397
1595-1613
A-147956
AGGUAGCGCAGCCGCCCCA
582
1595-1613

AD-73906
A-147957
CGCUACCUCAGCCUCAGGA
398
1606-1624
A-147958
UCCUGAGGCUGAGGUAGCG
583
1606-1624

AD-73907
A-147959
UCAGGAACAACUCACUGCA
399
1619-1637
A-147960
UGCAGUGAGUUGUUCCUGA
584
1619-1637

AD-73908
A-147961
CUCACUGCGGACCUUCACA
400
1629-1647
A-147962
UGUGAAGGUCCGCAGUGAG
585
1629-1647

AD-73909
A-147963
ACCUUCACGCCGCAGCCCA
401
1639-1657
A-147964
UGGGCUGCGGCGUGAAGGU
586
1639-1657

AD-73910
A-147965
CAGCCCCCGGGCCUGGAGA
402
1651-1669
A-147966
UCUCCAGGCCCGGGGGCUG
587
1651-1669

AD-73911
A-147967
GCCUGGAGCGCCUGUGGCU
403
1661-1679
A-147968
AGCCACAGGCGCUCCAGGC
588
1661-1679

AD-73912
A-147969
CUGUGGCUGGAGGGUAACA
404
1672-1690
A-147970
UGUUACCCUCCAGCCACAG
589
1672-1690

AD-73913
A-147971
GGUAACCCCUGGGACUGUA
405
1684-1702
A-147972
UACAGUCCCAGGGGUUACC
590
1684-1702

AD-73914
A-147973
GGGACUGUGGCUGCCCUCU
406
1694-1712
A-147974
AGAGGGCAGCCACAGUCCC
591
1694-1712

AD-73915
A-147975
UGCCCUCUCAAGGCGCUGA
407
1705-1723
A-147976
UCAGCGCCUUGAGAGGGCA
592
1705-1723

AD-73916
A-147977
CGCUGCGGGACUUCGCCCU
408
1718-1736
A-147978
AGGGCGAAGUCCCGCAGCG
593
1718-1736

AD-73917
A-147979
UUCGCCCUGCAGAACCCCA
409
1729-1747
A-147980
UGGGGUUCUGCAGGGCGAA
594
1729-1747

AD-73918
A-147981
CAGAACCCCAGUGCUGUGA
410
1738-1756
A-147982
UCACAGCACUGGGGUUCUG
595
1738-1756

AD-73919
A-147983
UGCUGUGCCCCGCUUCGUA
411
1749-1767
A-147984
UACGAAGCGGGGCACAGCA
596
1749-1767

AD-73920
A-147985
CUUCGUCCAGGCCAUCUGU
412
1761-1779
A-147986
ACAGAUGGCCUGGACGAAG
597
1761-1779

AD-73921
A-147987
CAUCUGUGAGGGGGACGAU
413
1773-1791
A-147988
AUCGUCCCCCUCACAGAUG
598
1773-1791

AD-73922
A-147989
GGGGACGAUUGCCAGCCGA
414
1783-1801
A-147990
UCGGCUGGCAAUCGUCCCC
599
1783-1801

AD-73923
A-147991
CAGCCGCCCGCGUACACCU
415
1795-1813
A-147992
AGGUGUACGCGGGCGGCUG
600
1795-1813

AD-73924
A-147993
CGUACACCUACAACAACAU
416
1805-1823
A-147994
AUGUUGUUGUAGGUGUACG
601
1805-1823

AD-73925
A-147995
AACAACAUCACCUGUGCCA
417
1816-1834
A-147996
UGGCACAGGUGAUGUUGUU
602
1816-1834

AD-73926
A-147997
UGUGCCAGCCCGCCCGAGA
418
1828-1846
A-147998
UCUCGGGCGGGCUGGCACA
603
1828-1846

AD-73927
A-147999
CGCCCGAGGUCGUGGGGCU
419
1838-1856
A-148000
AGCCCCACGACCUCGGGCG
604
1838-1856

AD-73928
A-148001
CGUGGGGCUCGACCUGCGA
420
1848-1866
A-148002
UCGCAGGUCGAGCCCCACG
605
1848-1866

AD-73929
A-148003
ACCUGCGGGACCUCAGCGA
421
1859-1877
A-148004
UCGCUGAGGUCCCGCAGGU
606
1859-1877

AD-73930
A-148005
UCAGCGAGGCCCACUUUGA
422
1871-1889
A-148006
UCAAAGUGGGCCUCGCUGA
607
1871-1889

AD-73931
A-148007
ACUUUGCUCCCUGCUGACA
423
1883-1901
A-148008
UGUCAGCAGGGAGCAAAGU
608
1883-1901

AD-73932
A-148009
CCUGCUGACCAGGUCCCCA
424
1892-1910
A-148010
UGGGGACCUGGUCAGCAGG
609
1892-1910

AD-73933
A-148011
UCCCCGGACUCAAGCCCCA
425
1905-1923
A-148012
UGGGGCUUGAGUCCGGGGA
610
1905-1923

AD-73934
A-148013
CAAGCCCCGGACUCAGGCA
426
1915-1933
A-148014
UGCCUGAGUCCGGGGCUUG
611
1915-1933

AD-73935
A-148015
UCAGGCCCCCACCUGGCUA
427
1927-1945
A-148016
UAGCCAGGUGGGGGCCUGA
612
1927-1945

AD-73936
A-148017
ACCUGGCUCACCUUGUGCU
428
1937-1955
A-148018
AGCACAAGGUGAGCCAGGU
613
1937-1955

AD-73937
A-148019
UUGUGCUGGGGACAGGUCA
429
1949-1967
A-148020
UGACCUGUCCCCAGCACAA
614
1949-1967

AD-73938
A-148021
GACAGGUCCUCAGUGUCCU
430
1959-1977
A-148022
AGGACACUGAGGACCUGUC
615
1959-1977

AD-73939
A-148023
CAGUGUCCUCAGGGGCCUA
431
1969-1987
A-148024
UAGGCCCCUGAGGACACUG
616
1969-1987

AD-73940
A-148025
GGGCCUGCCCAGUGCACUU
432
1981-1999
A-148026
AAGUGCACUGGGCAGGCCC
617
1981-1999

AD-73941
A-148027
UGCACUUGCUGGAAGACGA
433
1993-2011
A-148028
UCGUCUUCCAGCAAGUGCA
618
1993-2011

AD-73942
A-148029
UGGAAGACGCAAGGGCCUA
434
2002-2020
A-148030
UAGGCCCUUGCGUCUUCCA
619
2002-2020

AD-73943
A-148031
AGGGCCUGAUGGGGUGGAA
435
2013-2031
A-148032
UUCCACCCCAUCAGGCCCU
620
2013-2031

AD-73944
A-148033
GGGUGGAAGGCAUGGCGGA
436
2024-2042
A-148034
UCCGCCAUGCCUUCCACCC
621
2024-2042

AD-73945
A-148035
UGGCGGCCCCCCCAGCUGU
437
2036-2054
A-148036
ACAGCUGGGGGGGCCGCCA
622
2036-2054

AD-73946
A-148037
CAGCUGUCAUCAAUUAAAG
438
2048-2066
A-148038
CUUUAAUUGAUGACAGCUG
623
2048-2066

AD-73947
A-148039
AAUUAAAGGCAAAGGCAAU
439
2059-2077
A-148040
AUUGCCUUUGCCUUUAAUU
624
2059-2077

AD-73948
A-148041
AAGGCAAUCGAAUCUAAAA
440
2070-2088
A-148042
UUUUAGAUUCGAUUGCCUU
625
2070-2088

TABLE 7

Human IGFALS Dual-Glo ® in vitro 10 nM screen

Duplex Name
Average 10 nM
STDEV 10 nM

AD-73764
46.26
12.94

AD-73765
15.98
9.39

AD-73766
27.71
1.81

AD-73767
29.96
5.64

AD-73768
53.53
15.85

AD-73769
50.94
18.08

AD-73770
35.55
11.71

AD-73771
30.07
11.32

AD-73772
33.23
3.56

AD-73773
11.46
4.14

AD-73774
58.80
12.47

AD-73775
108.20
18.60

AD-73776
51.88
20.74

AD-73777
30.64
7.39

AD-73778
81.00
19.34

AD-73779
78.23
16.91

AD-73780
67.63
20.32

AD-73781
75.04
41.97

AD-73782
11.25
3.14

AD-73783
84.25
27.48

AD-73784
31.16
3.50

AD-73785
40.36
15.91

AD-73786
26.61
4.91

AD-73787
37.73
13.41

AD-73788
41.39
9.64

AD-73789
69.70
17.02

AD-73790
54.70
18.10

AD-73791
37.77
14.31

AD-73792
59.22
4.58

AD-73793
30.72
11.33

AD-73794
96.09
23.63

AD-73795
27.15
4.14

AD-73796
44.57
8.83

AD-73797
22.69
5.07

AD-73798
52.76
11.72

AD-73799
69.71
10.21

AD-73800
49.18
17.49

AD-73801
59.80
17.00

AD-73802
28.96
1.45

AD-73803
33.13
19.76

AD-73804
40.68
7.80

AD-73805
63.69
6.82

AD-73806
66.25
14.80

AD-73807
48.62
17.85

AD-73808
25.07
4.32

AD-73809
68.40
17.86

AD-73810
83.96
14.19

AD-73811
64.13
17.42

AD-73812
46.66
9.77

AD-73813
44.50
17.35

AD-73814
63.89
24.44

AD-73815
52.18
19.16

AD-73816
46.10
24.18

AD-73817
47.24
12.69

AD-73818
26.52
4.62

AD-73819
48.75
11.37

AD-73820
60.19
5.23

AD-73821
94.35
26.80

AD-73822
84.38
36.20

AD-73823
40.82
16.47

AD-73824
73.14
20.30

AD-73825
28.56
4.59

AD-73826
46.85
5.02

AD-73827
47.58
13.90

AD-73828
63.46
15.46

AD-73829
95.35
32.53

AD-73830
58.41
9.47

AD-73831
76.16
9.56

AD-73832
66.65
24.27

AD-73833
48.53
16.86

AD-73834
61.65
17.68

AD-73835
58.15
28.49

AD-73836
26.15
4.79

AD-73837
43.30
9.38

AD-73838
74.76
20.65

AD-73839
78.85
7.72

AD-73840
43.78
12.13

AD-73841
40.30
13.20

AD-73842
43.45
1.12

AD-73843
47.08
8.45

AD-73844
110.22
43.07

AD-73845
53.10
20.78

AD-73846
100.03
52.61

AD-73847
59.82
19.09

AD-73848
26.03
3.83

AD-73849
38.45
7.00

AD-73850
86.08
20.23

AD-73851
61.41
7.67

AD-73852
53.33
19.36

AD-73853
85.67
29.83

AD-73854
54.76
5.66

AD-73855
104.89
36.39

AD-73856
57.24
13.36

AD-73857
63.18
12.14

AD-73858
20.59
3.73

AD-73859
42.26
7.68

AD-73860
94.01
20.91

AD-73861
45.90
18.39

AD-73862
26.77
5.70

AD-73863
39.07
19.21

AD-73864
59.26
14.59

AD-73865
41.82
10.07

AD-73866
60.91
19.05

AD-73867
35.80
9.83

AD-73868
46.58
6.40

AD-73869
64.22
11.51

AD-73870
80.14
7.20

AD-73871
60.16
20.80

AD-73872
56.05
24.26

AD-73873
68.99
18.51

AD-73874
110.04
18.69

AD-73875
45.34
19.36

AD-73876
51.41
17.32

AD-73877
48.52
10.40

AD-73878
114.98
63.70

AD-73879
60.09
8.24

AD-73880
38.19
8.87

AD-73881
74.45
6.60

AD-73882
33.01
9.79

AD-73883
34.58
16.31

AD-73884
53.88
4.17

AD-73885
40.86
12.23

AD-73886
48.81
15.26

AD-73887
100.05
43.02

AD-73888
52.76
9.03

AD-73889
104.07
24.09

AD-73890
34.25
10.25

AD-73891
59.05
17.53

AD-73892
43.11
18.36

AD-73893
74.85
51.34

AD-73894
71.46
42.74

AD-73895
67.51
15.16

AD-73896
65.38
19.16

AD-73897
113.90
19.73

AD-73898
30.88
11.29

AD-73899
71.21
20.59

AD-73900
45.87
8.22

AD-73901
81.14
27.00

AD-73902
57.98
26.64

AD-73903
60.87
50.48

AD-73904
144.84
56.92

AD-73905
80.06
7.93

AD-73906
25.22
6.98

AD-73907
33.52
8.04

AD-73908
88.78
21.09

AD-73909
94.23
19.36

AD-73910
106.31
18.12

AD-73911
64.23
4.10

AD-73912
25.25
5.85

AD-73913
42.38
3.07

AD-73914
38.34
6.64

AD-73915
61.19
28.72

AD-73916
71.86
28.39

AD-73917
95.24
18.35

AD-73918
80.25
27.23

AD-73919
48.91
6.14

AD-73920
39.40
11.01

AD-73921
57.14
12.93

AD-73922
45.90
21.00

AD-73923
56.04
18.98

AD-73924
28.94
7.49

AD-73925
58.43
28.38

AD-73926
102.32
34.13

AD-73927
100.65
27.38

AD-73928
85.51
11.58

AD-73929
51.54
4.93

AD-73930
27.83
6.80

AD-73931
36.71
9.74

AD-73932
37.09
6.54

AD-73933
54.60
14.50

AD-73934
188.17
65.46

AD-73935
77.02
12.48

AD-73936
71.96
24.59

AD-73937
48.37
18.42

AD-73938
47.06
6.65

AD-73939
55.62
19.17

AD-73940
74.83
6.45

AD-73941
41.91
18.36

AD-73942
87.02
43.38

AD-73943
47.56
6.76

AD-73944
39.62
7.36

AD-73945
61.45
10.10

AD-73946
16.22
4.18

AD-73947
17.27
8.22

AD-73948
33.62
7.51

TABLE 8

Modified Sense and Antisense Strand Sequences of IGFALS dsRNAs

Sense

Antisense

Duplex
Oligo

Oligo

Name
Name
Sense Oligo Sequence
SEQ ID
Name
Antisense Oligo Seq
SEQ ID
mRNA target sequence
SEQ ID

AD-73764
A-147667
AGGGCAGGGGUGGCCGGCAdTdT
626
A-147668
UGCCGGCCACCCCUGCCCUdTdT
811
AGGGCAGGGGUGGCCGGCA
996

AD-73765
A-147669
CCGGCACAGCAGACGUACAdTdT
627
A-147670
UGUACGUCUGCUGUGCCGGdTdT
812
CCGGCACAGCAGACGUACC
997

AD-73766
A-147671
AGACGUACCCUCCCUCGCUdTdT
628
A-147672
AGCGAGGGAGGGUACGUCUdTdT
813
AGACGUACCCUCCCUCGCU
998

AD-73767
A-147673
UCCCUCGCUGCCUGCCUGAdTdT
629
A-147674
UCAGGCAGGCAGCGAGGGAdTdT
814
UCCCUCGCUGCCUGCCUGC
999

AD-73768
A-147675
UGCCUGCAGCCUGCCCUGAdTdT
630
A-147676
UCAGGGCAGGCUGCAGGCAdTdT
815
UGCCUGCAGCCUGCCCUGC
1000

AD-73769
A-147677
UGCCCUGCAUGCAGGAUGAdTdT
631
A-147678
UCAUCCUGCAUGCAGGGCAdTdT
816
UGCCCUGCAUGCAGGAUGG
1001

AD-73770
A-147679
AGGAUGGCCCUGAGGAAAGdTdT
632
A-147680
CUUUCCUCAGGGCCAUCCUdTdT
817
AGGAUGGCCCUGAGGAAAG
1002

AD-73771
A-147681
CUGAGGAAAGGAGGCCUGAdTdT
633
A-147682
UCAGGCCUCCUUUCCUCAGdTdT
818
CUGAGGAAAGGAGGCCUGG
1003

AD-73772
A-147683
AGGCCUGGCCCUGGCGCUAdTdT
634
A-147684
UAGCGCCAGGGCCAGGCCUdTdT
819
AGGCCUGGCCCUGGCGCUG
1004

AD-73773
A-147685
GCGCUGCUGCUGCUGUCCUdTdT
635
A-147686
AGGACAGCAGCAGCAGCGCdTdT
820
GCGCUGCUGCUGCUGUCCU
1005

AD-73774
A-147687
UGCUGUCCUGGGUGGCACUdTdT
636
A-147688
AGUGCCACCCAGGACAGCAdTdT
821
UGCUGUCCUGGGUGGCACU
1006

AD-73775
A-147689
UGGCACUGGGCCCCCGCAAdTdT
637
A-147690
UUGCGGGGGCCCAGUGCCAdTdT
822
UGGCACUGGGCCCCCGCAG
1007

AD-73776
A-147691
GCCCCCGCAGCCUGGAGGAdTdT
638
A-147692
UCCUCCAGGCUGCGGGGGCdTdT
823
GCCCCCGCAGCCUGGAGGG
1008

AD-73777
A-147693
UGGAGGGAGCAGACCCCGAdTdT
639
A-147694
UCGGGGUCUGCUCCCUCCAdTdT
824
UGGAGGGAGCAGACCCCGG
1009

AD-73778
A-147695
AGACCCCGGAACGCCGGGAdTdT
640
A-147696
UCCCGGCGUUCCGGGGUCUdTdT
825
AGACCCCGGAACGCCGGGG
1010

AD-73779
A-147697
CGCCGGGGGAAGCCGAGGAdTdT
641
A-147698
UCCUCGGCUUCCCCCGGCGdTdT
826
CGCCGGGGGAAGCCGAGGG
1011

AD-73780
A-147699
CGAGGGCCCAGCGUGCCCAdTdT
642
A-147700
UGGGCACGCUGGGCCCUCGdTdT
827
CGAGGGCCCAGCGUGCCCG
1012

AD-73781
A-147701
AGCGUGCCCGGCCGCCUGUdTdT
643
A-147702
ACAGGCGGCCGGGCACGCUdTdT
828
AGCGUGCCCGGCCGCCUGU
1013

AD-73782
A-147703
GCCUGUGUCUGCAGCUACAdTdT
644
A-147704
UGUAGCUGCAGACACAGGCdTdT
829
GCCUGUGUCUGCAGCUACG
1014

AD-73783
A-147705
UGCAGCUACGAUGACGACAdTdT
645
A-147706
UGUCGUCAUCGUAGCUGCAdTdT
830
UGCAGCUACGAUGACGACG
1015

AD-73784
A-147707
GACGACGCGGAUGAGCUCAdTdT
646
A-147708
UGAGCUCAUCCGCGUCGUCdTdT
831
GACGACGCGGAUGAGCUCA
1016

AD-73785
A-147709
AUGAGCUCAGCGUCUUCUAdTdT
647
A-147710
UAGAAGACGCUGAGCUCAUdTdT
832
AUGAGCUCAGCGUCUUCUG
1017

AD-73786
A-147711
UCUUCUGCAGCUCCAGGAAdTdT
648
A-147712
UUCCUGGAGCUGCAGAAGAdTdT
833
UCUUCUGCAGCUCCAGGAA
1018

AD-73787
A-147713
UCCAGGAACCUCACGCGCAdTdT
649
A-147714
UGCGCGUGAGGUUCCUGGAdTdT
834
UCCAGGAACCUCACGCGCC
1019

AD-73788
A-147715
UCACGCGCCUGCCUGAUGAdTdT
650
A-147716
UCAUCAGGCAGGCGCGUGAdTdT
835
UCACGCGCCUGCCUGAUGG
1020

AD-73789
A-147717
UGAUGGAGUCCCGGGCGGAdTdT
651
A-147718
UCCGCCCGGGACUCCAUCAdTdT
836
UGAUGGAGUCCCGGGCGGC
1021

AD-73790
A-147719
CGGGCGGCACCCAAGCCCUdTdT
652
A-147720
AGGGCUUGGGUGCCGCCCGdTdT
837
CGGGCGGCACCCAAGCCCU
1022

AD-73791
A-147721
CAAGCCCUGUGGCUGGACAdTdT
653
A-147722
UGUCCAGCCACAGGGCUUGdTdT
838
CAAGCCCUGUGGCUGGACG
1023

AD-73792
A-147723
UGGCUGGACGGCAACAACAdTdT
654
A-147724
UGUUGUUGCCGUCCAGCCAdTdT
839
UGGCUGGACGGCAACAACC
1024

AD-73793
A-147725
AACAACCUCUCGUCCGUCAdTdT
655
A-147726
UGACGGACGAGAGGUUGUUdTdT
840
AACAACCUCUCGUCCGUCC
1025

AD-73794
A-147727
UCCGUCCCCCCGGCAGCCUdTdT
656
A-147728
AGGCUGCCGGGGGGACGGAdTdT
841
UCCGUCCCCCCGGCAGCCU
1026

AD-73795
A-147729
CGGCAGCCUUCCAGAACCUdTdT
657
A-147730
AGGUUCUGGAAGGCUGCCGdTdT
842
CGGCAGCCUUCCAGAACCU
1027

AD-73796
A-147731
CAGAACCUCUCCAGCCUGAdTdT
658
A-147732
UCAGGCUGGAGAGGUUCUGdTdT
843
CAGAACCUCUCCAGCCUGG
1028

AD-73797
A-147733
AGCCUGGGCUUCCUCAACAdTdT
659
A-147734
UGUUGAGGAAGCCCAGGCUdTdT
844
AGCCUGGGCUUCCUCAACC
1029

AD-73798
A-147735
UUCCUCAACCUGCAGGGCAdTdT
660
A-147736
UGCCCUGCAGGUUGAGGAAdTdT
845
UUCCUCAACCUGCAGGGCG
1030

AD-73799
A-147737
CAGGGCGGCCAGCUGGGCAdTdT
661
A-147738
UGCCCAGCUGGCCGCCCUGdTdT
846
CAGGGCGGCCAGCUGGGCA
1031

AD-73800
A-147739
AGCUGGGCAGCCUGGAGCAdTdT
662
A-147740
UGCUCCAGGCUGCCCAGCUdTdT
847
AGCUGGGCAGCCUGGAGCC
1032

AD-73801
A-147741
CUGGAGCCACAGGCGCUGAdTdT
663
A-147742
UCAGCGCCUGUGGCUCCAGdTdT
848
CUGGAGCCACAGGCGCUGC
1033

AD-73802
A-147743
CGCUGCUGGGCCUAGAGAAdTdT
664
A-147744
UUCUCUAGGCCCAGCAGCGdTdT
849
CGCUGCUGGGCCUAGAGAA
1034

AD-73803
A-147745
CUAGAGAACCUGUGCCACAdTdT
665
A-147746
UGUGGCACAGGUUCUCUAGdTdT
850
CUAGAGAACCUGUGCCACC
1035

AD-73804
A-147747
UGUGCCACCUGCACCUGGAdTdT
666
A-147748
UCCAGGUGCAGGUGGCACAdTdT
851
UGUGCCACCUGCACCUGGA
1036

AD-73805
A-147749
ACCUGGAGCGGAACCAGCUdTdT
667
A-147750
AGCUGGUUCCGCUCCAGGUdTdT
852
ACCUGGAGCGGAACCAGCU
1037

AD-73806
A-147751
AACCAGCUGCGCAGCCUGAdTdT
668
A-147752
UCAGGCUGCGCAGCUGGUUdTdT
853
AACCAGCUGCGCAGCCUGG
1038

AD-73807
A-147753
CGCAGCCUGGCACUCGGCAdTdT
669
A-147754
UGCCGAGUGCCAGGCUGCGdTdT
854
CGCAGCCUGGCACUCGGCA
1039

AD-73808
A-147755
UCGGCACGUUUGCACACAAdTdT
670
A-147756
UUGUGUGCAAACGUGCCGAdTdT
855
UCGGCACGUUUGCACACAC
1040

AD-73809
A-147757
UUGCACACACGCCCGCGCUdTdT
671
A-147758
AGCGCGGGCGUGUGUGCAAdTdT
856
UUGCACACACGCCCGCGCU
1041

AD-73810
A-147759
CCCGCGCUGGCCUCGCUCAdTdT
672
A-147760
UGAGCGAGGCCAGCGCGGGdTdT
857
CCCGCGCUGGCCUCGCUCG
1042

AD-73811
A-147761
UCGCUCGGCCUCAGCAACAdTdT
673
A-147762
UGUUGCUGAGGCCGAGCGAdTdT
858
UCGCUCGGCCUCAGCAACA
1043

AD-73812
A-147763
AGCAACAACCGUCUGAGCAdTdT
674
A-147764
UGCUCAGACGGUUGUUGCUdTdT
859
AGCAACAACCGUCUGAGCA
1044

AD-73813
A-147767
CUGGAGGACGGGCUCUUCAdTdT
675
A-147768
UGAAGAGCCCGUCCUCCAGdTdT
860
CUGGAGGACGGGCUCUUCG
1045

AD-73814
A-147769
CUCUUCGAGGGCCUCGGCAdTdT
676
A-147770
UGCCGAGGCCCUCGAAGAGdTdT
861
CUCUUCGAGGGCCUCGGCA
1046

AD-73815
A-147771
GGCCUCGGCAGCCUCUGGAdTdT
677
A-147772
UCCAGAGGCUGCCGAGGCCdTdT
862
GGCCUCGGCAGCCUCUGGG
1047

AD-73816
A-147773
UCUGGGACCUCAACCUCGAdTdT
678
A-147774
UCGAGGUUGAGGUCCCAGAdTdT
863
UCUGGGACCUCAACCUCGG
1048

AD-73817
A-147775
AACCUCGGCUGGAAUAGCAdTdT
679
A-147776
UGCUAUUCCAGCCGAGGUUdTdT
864
AACCUCGGCUGGAAUAGCC
1049

AD-73818
A-147777
UGGAAUAGCCUGGCGGUGAdTdT
680
A-147778
UCACCGCCAGGCUAUUCCAdTdT
865
UGGAAUAGCCUGGCGGUGC
1050

AD-73819
A-147779
CGGUGCUCCCCGAUGCGGAdTdT
681
A-147780
UCCGCAUCGGGGAGCACCGdTdT
866
CGGUGCUCCCCGAUGCGGC
1051

AD-73820
A-147781
GAUGCGGCGUUCCGCGGCAdTdT
682
A-147782
UGCCGCGGAACGCCGCAUCdTdT
867
GAUGCGGCGUUCCGCGGCC
1052

AD-73821
A-147783
UUCCGCGGCCUGGGCAGCAdTdT
683
A-147784
UGCUGCCCAGGCCGCGGAAdTdT
868
UUCCGCGGCCUGGGCAGCC
1053

AD-73822
A-147785
GCAGCCUGCGCGAGCUGGUdTdT
684
A-147786
ACCAGCUCGCGCAGGCUGCdTdT
869
GCAGCCUGCGCGAGCUGGU
1054

AD-73823
A-147787
GAGCUGGUGCUGGCGGGCAdTdT
685
A-147788
UGCCCGCCAGCACCAGCUCdTdT
870
GAGCUGGUGCUGGCGGGCA
1055

AD-73824
A-147789
CUGGCGGGCAACAGGCUGAdTdT
686
A-147790
UCAGCCUGUUGCCCGCCAGdTdT
871
CUGGCGGGCAACAGGCUGG
1056

AD-73825
A-147791
AGGCUGGCCUACCUGCAGAdTdT
687
A-147792
UCUGCAGGUAGGCCAGCCUdTdT
872
AGGCUGGCCUACCUGCAGC
1057

AD-73826
A-147793
ACCUGCAGCCCGCGCUCUUdTdT
688
A-147794
AAGAGCGCGGGCUGCAGGUdTdT
873
ACCUGCAGCCCGCGCUCUU
1058

AD-73827
A-147795
CGCUCUUCAGCGGCCUGGAdTdT
689
A-147796
UCCAGGCCGCUGAAGAGCGdTdT
874
CGCUCUUCAGCGGCCUGGC
1059

AD-73828
A-147797
CGGCCUGGCCGAGCUCCGAdTdT
690
A-147798
UCGGAGCUCGGCCAGGCCGdTdT
875
CGGCCUGGCCGAGCUCCGG
1060

AD-73829
A-147799
AGCUCCGGGAGCUGGACCUdTdT
691
A-147800
AGGUCCAGCUCCCGGAGCUdTdT
876
AGCUCCGGGAGCUGGACCU
1061

AD-73830
A-147801
CUGGACCUGAGCAGGAACAdTdT
692
A-147802
UGUUCCUGCUCAGGUCCAGdTdT
877
CUGGACCUGAGCAGGAACG
1062

AD-73831
A-147803
AGGAACGCGCUGCGGGCCAdTdT
693
A-147804
UGGCCCGCAGCGCGUUCCUdTdT
878
AGGAACGCGCUGCGGGCCA
1063

AD-73832
A-147805
CGGGCCAUCAAGGCAAACAdTdT
694
A-147806
UGUUUGCCUUGAUGGCCCGdTdT
879
CGGGCCAUCAAGGCAAACG
1064

AD-73833
A-147807
AAGGCAAACGUGUUCGUGAdTdT
695
A-147808
UCACGAACACGUUUGCCUUdTdT
880
AAGGCAAACGUGUUCGUGC
1065

AD-73834
A-147809
UUCGUGCAGCUGCCCCGGAdTdT
696
A-147810
UCCGGGGCAGCUGCACGAAdTdT
881
UUCGUGCAGCUGCCCCGGC
1066

AD-73835
A-147813
AGAAACUCUACCUGGACCAdTdT
697
A-147814
UGGUCCAGGUAGAGUUUCUdTdT
882
AGAAACUCUACCUGGACCG
1067

AD-73836
A-147815
CCUGGACCGCAACCUCAUAdTdT
698
A-147816
UAUGAGGUUGCGGUCCAGGdTdT
883
CCUGGACCGCAACCUCAUC
1068

AD-73837
A-147817
CCUCAUCGCUGCCGUGGCAdTdT
699
A-147818
UGCCACGGCAGCGAUGAGGdTdT
884
CCUCAUCGCUGCCGUGGCC
1069

AD-73838
A-147819
CGUGGCCCCGGGCGCCUUAdTdT
700
A-147820
UAAGGCGCCCGGGGCCACGdTdT
885
CGUGGCCCCGGGCGCCUUC
1070

AD-73839
A-147821
GGCGCCUUCCUGGGCCUGAdTdT
701
A-147822
UCAGGCCCAGGAAGGCGCCdTdT
886
GGCGCCUUCCUGGGCCUGA
1071

AD-73840
A-147823
UGGGCCUGAAGGCGCUGCAdTdT
702
A-147824
UGCAGCGCCUUCAGGCCCAdTdT
887
UGGGCCUGAAGGCGCUGCG
1072

AD-73841
A-147825
CGCUGCGAUGGCUGGACCUdTdT
703
A-147826
AGGUCCAGCCAUCGCAGCGdTdT
888
CGCUGCGAUGGCUGGACCU
1073

AD-73842
A-147827
UGGACCUGUCCCACAACCAdTdT
704
A-147828
UGGUUGUGGGACAGGUCCAdTdT
889
UGGACCUGUCCCACAACCG
1074

AD-73843
A-147829
CACAACCGCGUGGCUGGCAdTdT
705
A-147830
UGCCAGCCACGCGGUUGUGdTdT
890
CACAACCGCGUGGCUGGCC
1075

AD-73844
A-147831
UGGCUGGCCUCCUGGAGGAdTdT
706
A-147832
UCCUCCAGGAGGCCAGCCAdTdT
891
UGGCUGGCCUCCUGGAGGA
1076

AD-73845
A-147833
CCUGGAGGACACGUUCCCAdTdT
707
A-147834
UGGGAACGUGUCCUCCAGGdTdT
892
CCUGGAGGACACGUUCCCC
1077

AD-73846
A-147835
UUCCCCGGUCUGCUGGGCAdTdT
708
A-147836
UGCCCAGCAGACCGGGGAAdTdT
893
UUCCCCGGUCUGCUGGGCC
1078

AD-73847
A-147837
UGCUGGGCCUGCGUGUGCUdTdT
709
A-147838
AGCACACGCAGGCCCAGCAdTdT
894
UGCUGGGCCUGCGUGUGCU
1079

AD-73848
A-147839
CGUGUGCUGCGGCUGUCCAdTdT
710
A-147840
UGGACAGCCGCAGCACACGdTdT
895
CGUGUGCUGCGGCUGUCCC
1080

AD-73849
A-147841
CUGUCCCACAACGCCAUCAdTdT
711
A-147842
UGAUGGCGUUGUGGGACAGdTdT
896
CUGUCCCACAACGCCAUCG
1081

AD-73850
A-147843
AACGCCAUCGCCAGCCUGAdTdT
712
A-147844
UCAGGCUGGCGAUGGCGUUdTdT
897
AACGCCAUCGCCAGCCUGC
1082

AD-73851
A-147845
AGCCUGCGGCCCCGCACCUdTdT
713
A-147846
AGGUGCGGGGCCGCAGGCUdTdT
898
AGCCUGCGGCCCCGCACCU
1083

AD-73852
A-147847
CGCACCUUCAAGGACCUGAdTdT
714
A-147848
UCAGGUCCUUGAAGGUGCGdTdT
899
CGCACCUUCAAGGACCUGC
1084

AD-73853
A-147849
AAGGACCUGCACUUCCUGAdTdT
715
A-147850
UCAGGAAGUGCAGGUCCUUdTdT
900
AAGGACCUGCACUUCCUGG
1085

AD-73854
A-147851
UUCCUGGAGGAGCUGCAGAdTdT
716
A-147852
UCUGCAGCUCCUCCAGGAAdTdT
901
UUCCUGGAGGAGCUGCAGC
1086

AD-73855
A-147853
CUGCAGCUGGGCCACAACAdTdT
717
A-147854
UGUUGUGGCCCAGCUGCAGdTdT
902
CUGCAGCUGGGCCACAACC
1087

AD-73856
A-147855
CCACAACCGCAUCCGGCAAdTdT
718
A-147856
UUGCCGGAUGCGGUUGUGGdTdT
903
CCACAACCGCAUCCGGCAG
1088

AD-73857
A-147857
UCCGGCAGCUGGCUGAGCAdTdT
719
A-147858
UGCUCAGCCAGCUGCCGGAdTdT
904
UCCGGCAGCUGGCUGAGCG
1089

AD-73858
A-147859
UGGCUGAGCGCAGCUUUGAdTdT
720
A-147860
UCAAAGCUGCGCUCAGCCAdTdT
905
UGGCUGAGCGCAGCUUUGA
1090

AD-73859
A-147861
AGCUUUGAGGGCCUGGGGAdTdT
721
A-147862
UCCCCAGGCCCUCAAAGCUdTdT
906
AGCUUUGAGGGCCUGGGGC
1091

AD-73860
A-147863
UGGGGCAGCUUGAGGUGCUdTdT
722
A-147864
AGCACCUCAAGCUGCCCCAdTdT
907
UGGGGCAGCUUGAGGUGCU
1092

AD-73861
A-147865
UUGAGGUGCUCACGCUAGAdTdT
723
A-147866
UCUAGCGUGAGCACCUCAAdTdT
908
UUGAGGUGCUCACGCUAGA
1093

AD-73862
A-147867
ACGCUAGACCACAACCAGAdTdT
724
A-147868
UCUGGUUGUGGUCUAGCGUdTdT
909
ACGCUAGACCACAACCAGC
1094

AD-73863
A-147869
AACCAGCUCCAGGAGGUCAdTdT
725
A-147870
UGACCUCCUGGAGCUGGUUdTdT
910
AACCAGCUCCAGGAGGUCA
1095

AD-73864
A-147871
AGGAGGUCAAGGCGGGCGAdTdT
726
A-147872
UCGCCCGCCUUGACCUCCUdTdT
911
AGGAGGUCAAGGCGGGCGC
1096

AD-73865
A-147873
CGGGCGCUUUCCUCGGCCUdTdT
727
A-147874
AGGCCGAGGAAAGCGCCCGdTdT
912
CGGGCGCUUUCCUCGGCCU
1097

AD-73866
A-147875
CUCGGCCUCACCAACGUGAdTdT
728
A-147876
UCACGUUGGUGAGGCCGAGdTdT
913
CUCGGCCUCACCAACGUGG
1098

AD-73867
A-147877
AACGUGGCGGUCAUGAACAdTdT
729
A-147878
UGUUCAUGACCGCCACGUUdTdT
914
AACGUGGCGGUCAUGAACC
1099

AD-73868
A-147879
UCAUGAACCUCUCUGGGAAdTdT
730
A-147880
UUCCCAGAGAGGUUCAUGAdTdT
915
UCAUGAACCUCUCUGGGAA
1100

AD-73869
A-147881
UCUGGGAACUGUCUCCGGAdTdT
731
A-147882
UCCGGAGACAGUUCCCAGAdTdT
916
UCUGGGAACUGUCUCCGGA
1101

AD-73870
A-147883
UCUCCGGAACCUUCCGGAAdTdT
732
A-147884
UUCCGGAAGGUUCCGGAGAdTdT
917
UCUCCGGAACCUUCCGGAG
1102

AD-73871
A-147885
UUCCGGAGCAGGUGUUCCAdTdT
733
A-147886
UGGAACACCUGCUCCGGAAdTdT
918
UUCCGGAGCAGGUGUUCCG
1103

AD-73872
A-147887
GGUGUUCCGGGGCCUGGGAdTdT
734
A-147888
UCCCAGGCCCCGGAACACCdTdT
919
GGUGUUCCGGGGCCUGGGC
1104

AD-73873
A-147889
CUGGGCAAGCUGCACAGCAdTdT
735
A-147890
UGCUGUGCAGCUUGCCCAGdTdT
920
CUGGGCAAGCUGCACAGCC
1105

AD-73874
A-147891
UGCACAGCCUGCACCUGGAdTdT
736
A-147892
UCCAGGUGCAGGCUGUGCAdTdT
921
UGCACAGCCUGCACCUGGA
1106

AD-73875
A-147895
CAGCUGCCUGGGACGCAUAdTdT
737
A-147896
UAUGCGUCCCAGGCAGCUGdTdT
922
CAGCUGCCUGGGACGCAUC
1107

AD-73876
A-147897
GACGCAUCCGCCCGCACAAdTdT
738
A-147898
UUGUGCGGGCGGAUGCGUCdTdT
923
GACGCAUCCGCCCGCACAC
1108

AD-73877
A-147899
CGCACACCUUCACCGGCCUdTdT
739
A-147900
AGGCCGGUGAAGGUGUGCGdTdT
924
CGCACACCUUCACCGGCCU
1109

AD-73878
A-147901
UCACCGGCCUCUCGGGGCUdTdT
740
A-147902
AGCCCCGAGAGGCCGGUGAdTdT
925
UCACCGGCCUCUCGGGGCU
1110

AD-73879
A-147903
UCGGGGCUCCGCCGACUCUdTdT
741
A-147904
AGAGUCGGCGGAGCCCCGAdTdT
926
UCGGGGCUCCGCCGACUCU
1111

AD-73880
A-147905
CGACUCUUCCUCAAGGACAdTdT
742
A-147906
UGUCCUUGAGGAAGAGUCGdTdT
927
CGACUCUUCCUCAAGGACA
1112

AD-73881
A-147907
CAAGGACAACGGCCUCGUAdTdT
743
A-147908
UACGAGGCCGUUGUCCUUGdTdT
928
CAAGGACAACGGCCUCGUG
1113

AD-73882
A-147909
GGCCUCGUGGGCAUUGAGAdTdT
744
A-147910
UCUCAAUGCCCACGAGGCCdTdT
929
GGCCUCGUGGGCAUUGAGG
1114

AD-73883
A-147911
UUGAGGAGCAGAGCCUGUAdTdT
745
A-147912
UACAGGCUCUGCUCCUCAAdTdT
930
UUGAGGAGCAGAGCCUGUG
1115

AD-73884
A-147913
AGAGCCUGUGGGGGCUGGAdTdT
746
A-147914
UCCAGCCCCCACAGGCUCUdTdT
931
AGAGCCUGUGGGGGCUGGC
1116

AD-73885
A-147915
GGGCUGGCGGAGCUGCUGAdTdT
747
A-147916
UCAGCAGCUCCGCCAGCCCdTdT
932
GGGCUGGCGGAGCUGCUGG
1117

AD-73886
A-147917
UGCUGGAGCUCGACCUGAAdTdT
748
A-147918
UUCAGGUCGAGCUCCAGCAdTdT
933
UGCUGGAGCUCGACCUGAC
1118

AD-73887
A-147919
GACCUGACCUCCAACCAGAdTdT
749
A-147920
UCUGGUUGGAGGUCAGGUCdTdT
934
GACCUGACCUCCAACCAGC
1119

AD-73888
A-147921
UCCAACCAGCUCACGCACAdTdT
750
A-147922
UGUGCGUGAGCUGGUUGGAdTdT
935
UCCAACCAGCUCACGCACC
1120

AD-73889
A-147923
ACGCACCUGCCCCACCGCAdTdT
751
A-147924
UGCGGUGGGGCAGGUGCGUdTdT
936
ACGCACCUGCCCCACCGCC
1121

AD-73890
A-147925
CACCGCCUCUUCCAGGGCAdTdT
752
A-147926
UGCCCUGGAAGAGGCGGUGdTdT
937
CACCGCCUCUUCCAGGGCC
1122

AD-73891
A-147927
UCCAGGGCCUGGGCAAGCUdTdT
753
A-147928
AGCUUGCCCAGGCCCUGGAdTdT
938
UCCAGGGCCUGGGCAAGCU
1123

AD-73892
A-147929
GCAAGCUGGAGUACCUGCUdTdT
754
A-147930
AGCAGGUACUCCAGCUUGCdTdT
939
GCAAGCUGGAGUACCUGCU
1124

AD-73893
A-147931
UACCUGCUGCUCUCCCGCAdTdT
755
A-147932
UGCGGGAGAGCAGCAGGUAdTdT
940
UACCUGCUGCUCUCCCGCA
1125

AD-73894
A-147933
CUCUCCCGCAACCGCCUGAdTdT
756
A-147934
UCAGGCGGUUGCGGGAGAGdTdT
941
CUCUCCCGCAACCGCCUGG
1126

AD-73895
A-147935
CCGCCUGGCAGAGCUGCCAdTdT
757
A-147936
UGGCAGCUCUGCCAGGCGGdTdT
942
CCGCCUGGCAGAGCUGCCG
1127

AD-73896
A-147937
AGCUGCCGGCGGACGCCCUdTdT
758
A-147938
AGGGCGUCCGCCGGCAGCUdTdT
943
AGCUGCCGGCGGACGCCCU
1128

AD-73897
A-147939
GACGCCCUGGGCCCCCUGAdTdT
759
A-147940
UCAGGGGGCCCAGGGCGUCdTdT
944
GACGCCCUGGGCCCCCUGC
1129

AD-73898
A-147941
CCCCUGCAGCGGGCCUUCUdTdT
760
A-147942
AGAAGGCCCGCUGCAGGGGdTdT
945
CCCCUGCAGCGGGCCUUCU
1130

AD-73899
A-147943
GGGCCUUCUGGCUGGACGUdTdT
761
A-147944
ACGUCCAGCCAGAAGGCCCdTdT
946
GGGCCUUCUGGCUGGACGU
1131

AD-73900
A-147945
UGGACGUCUCGCACAACCAdTdT
762
A-147946
UGGUUGUGCGAGACGUCCAdTdT
947
UGGACGUCUCGCACAACCG
1132

AD-73901
A-147947
ACAACCGCCUGGAGGCAUUdTdT
763
A-147948
AAUGCCUCCAGGCGGUUGUdTdT
948
ACAACCGCCUGGAGGCAUU
1133

AD-73902
A-147949
GAGGCAUUGCCCAACAGCAdTdT
764
A-147950
UGCUGUUGGGCAAUGCCUCdTdT
949
GAGGCAUUGCCCAACAGCC
1134

AD-73903
A-147951
CAACAGCCUCUUGGCACCAdTdT
765
A-147952
UGGUGCCAAGAGGCUGUUGdTdT
950
CAACAGCCUCUUGGCACCA
1135

AD-73904
A-147953
UUGGCACCACUGGGGCGGAdTdT
766
A-147954
UCCGCCCCAGUGGUGCCAAdTdT
951
UUGGCACCACUGGGGCGGC
1136

AD-73905
A-147955
UGGGGCGGCUGCGCUACCUdTdT
767
A-147956
AGGUAGCGCAGCCGCCCCAdTdT
952
UGGGGCGGCUGCGCUACCU
1137

AD-73906
A-147957
CGCUACCUCAGCCUCAGGAdTdT
768
A-147958
UCCUGAGGCUGAGGUAGCGdTdT
953
CGCUACCUCAGCCUCAGGA
1138

AD-73907
A-147959
UCAGGAACAACUCACUGCAdTdT
769
A-147960
UGCAGUGAGUUGUUCCUGAdTdT
954
UCAGGAACAACUCACUGCG
1139

AD-73908
A-147961
CUCACUGCGGACCUUCACAdTdT
770
A-147962
UGUGAAGGUCCGCAGUGAGdTdT
955
CUCACUGCGGACCUUCACG
1140

AD-73909
A-147963
ACCUUCACGCCGCAGCCCAdTdT
771
A-147964
UGGGCUGCGGCGUGAAGGUdTdT
956
ACCUUCACGCCGCAGCCCC
1141

AD-73910
A-147965
CAGCCCCCGGGCCUGGAGAdTdT
772
A-147966
UCUCCAGGCCCGGGGGCUGdTdT
957
CAGCCCCCGGGCCUGGAGC
1142

AD-73911
A-147967
GCCUGGAGCGCCUGUGGCUdTdT
773
A-147968
AGCCACAGGCGCUCCAGGCdTdT
958
GCCUGGAGCGCCUGUGGCU
1143

AD-73912
A-147969
CUGUGGCUGGAGGGUAACAdTdT
774
A-147970
UGUUACCCUCCAGCCACAGdTdT
959
CUGUGGCUGGAGGGUAACC
1144

AD-73913
A-147971
GGUAACCCCUGGGACUGUAdTdT
775
A-147972
UACAGUCCCAGGGGUUACCdTdT
960
GGUAACCCCUGGGACUGUG
1145

AD-73914
A-147973
GGGACUGUGGCUGCCCUCUdTdT
776
A-147974
AGAGGGCAGCCACAGUCCCdTdT
961
GGGACUGUGGCUGCCCUCU
1146

AD-73915
A-147975
UGCCCUCUCAAGGCGCUGAdTdT
777
A-147976
UCAGCGCCUUGAGAGGGCAdTdT
962
UGCCCUCUCAAGGCGCUGC
1147

AD-73916
A-147977
CGCUGCGGGACUUCGCCCUdTdT
778
A-147978
AGGGCGAAGUCCCGCAGCGdTdT
963
CGCUGCGGGACUUCGCCCU
1148

AD-73917
A-147979
UUCGCCCUGCAGAACCCCAdTdT
779
A-147980
UGGGGUUCUGCAGGGCGAAdTdT
964
UUCGCCCUGCAGAACCCCA
1149

AD-73918
A-147981
CAGAACCCCAGUGCUGUGAdTdT
780
A-147982
UCACAGCACUGGGGUUCUGdTdT
965
CAGAACCCCAGUGCUGUGC
1150

AD-73919
A-147983
UGCUGUGCCCCGCUUCGUAdTdT
781
A-147984
UACGAAGCGGGGCACAGCAdTdT
966
UGCUGUGCCCCGCUUCGUC
1151

AD-73920
A-147985
CUUCGUCCAGGCCAUCUGUdTdT
782
A-147986
ACAGAUGGCCUGGACGAAGdTdT
967
CUUCGUCCAGGCCAUCUGU
1152

AD-73921
A-147987
CAUCUGUGAGGGGGACGAUdTdT
783
A-147988
AUCGUCCCCCUCACAGAUGdTdT
968
CAUCUGUGAGGGGGACGAU
1153

AD-73922
A-147989
GGGGACGAUUGCCAGCCGAdTdT
784
A-147990
UCGGCUGGCAAUCGUCCCCdTdT
969
GGGGACGAUUGCCAGCCGC
1154

AD-73923
A-147991
CAGCCGCCCGCGUACACCUdTdT
785
A-147992
AGGUGUACGCGGGCGGCUGdTdT
970
CAGCCGCCCGCGUACACCU
1155

AD-73924
A-147993
CGUACACCUACAACAACAUdTdT
786
A-147994
AUGUUGUUGUAGGUGUACGdTdT
971
CGUACACCUACAACAACAU
1156

AD-73925
A-147995
AACAACAUCACCUGUGCCAdTdT
787
A-147996
UGGCACAGGUGAUGUUGUUdTdT
972
AACAACAUCACCUGUGCCA
1157

AD-73926
A-147997
UGUGCCAGCCCGCCCGAGAdTdT
788
A-147998
UCUCGGGCGGGCUGGCACAdTdT
973
UGUGCCAGCCCGCCCGAGG
1158

AD-73927
A-147999
CGCCCGAGGUCGUGGGGCUdTdT
789
A-148000
AGCCCCACGACCUCGGGCGdTdT
974
CGCCCGAGGUCGUGGGGCU
1159

AD-73928
A-148001
CGUGGGGCUCGACCUGCGAdTdT
790
A-148002
UCGCAGGUCGAGCCCCACGdTdT
975
CGUGGGGCUCGACCUGCGG
1160

AD-73929
A-148003
ACCUGCGGGACCUCAGCGAdTdT
791
A-148004
UCGCUGAGGUCCCGCAGGUdTdT
976
ACCUGCGGGACCUCAGCGA
1161

AD-73930
A-148005
UCAGCGAGGCCCACUUUGAdTdT
792
A-148006
UCAAAGUGGGCCUCGCUGAdTdT
977
UCAGCGAGGCCCACUUUGC
1162

AD-73931
A-148007
ACUUUGCUCCCUGCUGACAdTdT
793
A-148008
UGUCAGCAGGGAGCAAAGUdTdT
978
ACUUUGCUCCCUGCUGACC
1163

AD-73932
A-148009
CCUGCUGACCAGGUCCCCAdTdT
794
A-148010
UGGGGACCUGGUCAGCAGGdTdT
979
CCUGCUGACCAGGUCCCCG
1164

AD-73933
A-148011
UCCCCGGACUCAAGCCCCAdTdT
795
A-148012
UGGGGCUUGAGUCCGGGGAdTdT
980
UCCCCGGACUCAAGCCCCG
1165

AD-73934
A-148013
CAAGCCCCGGACUCAGGCAdTdT
796
A-148014
UGCCUGAGUCCGGGGCUUGdTdT
981
CAAGCCCCGGACUCAGGCC
1166

AD-73935
A-148015
UCAGGCCCCCACCUGGCUAdTdT
797
A-148016
UAGCCAGGUGGGGGCCUGAdTdT
982
UCAGGCCCCCACCUGGCUC
1167

AD-73936
A-148017
ACCUGGCUCACCUUGUGCUdTdT
798
A-148018
AGCACAAGGUGAGCCAGGUdTdT
983
ACCUGGCUCACCUUGUGCU
1168

AD-73937
A-148019
UUGUGCUGGGGACAGGUCAdTdT
799
A-148020
UGACCUGUCCCCAGCACAAdTdT
984
UUGUGCUGGGGACAGGUCC
1169

AD-73938
A-148021
GACAGGUCCUCAGUGUCCUdTdT
800
A-148022
AGGACACUGAGGACCUGUCdTdT
985
GACAGGUCCUCAGUGUCCU
1170

AD-73939
A-148023
CAGUGUCCUCAGGGGCCUAdTdT
801
A-148024
UAGGCCCCUGAGGACACUGdTdT
986
CAGUGUCCUCAGGGGCCUG
1171

AD-73940
A-148025
GGGCCUGCCCAGUGCACUUdTdT
802
A-148026
AAGUGCACUGGGCAGGCCCdTdT
987
GGGCCUGCCCAGUGCACUU
1172

AD-73941
A-148027
UGCACUUGCUGGAAGACGAdTdT
803
A-148028
UCGUCUUCCAGCAAGUGCAdTdT
988
UGCACUUGCUGGAAGACGC
1173

AD-73942
A-148029
UGGAAGACGCAAGGGCCUAdTdT
804
A-148030
UAGGCCCUUGCGUCUUCCAdTdT
989
UGGAAGACGCAAGGGCCUG
1174

AD-73943
A-148031
AGGGCCUGAUGGGGUGGAAdTdT
805
A-148032
UUCCACCCCAUCAGGCCCUdTdT
990
AGGGCCUGAUGGGGUGGAA
1175

AD-73944
A-148033
GGGUGGAAGGCAUGGCGGAdTdT
806
A-148034
UCCGCCAUGCCUUCCACCCdTdT
991
GGGUGGAAGGCAUGGCGGC
1176

AD-73945
A-148035
UGGCGGCCCCCCCAGCUGUdTdT
807
A-148036
ACAGCUGGGGGGGCCGCCAdTdT
992
UGGCGGCCCCCCCAGCUGU
1177

AD-73946
A-148037
CAGCUGUCAUCAAUUAAAGdTdT
808
A-148038
CUUUAAUUGAUGACAGCUGdTdT
993
CAGCUGUCAUCAAUUAAAG
1178

AD-73947
A-148039
AAUUAAAGGCAAAGGCAAUdTdT
809
A-148040
AUUGCCUUUGCCUUUAAUUdTdT
994
AAUUAAAGGCAAAGGCAAU
1179

AD-73948
A-148041
AAGGCAAUCGAAUCUAAAAdTdT
810
A-148042
UUUUAGAUUCGAUUGCCUUdTdT
995
AAGGCAAUCGAAUCUAAAA
1180

Example 5—In Vitro Screening
Cell Culture and Plasmids/Transfections for Dual-Glo® Assay:

HeLa cells (ATCC) were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 50 of siRNA duplexes per well into a 384-well plate and incubated at room temperature for 15 minutes. Forty μl of Dulbecco's Modified Eagle Medium (Life Tech) containing ˜5×10³cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

Real time PCR

Two μl of cDNA were added to a master mix containing 0.5 μl of Human GAPDH TaqMan Probe (4326317E), 0.5 μl IGF-1 human probe (Hs01547656_ml) and 50 Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche). Each duplex was tested in duplicate and data were normalized to cells transfected with a non-targeting control siRNA.

To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with a non-targeting control siRNA.

TABLE 9

Unmodified Sense and Antisense Strand Sequences of IGF-1 dsRNAs

Range in

Range in

Duplex
Sense Oligo

SEQ ID
SEQ
Antisense

SEQ ID
SEQ ID

Name
Name
Sense sequence
No: 11
ID NO
Oligo Name
Antisense sequence
No: 11
ID NO

AD-66716
A-133440
GCUGCUUCCGGAGCUGUGAUA
548-568
1181
A-133441
UAUCACAGCUCCGGAAGCAGCAC
546-568
1247

AD-66717
A-133442
UCUGCGGGGCUGAGCUGGUGA
422-442
1182
A-133443
UCACCAGCUCAGCCCCGCAGAGC
420-442
1248

AD-66718
A-133444
CCUGCUCACCUUCACCAGCUA
378-398
1183
A-133445
UAGCUGGUGAAGGUGAGCAGGCA
376-398
1249

AD-66719
A-133446
GUGGAGACAGGGGCUUUUAUU
461-481
1184
A-133447
AAUAAAAGCCCCUGUCUCCACAC
459-481
1250

AD-66720
A-133448
UGGAGACAGGGGCUUUUAUUU
462-482
1185
A-133449
AAAUAAAAGCCCCUGUCUCCACA
460-482
1251

AD-66721
A-133450
GAGACAGGGGCUUUUAUUUCA
464-484
1186
A-133451
UGAAAUAAAAGCCCCUGUCUCCA
462-484
1252

AD-66722
A-133452
CAUGUCCUCCUCGCAUCUCUU
342-362
1187
A-133453
AAGAGAUGCGAGGAGGACAUGGU
340-362
1253

AD-66723
A-133454
UUUUAUUUCAACAAGCCCACA
475-495
1188
A-133455
UGUGGGCUUGUUGAAAUAAAAGC
473-495
1254

AD-66724
A-133456
UGUGGAGACAGGGGCUUUUAU
460-480
1189
A-133457
AUAAAAGCCCCUGUCUCCACACA
458-480
1255

AD-66725
A-133458
UGGAUGAGUGCUGCUUCCGGA
539-559
1190
A-133459
UCCGGAAGCAGCACUCAUCCACG
537-559
1256

AD-66726
A-133460
UCGUGUGUGGAGACAGGGGCU
455-475
1191
A-133461
AGCCCCUGUCUCCACACACGAAC
453-475
1257

AD-66727
A-133462
GAUGUAUUGCGCACCCCUCAA
582-602
1192
A-133463
UUGAGGGGUGCGCAAUACAUCUC
580-602
1258

AD-66728
A-133464
UUCAGUUCGUGUGUGGAGACA
449-469
1193
A-133465
UGUCUCCACACACGAACUGAAGA
447-469
1259

AD-66729
A-133466
CUCCUCGCAUCUCUUCUACCU
348-368
1194
A-133467
AGGUAGAAGAGAUGCGAGGAGGA
346-368
1260

AD-66730
A-133468
AGAUGUAUUGCGCACCCCUCA
581-601
1195
A-133469
UGAGGGGUGCGCAAUACAUCUCC
579-601
1261

AD-66731
A-133470
GCCACACCGACAUGCCCAAGA
638-658
1196
A-133471
UCUUGGGCAUGUCGGUGUGGCGC
636-658
1262

AD-66732
A-133472
GGAGAUGUAUUGCGCACCCCU
579-599
1197
A-133473
AGGGGUGCGCAAUACAUCUCCAG
577-599
1263

AD-66733
A-133474
UUCAACAAGCCCACAGGGUAU
481-501
1198
A-133475
AUACCCUGUGGGCUUGUUGAAAU
479-501
1264

AD-66734
A-133476
UGCCCAGCGCCACACCGACAU
630-650
1199
A-133478
AUGUCGGUGUGGCGCUGGGCACG
628-650
1265

AD-66735
A-133480
GCAUCGUGGAUGAGUGCUGCU
533-553
1200
A-133482
AGCAGCACUCAUCCACGAUGCCU
531-553
1266

AD-66736
A-133484
GGAGACAGGGGCUUUUAUUUA
463-483
1201
A-133486
UAAAUAAAAGCCCCUGUCUCCAC
461-483
1267

AD-66737
A-133488
GGGCUUUUAUUUCAACAAGCA
471-491
1202
A-133490
UGCUUGUUGAAAUAAAAGCCCCU
469-491
1268

AD-66738
A-133492
UCGUGGAUGAGUGCUGCUUCA
536-556
1203
A-133494
UGAAGCAGCACUCAUCCACGAUG
534-556
1269

AD-66739
A-133496
UCCUCGCAUCUCUUCUACCUA
349-369
1204
A-133498
UAGGUAGAAGAGAUGCGAGGAGG
347-369
1270

AD-66740
A-133500
GGGGCUUUUAUUUCAACAAGA
470-490
1205
A-133502
UCUUGUUGAAAUAAAAGCCCCUG
468-490
1271

AD-66741
A-133504
UUUAUUUCAACAAGCCCACAA
476-496
1206
A-133506
UUGUGGGCUUGUUGAAAUAAAAG
474-496
1272

AD-66742
A-133508
GCUGGAGAUGUAUUGCGCACA
576-596
1207
A-133510
UGUGCGCAAUACAUCUCCAGCCU
574-596
1273

AD-66743
A-133512
GGGUAUGGCUCCAGCAGUCGA
496-516
1208
A-133513
UCGACUGCUGGAGCCAUACCCUG
494-516
1274

AD-66744
A-133514
CUGGAGAUGUAUUGCGCACCA
577-597
1209
A-133515
UGGUGCGCAAUACAUCUCCAGCC
575-597
1275

AD-66745
A-133516
GAAGAUGCACACCAUGUCCUA
330-350
1210
A-133517
UAGGACAUGGUGUGCAUCUUCAC
328-350
1276

AD-66746
A-133477
GAUGCUCUUCAGUUCGUGUGU
442-462
1211
A-133479
ACACACGAACUGAAGAGCAUCCA
440-462
1277

AD-66747
A-133481
UGGAUGCUCUUCAGUUCGUGU
440-460
1212
A-133483
ACACGAACUGAAGAGCAUCCACC
438-460
1278

AD-66748
A-133485
GGUGGAUGCUCUUCAGUUCGU
438-458
1213
A-133487
ACGAACUGAAGAGCAUCCACCAG
436-458
1279

AD-66749
A-133489
UGAGCUGGUGGAUGCUCUUCA
432-452
1214
A-133491
UGAAGAGCAUCCACCAGCUCAGC
430-452
1280

AD-66750
A-133493
GCUGGUGGAUGCUCUUCAGUU
435-455
1215
A-133495
AACUGAAGAGCAUCCACCAGCUC
433-455
1281

AD-66751
A-133497
GGAUGCUCUUCAGUUCGUGUA
441-461
1216
A-133499
UACACGAACUGAAGAGCAUCCAC
439-461
1282

AD-66752
A-133501
CUGGUGGAUGCUCUUCAGUUA
436-456
1217
A-133503
UAACUGAAGAGCAUCCACCAGCU
434-456
1283

AD-66753
A-133505
GGCUGAGCUGGUGGAUGCUCU
429-449
1218
A-133507
AGAGCAUCCACCAGCUCAGCCCC
427-449
1284

AD-66754
A-133509
CUGAGCUGGUGGAUGCUCUUA
431-451
1219
A-133511
UAAGAGCAUCCACCAGCUCAGCC
429-451
1285

AD-66755
A-133518
CAUUGUGGAUGAGUGUUGCUU
534-554
1220
A-133519
AAGCAACACUCAUCCACAAUGCC
532-554
1286

AD-66756
A-133520
AGAUACACAUCAUGUCGUCUU
*
1221
A-133521
AAGACGACAUGAUGUGUAUCUUU
*
1287

AD-66757
A-133522
UGGAUGAGUGUUGCUUCCGGA
539-559
1222
A-133523
UCCGGAAGCAACACUCAUCCACA
537-559
1288

AD-66758
A-133524
UGUUGCUUCCGGAGCUGUGAU
547-567
1223
A-133525
AUCACAGCUCCGGAAGCAACACU
545-567
1289

AD-66759
A-133526
GCUUUUACUUCAACAAGCCCA
473-493
1224
A-133527
UGGGCUUGUUGAAGUAAAAGCCC
471-493
1290

AD-66760
A-133528
AUGAGUGUUGCUUCCGGAGCU
542-562
1225
A-133529
AGCUCCGGAAGCAACACUCAUCC
540-562
1291

AD-66761
A-133530
CACACUGACAUGCCCAAGACU
640-660
1226
A-133531
AGUCUUGGGCAUGUCAGUGUGGC
638-660
1292

AD-66762
A-133532
GCUAUGGCUCCAGCAUUCGGA
497-517
1227
A-133533
UCCGAAUGCUGGAGCCAUAGCCU
495-517
1293

AD-66763
A-133534
AAGAUACACAUCAUGUCGUCU
*
1228
A-133535
AGACGACAUGAUGUGUAUCUUUA
*
1294

AD-66764
A-133536
UUGCUUCCGGAGCUGUGAUCU
549-569
1229
A-133537
AGAUCACAGCUCCGGAAGCAACA
547-569
1295

AD-66765
A-133538
UCCGGAGCUGUGAUCUGAGGA
554-574
1230
A-133539
UCCUCAGAUCACAGCUCCGGAAG
552-574
1296

AD-66766
A-133540
GUGGAUGAGUGUUGCUUCCGA
538-558
1231
A-133541
UCGGAAGCAACACUCAUCCACAA
536-558
1297

AD-66767
A-133542
UACACAUCAUGUCGUCUUCAA
*
1232
A-133543
UUGAAGACGACAUGAUGUGUAUC
*
1298

AD-66768
A-133544
AAAGAUACACAUCAUGUCGUA
*
1233
A-133545
UACGACAUGAUGUGUAUCUUUAU
*
1299

AD-66769
A-133546
GGCUAUGGCUCCAGCAUUCGA
496-516
1234
A-133547
UCGAAUGCUGGAGCCAUAGCCUG
494-516
1300

AD-66770
A-133548
ACACUGACAUGCCCAAGACUA
641-661
1235
A-133549
UAGUCUUGGGCAUGUCAGUGUGG
639-661
1301

AD-66771
A-133550
AGUGUUGCUUCCGGAGCUGUA
545-565
1236
A-133551
UACAGCUCCGGAAGCAACACUCA
543-565
1302

AD-66772
A-133552
GAGACCCUUUGCGGGGCUGAA
*
1237
A-133553
UUCAGCCCCGCAAAGGGUCUCUG
*
1303

AD-66773
A-133554
ACUGACAUGCCCAAGACUCAA
643-663
1238
A-133555
UUGAGUCUUGGGCAUGUCAGUGU
641-663
1304

AD-66774
A-133556
GAUACACAUCAUGUCGUCUUA
*
1239
A-133557
UAAGACGACAUGAUGUGUAUCUU
*
1305

AD-66775
A-133558
AAGCCCACAGGCUAUGGCUCA
487-507
1240
A-133559
UGAGCCAUAGCCUGUGGGCUUGU
485-507
1306

Range in

Range in

Duplex
Sense Oligo

SEQ ID
SEQ
Antisense

SEQ ID
SEQ ID

Name
Name
Sense sequence
No: 17
ID NO
Oligo Name
Antisense sequence
No: 17
ID NO

AD-66756
A-133520
AGAUACACAUCAUGUCGUCUU
366-368
1241
A-133521
AAGACGACAUGAUGUGUAUCUUU
364-368
1307

AD-66763
A-133534
AAGAUACACAUCAUGUCGUCU
365-385
1242
A-133535
AGACGACAUGAUGUGUAUCUUUA
363-385
1308

AD-66767
A-133542
UACACAUCAUGUCGUCUUCAA
369-389
1243
A-133543
UUGAAGACGACAUGAUGUGUAUC
367-389
1309

AD-66768
A-133544
AAAGAUACACAUCAUGUCGUA
364-384
1244
A-133545
UACGACAUGAUGUGUAUCUUUAU
362-384
1310

AD-66772
A-133552
GAGACCCUUUGCGGGGCUGAA
449-469
1245
A-133553
UUCAGCCCCGCAAAGGGUCUCUG
447-469
1311

AD-66774
A-133556
GAUACACAUCAUGUCGUCUUA
367-387
1246
A-133557
UAAGACGACAUGAUGUGUAUCUU
365-387
1312

*Targeting sequence in NM_010512 (SEQ ID NO: 7).

TABLE 10

IGF-1 Screen in HeLa cells

Each duplex was tested in duplicate and data were normalized to

cells transfected with a non-targeting control siRNA AD-1955.

Duplex ID
10 nM Avg
STDEV
0.1 nM Avg
STDEV

AD-66716
58.4
3.1
90.3
24.0

AD-66717
82.2
1.6
81.1
10.3

AD-66718
62.7
7.0
76.4
10.1

AD-66719
51.0
6.7
83.2
8.1

AD-66720
30.3
0.9
74.9
15.7

AD-66721
58.0
8.5
82.5
23.9

AD-66722
4.7
1.0
29.2
9.0

AD-66723
70.5
7.3
85.4
11.3

AD-66724
32.4
8.0
82.7
9.7

AD-66725
22.6
6.4
72.8
12.1

AD-66726
32.8
2.1
76.4
25.0

AD-66727
53.5
1.1
83.1
6.5

AD-66728
59.1
11.5
91.5
15.2

AD-66729
27.9
4.2
75.3
27.4

AD-66730
79.1
12.7
87.8
18.4

AD-66731
86.4
15.9
97.0
25.4

AD-66732
81.0
8.3
80.9
15.0

AD-66733
8.7
3.7
43.7
1.9

AD-66734
65.4
3.2
83.7
15.5

AD-66735
62.0
6.7
82.6
8.1

AD-66736
71.9
4.5
83.5
23.4

AD-66737
68.7
4.0
92.0
14.4

AD-66738
19.2
3.3
79.4
14.3

AD-66739
10.6
2.7
61.8
23.6

AD-66740
23.2
4.4
68.2
14.6

AD-66741
83.6
0.5
76.5
14.2

AD-66742
73.1
2.2
86.2
10.1

AD-66743
58.9
1.8
88.1
11.2

AD-66744
53.9
2.1
96.8
7.6

AD-66745
28.3
5.5
76.8
7.9

AD-66746
6.3
0.7
50.1
3.9

AD-66747
8.5
2.8
50.0
0.0

AD-66748
6.2
1.3
34.8
4.1

AD-66749
30.0
0.5
92.4
3.2

AD-66750
27.6
0.7
74.8
1.5

AD-66751
50.1
0.3
89.3
15.2

AD-66752
9.6
1.3
55.5
12.4

AD-66753
54.6
2.1
89.0
0.9

AD-66754
78.6
16.8
104.3
2.0

AD-66755
46.8
4.8
103.4
18.6

AD-66756
86.3
2.5
95.9
18.2

AD-66757
69.1
0.7
103.0
5.0

AD-66758
67.5
3.6
86.5
2.5

AD-66759
106.5
16.2
91.4
20.4

AD-66760
54.2
1.6
86.8
0.4

AD-66761
40.8
3.8
92.2
7.2

AD-66762
96.8
7.1
100.6
8.4

AD-66763
81.2
11.4
92.1
0.0

AD-66764
86.0
2.2
101.2
12.9

AD-66765
100.9
13.7
93.2
25.3

AD-66766
36.6
3.9
78.5
15.7

AD-66767
124.0
12.2
89.9
1.3

AD-66768
113.9
15.1
92.7
15.4

AD-66769
92.0
7.1
93.6
8.2

AD-66770
79.7
3.6
98.7
1.9

AD-66771
60.6
12.1
97.6
10.5

AD-66772
95.5
7.0
95.9
9.9

AD-66773
61.3
3.9
90.7
7.5

AD-66774
95.6
8.9
81.9
21.8

AD-66775
113.1
13.9
99.5
6.8

AD-1955
100.0
8.0

TABLE 11

Modified Sense and Antisense Sequences of IGF-1

SEQ

SEQ

Duplex
Snese Oligo

ID
Antisense

ID

Name
Name
Modified Sense Sequence
NO
Oligo Name
Modified Antisense Sequence
NO

AD-66716
A-133440
GfscsUfgCfuUfcCfGfGfaGfcUfgUfgAfuAfL96
1313
A-133441
usAfsuCfaCfaGfcUfccgGfaAfgCfaGfcsasc
1373

AD-66717
A-133442
UfscsUfgCfgGfgGfCfUfgAfgCfuGfgUfgAfL96
1314
A-133443
usCfsaCfcAfgCfuCfagcCfcCfgCfaGfasgsc
1374

AD-66718
A-133444
CfscsUfgCfuCfaCfCfUfuCfaCfcAfgCfuAfL96
1315
A-133445
usAfsgCfuGfgUfgAfaggUfgAfgCfaGfgscsa
1375

AD-66719
A-133446
GfsusGfgAfgAfcAfGfGfgGfcUfuUfuAfuUfL96
1316
A-133447
asAfsuAfaAfaGfcCfccuGfuCfuCfcAfcsasc
1376

AD-66720
A-133448
UfsgsGfaGfaCfaGfGfGfgCfuUfuUfaUfuUfL96
1317
A-133449
asAfsaUfaAfaAfgCfcccUfgUfcUfcCfascsa
1377

AD-66721
A-133450
GfsasGfaCfaGfgGfGfCfuUfuUfaUfuUfcAfL96
1318
A-133451
usGfsaAfaUfaAfaAfgccCfcUfgUfcUfcscsa
1378

AD-66722
A-133452
CfsasUfgUfcCfuCfCfUfcGfcAfuCfuCfuUfL96
1319
A-133453
asAfsgAfgAfuGfcGfaggAfgGfaCfaUfgsgsu
1379

AD-66723
A-133454
UfsusUfuAfuUfuCfAfAfcAfaGfcCfcAfcAfL96
1320
A-133455
usGfsuGfgGfcUfuGfuugAfaAfuAfaAfasgsc
1380

AD-66724
A-133456
UfsgsUfgGfaGfaCfAfGfgGfgCfuUfuUfaUfL96
1321
A-133457
asUfsaAfaAfgCfcCfcugUfcUfcCfaCfascsa
1381

AD-66725
A-133458
UfsgsGfaUfgAfgUfGfCfuGfcUfuCfcGfgAfL96
1322
A-133459
usCfscGfgAfaGfcAfgcaCfuCfaUfcCfascsg
1382

AD-66726
A-133460
UfscsGfuGfuGfuGfGfAfgAfcAfgGfgGfcUfL96
1323
A-133461
asGfscCfcCfuGfuCfuccAfcAfcAfcGfasasc
1383

AD-66727
A-133462
GfsasUfgUfaUfuGfCfGfcAfcCfcCfuCfaAfL96
1324
A-133463
usUfsgAfgGfgGfuGfcgcAfaUfaCfaUfcsusc
1384

AD-66728
A-133464
UfsusCfaGfuUfcGfUfGfuGfuGfgAfgAfcAfL96
1325
A-133465
usGfsuCfuCfcAfcAfcacGfaAfcUfgAfasgsa
1385

AD-66729
A-133466
CfsusCfcUfcGfcAfUfCfuCfuUfcUfaCfcUfL96
1326
A-133467
asGfsgUfaGfaAfgAfgauGfcGfaGfgAfgsgsa
1386

AD-66730
A-133468
AfsgsAfuGfuAfuUfGfCfgCfaCfcCfcUfcAfL96
1327
A-133469
usGfsaGfgGfgUfgCfgcaAfuAfcAfuCfuscsc
1387

AD-66731
A-133470
GfscsCfaCfaCfcGfAfCfaUfgCfcCfaAfgAfL96
1328
A-133471
usCfsuUfgGfgCfaUfgucGfgUfgUfgGfcsgsc
1388

AD-66732
A-133472
GfsgsAfgAfuGfuAfUfUfgCfgCfaCfcCfcUfL96
1329
A-133473
asGfsgGfgUfgCfgCfaauAfcAfuCfuCfcsasg
1389

AD-66733
A-133474
UfsusCfaAfcAfaGfCfCfcAfcAfgGfgUfaUfL96
1330
A-133475
asUfsaCfcCfuGfuGfggcUfuGfuUfgAfasasu
1390

AD-66734
A-133476
UfsgsCfcCfaGfcGfCfCfaCfaCfcGfaCfaUfL96
1331
A-133478
asUfsgUfcGfgUfgUfggcGfcUfgGfgCfascsg
1391

AD-66735
A-133480
GfscsAfuCfgUfgGfAfUfgAfgUfgCfuGfcUfL96
1332
A-133482
asGfscAfgCfaCfuCfaucCfaCfgAfuGfcscsu
1392

AD-66736
A-133484
GfsgsAfgAfcAfgGfGfGfcUfuUfuAfuUfuAfL96
1333
A-133486
usAfsaAfuAfaAfaGfcccCfuGfuCfuCfcsasc
1393

AD-66737
A-133488
GfsgsGfcUfuUfuAfUfUfuCfaAfcAfaGfcAfL96
1334
A-133490
usGfscUfuGfuUfgAfaauAfaAfaGfcCfcscsu
1394

AD-66738
A-133492
UfscsGfuGfgAfuGfAfGfuGfcUfgCfuUfcAfL96
1335
A-133494
usGfsaAfgCfaGfcAfcucAfuCfcAfcGfasusg
1395

AD-66739
A-133496
UfscsCfuCfgCfaUfCfUfcUfuCfuAfcCfuAfL96
1336
A-133498
usAfsgGfuAfgAfaGfagaUfgCfgAfgGfasgsg
1396

AD-66740
A-133500
GfsgsGfgCfuUfuUfAfUfuUfcAfaCfaAfgAfL96
1337
A-133502
usCfsuUfgUfuGfaAfauaAfaAfgCfcCfcsusg
1397

AD-66741
A-133504
UfsusUfaUfuUfcAfAfCfaAfgCfcCfaCfaAfL96
1338
A-133506
usUfsgUfgGfgCfuUfguuGfaAfaUfaAfasasg
1398

AD-66742
A-133508
GfscsUfgGfaGfaUfGfUfaUfuGfcGfcAfcAfL96
1339
A-133510
usGfsuGfcGfcAfaUfacaUfcUfcCfaGfcscsu
1399

AD-66743
A-133512
GfsgsGfuAfuGfgCfUfCfcAfgCfaGfuCfgAfL96
1340
A-133513
usCfsgAfcUfgCfuGfgagCfcAfuAfcCfcsusg
1400

AD-66744
A-133514
CfsusGfgAfgAfuGfUfAfuUfgCfgCfaCfcAfL96
1341
A-133515
usGfsgUfgCfgCfaAfuacAfuCfuCfcAfgscsc
1401

AD-66745
A-133516
GfsasAfgAfuGfcAfCfAfcCfaUfgUfcCfuAfL96
1342
A-133517
usAfsgGfaCfaUfgGfuguGfcAfuCfuUfcsasc
1402

AD-66746
A-133477
GfsasUfgCfuCfuUfCfAfgUfuCfgUfgUfgUfL96
1343
A-133479
asCfsaCfaCfgAfaCfugaAfgAfgCfaUfcscsa
1403

AD-66747
A-133481
UfsgsGfaUfgCfuCfUfUfcAfgUfuCfgUfgUfL96
1344
A-133483
asCfsaCfgAfaCfuGfaagAfgCfaUfcCfascsc
1404

AD-66748
A-133485
GfsgsUfgGfaUfgCfUfCfuUfcAfgUfuCfgUfL96
1345
A-133487
asCfsgAfaCfuGfaAfgagCfaUfcCfaCfcsasg
1405

AD-66749
A-133489
UfsgsAfgCfuGfgUfGfGfaUfgCfuCfuUfcAfL96
1346
A-133491
usGfsaAfgAfgCfaUfccaCfcAfgCfuCfasgsc
1406

AD-66750
A-133493
GfscsUfgGfuGfgAfUfGfcUfcUfuCfaGfuUfL96
1347
A-133495
asAfscUfgAfaGfaGfcauCfcAfcCfaGfcsusc
1407

AD-66751
A-133497
GfsgsAfuGfcUfcUfUfCfaGfuUfcGfuGfuAfL96
1348
A-133499
usAfscAfcGfaAfcUfgaaGfaGfcAfuCfcsasc
1408

AD-66752
A-133501
CfsusGfgUfgGfaUfGfCfuCfuUfcAfgUfuAfL96
1349
A-133503
usAfsaCfuGfaAfgAfgcaUfcCfaCfcAfgscsu
1409

AD-66753
A-133505
GfsgsCfuGfaGfcUfGfGfuGfgAfuGfcUfcUfL96
1350
A-133507
asGfsaGfcAfuCfcAfccaGfcUfcAfgCfcscsc
1410

AD-66754
A-133509
CfsusGfaGfcUfgGfUfGfgAfuGfcUfaffuAfL96
1351
A-133511
usAfsaGfaGfcAfuCfcacCfaGfcUfcAfgscsc
1411

AD-66755
A-133518
CfsasUfuGfuGfgAfUfGfaGfuGfuUfgCfuUfL96
1352
A-133519
asAfsgCfaAfcAfcUfcauCfcAfcAfaUfgscsc
1412

AD-66756
A-133520
AfsgsAfuAfcAfcAfUfCfaUfgUfcGfuCfuUfL96
1353
A-133521
asAfsgAfcGfaCfaUfgauGfuGfuAfuCfususu
1413

AD-66757
A-133522
UfsgsGfaUfgAfgUfGfUfuGfcUfuCfcGfgAfL96
1354
A-133523
usCfscGfgAfaGfcAfacaCfuCfaUfcCfascsa
1414

AD-66758
A-133524
UfsgsUfuGfcUfuCfCfGfgAfgCfuGfuGfaUfL96
1355
A-133525
asUfscAfcAfgCfuCfcggAfaGfcAfaCfascsu
1415

AD-66759
A-133526
GfscsUfuUfuAfcUfUfCfaAfcAfaGfcCfcAfL96
1356
A-133527
usGfsgGfcUfuGfuUfgaaGfuAfaAfaGfcscsc
1416

AD-66760
A-133528
AfsusGfaGfuGfuUfGfCfuUfcCfgGfaGfcUfL96
1357
A-133529
asGfscUfcCfgGfaAfgcaAfcAfcUfcAfuscsc
1417

AD-66761
A-133530
CfsasCfaCfuGfaCfAfUfgCfcCfaAfgAfcUfL96
1358
A-133531
asGfsuCfuUfgGfgCfaugUfcAfgUfgUfgsgsc
1418

AD-66762
A-133532
GfscsUfaUfgGfcUfCfCfaGfcAfuUfcGfgAfL96
1359
A-133533
usCfscGfaAfuGfcUfggaGfcCfaUfaGfcscsu
1419

AD-66763
A-133534
AfsasGfaUfaCfaCfAfUfcAfuGfuCfgUfcUfL96
1360
A-133535
asGfsaCfgAfcAfuGfaugUfgUfaUfaffususa
1420

AD-66764
A-133536
UfsusGfcUfuCfcGfGfAfgCfuGfuGfaUfcUfL96
1361
A-133537
asGfsaUfcAfcAfgCfuccGfgAfaGfcAfascsa
1421

AD-66765
A-133538
UfscsCfgGfaGfcUfGfUfgAfuCfuGfaGfgAfL96
1362
A-133539
usCfscUfcAfgAfuCfacaGfcUfcCfgGfasasg
1422

AD-66766
A-133540
GfsusGfgAfuGfaGfUfGfuUfgCfuUfcCfgAfL96
1363
A-133541
usCfsgGfaAfgCfaAfcacUfcAfuCfcAfcsasa
1423

AD-66767
A-133542
UfsasCfaCfaUfcAfUfGfuCfgUfcUfuCfaAfL96
1364
A-133543
usUfsgAfaGfaCfgAfcauGfaUfgUfgUfasusc
1424

AD-66768
A-133544
AfsasAfgAfuAfcAfCfAfuCfaUfgUfcGfuAfL96
1365
A-133545
usAfscGfaCfaUfgAfuguGfuAfuCfuUfusasu
1425

AD-66769
A-133546
GfsgsCfuAfuGfgCfUfCfcAfgCfaUfuCfgAfL96
1366
A-133547
usCfsgAfaUfgCfuGfgagCfcAfuAfgCfcsusg
1426

AD-66770
A-133548
AfscsAfcUfgAfcAfUfGfcCfcAfaGfaCfuAfL96
1367
A-133549
usAfsgUfcUfuGfgGfcauGfuCfaGfuGfusgsg
1427

AD-66771
A-133550
AfsgsUfgUfuGfcUfUfCfcGfgAfgCfuGfuAfL96
1368
A-133551
usAfscAfgCfuCfcGfgaaGfcAfaCfaCfuscsa
1428

AD-66772
A-133552
GfsasGfaCfcCfuUfUfGfcGfgGfgCfuGfaAfL96
1369
A-133553
usUfscAfgCfcCfcGfcaaAfgGfgUfcUfcsusg
1429

AD-66773
A-133554
AfscsUfgAfcAfuGfCfCfcAfaGfaCfuCfaAfL96
1370
A-133555
usUfsgAfgUfcUfuGfggcAfuGfuCfaGfusgsu
1430

AD-66774
A-133556
GfsasUfaCfaCfaUfCfAfuGfuCfgUfcUfuAfL96
1371
A-133557
usAfsaGfaCfgAfcAfugaUfgUfgUfaUfcsusu
1431

AD-66775
A-133558
AfsasGfcCfcAfcAfGfGfcUfaUfgGfcUfcAfL96
1372
A-133559
usGfsaGfcCfaUfaGfccuGfuGfgGfcUfusgsu
1432

Example 6—Knockdown of IGF-1 Expression with an IGF-1 siRNA Decreases Expression of IGF-1

A series of siRNAs targeting mouse IGF-1 were designed and tested for the ability to knockdown expression of IGF-1 mRNA in 6-8 week old C57Bl/6 female mice. Duplexes were selected for further optimization using chemical modifications. Analysis for IGF-1 knockdown in mice using the same assay identified the AD-68112 duplex (sense sequence gsasuacaCfaUfCfAfugucgucuuaL96 (SEQ ID NO:1433); antisense sequence usAfsagaCfgAfCfaugaUfgUfguaucsusu (SEQ ID NO: 1434), based on the sequence of the AD-66774 duplex, for use in further studies.

A single 3 mg/kg or 10 mg/kg dose of AD-68112; or PBS control, was administered subcutaneously on day 0 to 6-8 week old C57Bl/6 female mice (n=3 per group). On days 7, 14, and 21 the mice were sacrificed to assess knockdown of IGF-1 mRNA in liver by qPCR.

AD-68112 was found to be effective in decreasing expression of IGF-1 mRNA. The results are shown in the table below. Results are expressed as mRNA levels relative to control.

Dose
Day 7
Day 14
Day 21

3 mg/kg
7.4
17.8
33.3

10 mg/kg
3.4
7.4
12.7

In a separate study, a decrease in serum IGF-1 was observed in 6-8 week old C57Bl/6 female mice (n=3) response to treatment with AD-68112 in a single dose of 3 mg/kg. Specifically, AD-68112 decreased the serum IGF-1 protein level to about 17%, 70%, and 55% on days 7, 14, and 21, respectively.

Further, in a dose-response study, AD-68112 was demonstrated to be effective in knocking down the expression of IGF-1 mRNA in the liver in a dose-response manner. Specifically, C57Bl/6 female mice, 6-8 weeks of age (n=3 per group) were administered a single 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 10 mg/kg AD-68112 at; or a PBS control. IGF-1 serum protein level reduction was observed in a dose response manner.

Example 7—Knockdown of IGFALS and IGF-1 Expression with IGFALS and IGF-1 siRNAs Alone and in Combination

A series of siRNAs targeted each to mouse IGFALS (AD-66807, sense sequence, ascsagauGfaGfCfUfcagcgucuuuL96, SEQ ID NO: 1435; and antisense sequence asAfsagaCfgCfUfgagcUfcAfucugusgsu, SEQ ID NO:1436) and mouse IGF-1 (AD-68112) were tested for the ability to knockdown expression of IGFALS and IGF-1 mRNA and protein expression in 6-8 week old C57Bl/6 female mice, either alone or in combination.

Weekly 3 mg/kg doses of AD-66807 and AD-68112, either alone or in combination; or PBS control, were administered to 4 week old C57Bl/6 female mice (n=8 per group) subcutaneously starting at day 0 for 8 weeks. On day 58 or 59, the mice were sacrificed to assess knockdown of IGFALS and IGF-1 mRNA in liver by qPCR. Serum IGFALS levels were assayed by western blot and serum IGF-1 levels were assayed by ELISA.

AD-66807 and AD-68112 were found to be effective in decreasing expression of IGFALS and IGF-1 mRNA and protein. The results are shown in the tables below. RNA and protein levels are expressed as levels relative to control.

IGFALS and IGF-1 mRNA Levels Relative to Control

Treatment
IGFALS
IGF-1

Control (PBS)
1.16
1.05

SiRNA-IGFALS (3 mg/kg)
0.11
1.10

SiRNA-IGF-1 (3 mg/kg)
0.70
0.03

SiRNA-ALS (3 mg/kg) + IGF-1 (3 mg/kg)
0.09
0.05

IGFALS and IGF-1 Protein Levels Relative to Control.

Treatment
IGFALS
IGF-1

Control (PBS)
1.0
1.0

SiRNA-IGFALS (3 mg/kg)
0.03
0.26

SiRNA-IGF-1 (3 mg/kg)
0.61
0.13

SiRNA-ALS (3 mg/kg) + IGF-1 (3 mg/kg)
0.01
0.05

A dose response study was performed. AD-66807 and AD-68112 were subcutaneously administered to 6-8 week old C57Bl/6 female mice (n=3 per group) at 0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10 mg/kg, either alone or in combination; or PBS control starting at day 0. On day 14, the mice were sacrificed to assess knockdown of IGFALS and IGF-1 mRNA in liver by qPCR. Serum IGF-1 levels were assayed by ELISA.

AD-66807 and AD-68112 were found to be effective in decreasing expression of serum IGF-1 protein. The results are shown in the table below. Protein levels are expressed as levels relative to control.

Serum IGF-1 Protein Levels Relative to Control at Day 7.

0
0.3
1.0
3.0
10.0

Treatment
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg

SiRNA-IGFALS
100
0.81
0.54
0.37
0.31

SiRNA-IGF-1
100
1.13
0.83
0.52
0.49

SiRNA-IGFALS +
100
0.61
0.34
0.14
0.06

SiRNA-IGF-1

Example 8—Knockdown of IGFALS and IGF-1 Expression with IGFALS and IGF-1 siRNAs Alone and in Combination in a Transgenic Mouse Expressing Bovine Growth Hormone

Similar knockdown of serum IGFALS and IGF-1 levels were observed in a transgenic mouse that constitutively expresses bovine growth hormone and recapitulates some of the features of acromegaly (Olsson et al, Am J Physiol Endocrinol Metab. 2003; 285:E504-11). Specifically, AD-66807 and AD-68112 were demonstrated to decrease serum levels of IGF-1 protein. At least a trend in decreased weight gain was observed in male mice treated with either AD-66807 or AD-68112 alone, and in female mice treated with a combination of AD-66807 and AD-68112.

Example 9—IGFALS Transcripts, siRNA Design, and siRNA Screening

A set of siRNAs targeting the human IGFALS, “insulin like growth factor binding protein acid labile subunit” (human: NCBI refseqID NM_004970 (SEQ ID NO: 1); NCBI GeneID: 3483), as well as toxicology-species IGFALS orthologs (cynomolgus monkey: XM_005590898) were designed using custom R and Python scripts. The human NM_004970 REFSEQ mRNA, version 2, has a length of 2168 bases.

The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 19mer siRNA from position 10 through the end was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct siRNA designs targeting a large number of vertebrate genes. Subsets of the IGFALS siRNAs were designed with perfect or near-perfect matches between human and cynomolgus monkey. For each strand of the siRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the siRNA and all potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed region, defined here as positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the siRNA, defined here as positions 10-11 of the antisense oligonucleotide. The relative weight of the mismatches was 2.8; 1.2:1 for seed mismatches, cleavage site, and other positions up through antisense position 19. Mismatches in the first position were ignored. A specificity score was calculated for each strand by summing the value of each weighted mismatch. Preference was given to siRNAs whose antisense score in human was >=2.2 and predicted efficacy was >=50% knockdown of the transcript.

In Vitro Dual-Glo® Screening

Cell Culture and Transfections

Cos7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂in DMEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. Human IGFALS (NM_004970 (SEQ ID NO:1) was cloned into the psicheck2 vector to generate the Dual-Glo® Luciferase construct. The Dual-luciferase plasmid was co-transfected with siRNA into 5000 cells using Lipofectamine RNAiMax (Invitrogen, Carlsbad Calif. cat #13778-150). For each well of a 384 well plate, 0.1 μl of Lipofectamine was added to 5 ng of plasmid vector and siRNA in 15 μl of Opti-MEM and allowed to complex at room temperature for 15 minutes. The mixture was then added to the cells resuspended in 35 ul of fresh complete media. Cells were incubated for 48 hours before luciferase was measured. Screens were performed at 10 nM and 0.1 nM final duplex concentration.

Dual-Glo® Luciferase Assay

Forty-eight hours after the siRNAs were transfected, Firefly (transfection control) and Renilla (fused to IGFALS target sequence in 3′ UTR) luciferase were measured. First, media was removed from cells. Then Firefly luciferase activity was measured by adding 20 ul of Dual-Glo® Luciferase Reagent mixed with 20 ul of complete media to each well. The mixture was incubated at room temperature for 30 minutes before luminescense (500 nm) was measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 20 ul of room temperature of Dual-Glo® Stop & Glo® Reagent to each well and the plates were incubated for 20 minutes before luminescence was again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent quenched the firefly luciferase signal and sustained luminescence for the Renilla luciferase reaction. siRNA activity was determined by normalizing the Renilla (IGFALS) signal to the Firefly (control) signal within each well. The magnitude of siRNA activity was then assessed relative to cells that were transfected with the same vector but were not treated with siRNA or were treated with a non-targeting siRNA. All transfections were done in quadruplicates.

TABLE 12

Unmodified Sense and Antisense Strand Sequences of IGFALS dsRNAs

Sense

Position

Antisense

Position

Duplex
Oligo

in NM_
SEQ ID
Oligo

in NM_
SEQ ID

Name
Name
Sense Sequence
004970.2
NO
Name
Antisense Sequence
004970.2
NO

AD-76171
A-152158
CAAUUAAAGGCAAAGGCAAUA
2058-2078
1437
A-152159
UAUUGCCUUUGCCUUUAAUUGAU
2056-2078
1484

AD-76172
A-152160
ACACCUACAACAACAUCACCA
1808-1828
1438
A-152161
UGGUGAUGUUGUUGUAGGUGUAC
1806-1828
1485

AD-76173
A-152162
UACACCUACAACAACAUCACA
1807-1827
1439
A-152163
UGUGAUGUUGUUGUAGGUGUACG
1805-1827
1486

AD-76174
A-152164
CAUCAAGGCAAACGUGUUCGA
777-797
1440
A-152165
UCGAACACGUUUGCCUUGAUGGC
775-797
1487

AD-76175
A-152166
CACCUACAACAACAUCACCUA
1809-1829
1441
A-152167
UAGGUGAUGUUGUUGUAGGUGUA
1807-1829
1488

AD-76176
A-152168
CGUACACCUACAACAACAUCA
1805-1825
1442
A-152169
UGAUGUUGUUGUAGGUGUACGCG
1803-1825
1489

AD-76177
A-152170
GUACACCUACAACAACAUCAA
1806-1826
1443
A-152171
UUGAUGUUGUUGUAGGUGUACGC
1804-1826
1490

AD-76178
A-152172
GACUCUUCCUCAAGGACAACA
1322-1342
1444
A-152173
UGUUGUCCUUGAGGAAGAGUCGG
1320-1342
1491

AD-76179
A-152174
AGCCUUCCAGAACCUCUCCAA
357-377
1445
A-152175
UUGGAGAGGUUCUGGAAGGCUGC
355-377
1492

AD-76180
A-152176
UCACGCUAGACCACAACCAGA
1109-1129
1446
A-152177
UCUGGUUGUGGUCUAGCGUGAGC
1107-1129
1493

AD-76181
A-152178
UGGACGUCUCGCACAACCGCA
1541-1561
1447
A-152179
UGCGGUUGUGCGAGACGUCCAGC
1539-1561
1494

AD-76182
A-152180
ACCUGGACCGCAACCUCAUCA
824-844
1448
A-152181
UGAUGAGGUUGCGGUCCAGGUAG
822-844
1495

AD-76183
A-152182
UGGACCUGUCCCACAACCGCA
893-913
1449
A-152183
UGCGGUUGUGGGACAGGUCCAGC
891-913
1496

AD-76184
A-152184
UGCGGCUGUCCCACAACGCCA
965-985
1450
A-152185
UGGCGUUGUGGGACAGCCGCAGC
963-985
1497

AD-76185
A-152186
CGCUCGGCCUCAGCAACAACA
530-550
1451
A-152187
UGUUGUUGCUGAGGCCGAGCGAG
528-550
1498

AD-76186
A-152188
CCUCAGGAACAACUCACUGCA
1617-1637
1452
A-152189
UGCAGUGAGUUGUUCCUGAGGCU
1615-1637
1499

AD-76187
A-152190
CUGCGGACCUUCACGCCGCAA
1633-1653
1453
A-152191
UUGCGGCGUGAAGGUCCGCAGUG
1631-1653
1500

AD-76188
A-152192
CCUCUCUGGGAACUGUCUCCA
1185-1205
1454
A-152193
UGGAGACAGUUCCCAGAGAGGUU
1183-1205
1501

AD-76189
A-152194
GCUCUCCCGCAACCGCCUGGA
1473-1493
1455
A-152195
UCCAGGCGGUUGCGGGAGAGCAG
1471-1493
1502

AD-76190
A-152196
CGUCUCGCACAACCGCCUGGA
1545-1565
1456
A-152197
UCCAGGCGGUUGUGCGAGACGUC
1543-1565
1503

AD-76191
A-152198
CACGCUAGACCACAACCAGCA
1110-1130
1457
A-152199
UGCUGGUUGUGGUCUAGCGUGAG
1108-1130
1504

AD-76192
A-152200
UGCUCUCCCGCAACCGCCUGA
1472-1492
1458
A-152201
UCAGGCGGUUGCGGGAGAGCAGC
1470-1492
1505

AD-76193
A-152202
ACCUCAGCCUCAGGAACAACA
1610-1630
1459
A-152203
UGUUGUUCCUGAGGCUGAGGUAG
1608-1630
1506

AD-76194
A-152204
CACCUUCAAGGACCUGCACUA
1005-1025
1460
A-152205
UAGUGCAGGUCCUUGAAGGUGCG
1003-1025
1507

AD-76195
A-152206
GGCCUCGUGGGCAUUGAGGAA
1342-1362
1461
A-152207
UUCCUCAAUGCCCACGAGGCCGU
1340-1362
1508

AD-76196
A-152208
ACCUUCAAGGACCUGCACUUA
1006-1026
1462
A-152209
UAAGUGCAGGUCCUUGAAGGUGC
1004-1026
1509

AD-76197
A-152210
GGCCUUCUGGCUGGACGUCUA
1530-1550
1463
A-152211
UAGACGUCCAGCCAGAAGGCCCG
1528-1550
1510

AD-76198
A-152212
GGCAUUGAGGAGCAGAGCCUA
1351-1371
1464
A-152213
UAGGCUCUGCUCCUCAAUGCCCA
1349-1371
1511

AD-76199
A-152214
GCCUUCCAGAACCUCUCCAGA
358-378
1465
A-152215
UCUGGAGAGGUUCUGGAAGGCUG
356-378
1512

AD-76200
A-152216
GCGGUCAUGAACCUCUCUGGA
1174-1194
1466
A-152217
UCCAGAGAGGUUCAUGACCGCCA
1172-1194
1513

AD-76201
A-152218
AGGCUGGAGGACGGGCUCUUA
559-579
1467
A-152219
UAAGAGCCCGUCCUCCAGCCUGC
557-579
1514

AD-76202
A-152220
CUCUACCUGGACCGCAACCUA
820-840
1468
A-152221
UAGGUUGCGGUCCAGGUAGAGUU
818-840
1515

AD-76203
A-152222
GCGGAUGAGCUCAGCGUCUUA
238-258
1469
A-152223
UAAGACGCUGAGCUCAUCCGCGU
236-258
1516

AD-76204
A-152224
CCGUCUGAGCAGGCUGGAGGA
549-569
1470
A-152225
UCCUCCAGCCUGCUCAGACGGUU
547-569
1517

AD-76205
A-152226
GGUCAUGAACCUCUCUGGGAA
1176-1196
1471
A-152227
UUCCCAGAGAGGUUCAUGACCGC
1174-1196
1518

AD-76206
A-152228
CUGCAGCUGGGCCACAACCGA
1036-1056
1472
A-152229
UCGGUUGUGGCCCAGCUGCAGCU
1034-1056
1519

AD-76207
A-152230
CGGGAGCUGGACCUGAGCAGA
742-762
1473
A-152231
UCUGCUCAGGUCCAGCUCCCGGA
740-762
1520

AD-76208
A-152232
CCGCCUGUGUCUGCAGCUACA
209-229
1474
A-152233
UGUAGCUGCAGACACAGGCGGCC
207-229
1521

AD-76209
A-152234
UCUUCUGCAGCUCCAGGAACA
254-274
1475
A-152235
UGUUCCUGGAGCUGCAGAAGACG
252-274
1522

AD-76210
A-152236
CUGGCUGAGCGCAGCUUUGAA
1066-1086
1476
A-152237
UUCAAAGCUGCGCUCAGCCAGCU
1064-1086
1523

AD-76211
A-152238
CUCGACCUGACCUCCAACCAA
1396-1416
1477
A-152239
UUGGUUGGAGGUCAGGUCGAGCU
1394-1416
1524

AD-76212
A-152240
CUCAACCUCGGCUGGAAUAGA
604-624
1478
A-152241
UCUAUUCCAGCCGAGGUUGAGGU
602-624
1525

AD-76213
A-152242
GCCUAGAGAACCUGUGCCACA
440-460
1479
A-152243
UGUGGCACAGGUUCUCUAGGCCC
438-460
1526

AD-76214
A-152244
CAGCUUGAGGUGCUCACGCUA
1096-1116
1480
A-152245
UAGCGUGAGCACCUCAAGCUGCC
1094-1116
1527

AD-76215
A-152246
CAGGUCCUCAGUGUCCUCAGA
1961-1981
1481
A-152247
UCUGAGGACACUGAGGACCUGUC
1959-1981
1528

AD-76216
A-152248
GCUGCGAUGGCUGGACCUGUA
882-902
1482
A-152249
UACAGGUCCAGCCAUCGCAGCGC
880-902
1529

AD-76217
A-152250
GGCAAGCUGGAGUACCUGCUA
1453-1473
1483
A-152251
UAGCAGGUACUCCAGCUUGCCCA
1451-1473
1530

TABLE 13

IGFALS in vitro 10 nM and 0.1 nM screen

10 nM
10 nM
0.1 nM
0.1 nM
Position in

Duplex Name
AVG
STD
AVG
STD
NM_004970.2

AD-76171
29.9
0.1
55.8
5.4
2058-2078

AD-76172
42.7
2.3
101.9
7.8
1808-1828

AD-76173
32.8
3.1
81.2
7.1
1807-1827

AD-76174
45.3
10.5
86.2
9.7
777-797

AD-76175
42.8
7.6
97.3
8.7
1809-1829

AD-76176
69
5.8
93.9
11.3
1805-1825

AD-76177
53.6
11
99.2
15.8
1806-1826

AD-76178
72.6
4.9
92.6
6.9
1322-1342

AD-76179
80.1
8.2
113.3
8
357-377

AD-76180
132
13.9
124.2
16.8
1109-1129

AD-76181
67.1
5.9
99.1
0.3
1541-1561

AD-76182
110.4
17.9
102
5.1
824-844

AD-76183
69.8
9.9
100.3
6.9
893-913

AD-76184
62
6.3
99.4
7.1
965-985

AD-76185
119.5
33.1
92.8
4.4
530-550

AD-76186
52.4
4.6
94.7
2.7
1617-1637

AD-76187
116.7
6.4
117.7
9.4
1633-1653

AD-76188
60.4
5.8
106.2
4.8
1185-1205

AD-76189
89.9
2.1
102.9
8.4
1473-1493

AD-76190
83.5
3.5
104.5
6.5
1545-1565

AD-76191
80.9
3.3
96.4
7.2
1110-1130

AD-76192
93.9
3.9
103
5.4
1472-1492

AD-76193
99.3
3.5
95.1
10.2
1610-1630

AD-76194
192.1
4.9
118
12.1
1005-1025

AD-76195
86.1
3.3
106.7
8.4
1342-1362

AD-76196
184
23.4
114.8
6.7
1006-1026

AD-76197
55.3
4.4
111.4
14.2
1530-1550

AD-76198
66.7
3.6
97.1
13.1
1351-1371

AD-76199
54.5
2
91.8
4.2
358-378

AD-76200
63.9
10.1
88
10.6
1174-1194

AD-76201
150.6
4.6
113.6
18.3
559-579

AD-76202
64.7
1.4
95.7
13.7
820-840

AD-76203
41.3
1.7
93
2.1
238-258

AD-76204
67.5
6.9
101.3
3.6
549-569

AD-76205
73.1
9.5
87.6
7.8
1176-1196

AD-76206
86.4
3.7
107.7
17
1036-1056

AD-76207
131.4
22.4
99.7
10.5
742-762

AD-76208
46.5
12
103
13.2
209-229

AD-76209
43.8
5.2
98.1
13.9
254-274

AD-76210
41.6
7.3
94.4
6
1066-1086

AD-76211
152.3
9.4
125.1
3.4
1396-1416

AD-76212
59.2
12.7
82.3
23.4
604-624

AD-76213
71.1
4.9
94.9
11.6
440-460

AD-76214
109.8
5.9
102.8
8.4
1096-1116

AD-76215
70.2
7.5
103.2
25.4
1961-1981

AD-76216
62.8
9.5
107.7
15.1
882-902

AD-76217
68.8
1.7
94.5
2.4
1453-1473

Mock
117.3
16.3
106.9
9.5

TABLE 14

Modified Sense and Antisense Strand Sequences of IGFALS dsRNAs

Sense

Antisense

Duplex
Oligo

SEQ ID
Oligo

SEQ ID

SEQ ID

Name
Name
Sense OligoSequence
NO
Name
Antisense Oligo Sequence
NO
mRNA target sequence
NO

AD-76171
A-152158
csasauuaAfaGfGfCfaaaggcaauaL96
1531
A-152159
VPusAfsuugCfcUfUfugccUfuUfaauugsasu
1578
AUCAAUUAAAGGCAAAGGCAAUC
1625

AD-76172
A-152160
ascsaccuAfcAfAfCfaacaucaccaL96
1532
A-152161
VPusGfsgugAfuGfUfuguuGfuAfggugusasc
1579
GUACACCUACAACAACAUCACCU
1626

AD-76173
A-152162
usascaccUfaCfAfAfcaacaucacaL96
1533
A-152163
VPusGfsugaUfgUfUfguugUfaGfguguascsg
1580
CGUACACCUACAACAACAUCACC
1627

AD-76174
A-152164
csasucaaGfgCfAfAfacguguucgaL96
1534
A-152165
VPusCfsgaaCfaCfGfuuugCfcUfugaugsgsc
1581
GCCAUCAAGGCAAACGUGUUCGU
1628

AD-76175
A-152166
csasccuaCfaAfCfAfacaucaccuaL96
1535
A-152167
VPusAfsgguGfaUfGfuuguUfgUfaggugsusa
1582
UACACCUACAACAACAUCACCUG
1629

AD-76176
A-152168
csgsuacaCfcUfAfCfaacaacaucaL96
1536
A-152169
VPusGfsaugUfuGfUfuguaGfgUfguacgscsg
1583
CGCGUACACCUACAACAACAUCA
1630

AD-76177
A-152170
gsusacacCfuAfCfAfacaacaucaaL96
1537
A-152171
VPusUfsgauGfuUfGfuuguAfgGfuguacsgsc
1584
GCGUACACCUACAACAACAUCAC
1631

AD-76178
A-152172
gsascucuUfcCfUfCfaaggacaacaL96
1538
A-152173
VPusGfsuugUfcCfUfugagGfaAfgagucsgsg
1585
CCGACUCUUCCUCAAGGACAACG
1632

AD-76179
A-152174
asgsccuuCfcAfGfAfaccucuccaaL96
1539
A-152175
VPusUfsggaGfaGfGfuucuGfgAfaggcusgsc
1586
GCAGCCUUCCAGAACCUCUCCAG
1633

AD-76180
A-152176
uscsacgcUfaGfAfCfcacaaccagaL96
1540
A-152177
VPusCfsuggUfuGfUfggucUfaGfcgugasgsc
1587
GCUCACGCUAGACCACAACCAGC
1634

AD-76181
A-152178
usgsgacgUfcUfCfGfcacaaccgcaL96
1541
A-152179
VPusGfscggUfuGfUfgcgaGfaCfguccasgsc
1588
GCUGGACGUCUCGCACAACCGCC
1635

AD-76182
A-152180
ascscuggAfcCfGfCfaaccucaucaL96
1542
A-152181
VPusGfsaugAfgGfUfugcgGfuCfcaggusasg
1589
CUACCUGGACCGCAACCUCAUCG
1636

AD-76183
A-152182
usgsgaccUfgUfCfCfcacaaccgcaL96
1543
A-152183
VPusGfscggUfuGfUfgggaCfaGfguccasgsc
1590
GCUGGACCUGUCCCACAACCGCG
1637

AD-76184
A-152184
usgscggcUfgUfCfCfcacaacgccaL96
1544
A-152185
VPusGfsgcgUfuGfUfgggaCfaGfccgcasgsc
1591
GCUGCGGCUGUCCCACAACGCCA
1638

AD-76185
A-152186
csgscucgGfcCfUfCfagcaacaacaL96
1545
A-152187
VPusGfsuugUfuGfCfugagGfcCfgagcgsasg
1592
CUCGCUCGGCCUCAGCAACAACC
1639

AD-76186
A-152188
cscsucagGfaAfCfAfacucacugcaL96
1546
A-152189
VPusGfscagUfgAfGfuuguUfcCfugaggscsu
1593
AGCCUCAGGAACAACUCACUGCG
1640

AD-76187
A-152190
csusgcggAfcCfUfUfcacgccgcaaL96
1547
A-152191
VPusUfsgcgGfcGfUfgaagGfuCfcgcagsusg
1594
CACUGCGGACCUUCACGCCGCAG
1641

AD-76188
A-152192
cscsucucUfgGfGfAfacugucuccaL96
1548
A-152193
VPusGfsgagAfcAfGfuuccCfaGfagaggsusu
1595
AACCUCUCUGGGAACUGUCUCCG
1642

AD-76189
A-152194
gscsucucCfcGfCfAfaccgccuggaL96
1549
A-152195
VPusCfscagGfcGfGfuugcGfgGfagagcsasg
1596
CUGCUCUCCCGCAACCGCCUGGC
1643

AD-76190
A-152196
csgsucucGfcAfCfAfaccgccuggaL96
1550
A-152197
VPusCfscagGfcGfGfuuguGfcGfagacgsusc
1597
GACGUCUCGCACAACCGCCUGGA
1644

AD-76191
A-152198
csascgcuAfgAfCfCfacaaccagcaL96
1551
A-152199
VPusGfscugGfuUfGfugguCfuAfgcgugsasg
1598
CUCACGCUAGACCACAACCAGCU
1645

AD-76192
A-152200
usgscucuCfcCfGfCfaaccgccugaL96
1552
A-152201
VPusCfsaggCfgGfUfugcgGfgAfgagcasgsc
1599
GCUGCUCUCCCGCAACCGCCUGG
1646

AD-76193
A-152202
ascscucaGfcCfUfCfaggaacaacaL96
1553
A-152203
VPusGfsuugUfuCfCfugagGfcUfgaggusasg
1600
CUACCUCAGCCUCAGGAACAACU
1647

AD-76194
A-152204
csasccuuCfaAfGfGfaccugcacuaL96
1554
A-152205
VPusAfsgugCfaGfGfuccuUfgAfaggugscsg
1601
CGCACCUUCAAGGACCUGCACUU
1648

AD-76195
A-152206
gsgsccucGfuGfGfGfcauugaggaaL96
1555
A-152207
VPusUfsccuCfaAfUfgcccAfcGfaggccsgsu
1602
ACGGCCUCGUGGGCAUUGAGGAG
1649

AD-76196
A-152208
ascscuucAfaGfGfAfccugcacuuaL96
1556
A-152209
VPusAfsaguGfcAfGfguccUfuGfaaggusgsc
1603
GCACCUUCAAGGACCUGCACUUC
1650

AD-76197
A-152210
gsgsccuuCfuGfGfCfuggacgucuaL96
1557
A-152211
VPusAfsgacGfuCfCfagccAfgAfaggccscsg
1604
CGGGCCUUCUGGCUGGACGUCUC
1651

AD-76198
A-152212
gsgscauuGfaGfGfAfgcagagccuaL96
1558
A-152213
VPusAfsggcUfcUfGfcuccUfcAfaugccscsa
1605
UGGGCAUUGAGGAGCAGAGCCUG
1652

AD-76199
A-152214
gscscuucCfaGfAfAfccucuccagaL96
1559
A-152215
VPusCfsuggAfgAfGfguucUfgGfaaggcsusg
1606
CAGCCUUCCAGAACCUCUCCAGC
1653

AD-76200
A-152216
gscsggucAfuGfAfAfccucucuggaL96
1560
A-152217
VPusCfscagAfgAfGfguucAfuGfaccgcscsa
1607
UGGCGGUCAUGAACCUCUCUGGG
1654

AD-76201
A-152218
asgsgcugGfaGfGfAfcgggcucuuaL96
1561
A-152219
VPusAfsagaGfcCfCfguccUfcCfagccusgsc
1608
GCAGGCUGGAGGACGGGCUCUUC
1655

AD-76202
A-152220
csuscuacCfuGfGfAfccgcaaccuaL96
1562
A-152221
VPusAfsgguUfgCfGfguccAfgGfuagagsusu
1609
AACUCUACCUGGACCGCAACCUC
1656

AD-76203
A-152222
gscsggauGfaGfCfUfcagcgucuuaL96
1563
A-152223
VPusAfsagaCfgCfUfgagcUfcAfuccgcsgsu
1610
ACGCGGAUGAGCUCAGCGUCUUC
1657

AD-76204
A-152224
cscsgucuGfaGfCfAfggcuggaggaL96
1564
A-152225
VPusCfscucCfaGfCfcugcUfcAfgacggsusu
1611
AACCGUCUGAGCAGGCUGGAGGA
1658

AD-76205
A-152226
gsgsucauGfaAfCfCfucucugggaaL96
1565
A-152227
VPusUfscccAfgAfGfagguUfcAfugaccsgsc
1612
GCGGUCAUGAACCUCUCUGGGAA
1659

AD-76206
A-152228
csusgcagCfuGfGfGfccacaaccgaL96
1566
A-152229
VPusCfsgguUfgUfGfgcccAfgCfugcagscsu
1613
AGCUGCAGCUGGGCCACAACCGC
1660

AD-76207
A-152230
csgsggagCfuGfGfAfccugagcagaL96
1567
A-152231
VPusCfsugcUfcAfGfguccAfgCfucccgsgsa
1614
UCCGGGAGCUGGACCUGAGCAGG
1661

AD-76208
A-152232
cscsgccuGfuGfUfCfugcagcuacaL96
1568
A-152233
VPusGfsuagCfuGfCfagacAfcAfggcggscsc
1615
GGCCGCCUGUGUCUGCAGCUACG
1662

AD-76209
A-152234
uscsuucuGfcAfGfCfuccaggaacaL96
1569
A-152235
VPusGfsuucCfuGfGfagcuGfcAfgaagascsg
1616
CGUCUUCUGCAGCUCCAGGAACC
1663

AD-76210
A-152236
csusggcuGfaGfCfGfcagcuuugaaL96
1570
A-152237
VPusUfscaaAfgCfUfgcgcUfcAfgccagscsu
1617
AGCUGGCUGAGCGCAGCUUUGAG
1664

AD-76211
A-152238
csuscgacCfuGfAfCfcuccaaccaaL96
1571
A-152239
VPusUfsgguUfgGfAfggucAfgGfucgagscsu
1618
AGCUCGACCUGACCUCCAACCAG
1665

AD-76212
A-152240
csuscaacCfuCfGfGfcuggaauagaL96
1572
A-152241
VPusCfsuauUfcCfAfgccgAfgGfuugagsgsu
1619
ACCUCAACCUCGGCUGGAAUAGC
1666

AD-76213
A-152242
gscscuagAfgAfAfCfcugugccacaL96
1573
A-152243
VPusGfsuggCfaCfAfgguuCfuCfuaggcscsc
1620
GGGCCUAGAGAACCUGUGCCACC
1667

AD-76214
A-152244
csasgcuuGfaGfGfUfgcucacgcuaL96
1574
A-152245
VPusAfsgcgUfgAfGfcaccUfcAfagcugscsc
1621
GGCAGCUUGAGGUGCUCACGCUA
1668

AD-76215
A-152246
csasggucCfuCfAfGfuguccucagaL96
1575
A-152247
VPusCfsugaGfgAfCfacugAfgGfaccugsusc
1622
GACAGGUCCUCAGUGUCCUCAGG
1669

AD-76216
A-152248
gscsugcgAfuGfGfCfuggaccuguaL96
1576
A-152249
VPusAfscagGfuCfCfagccAfuCfgcagcsgsc
1623
GCGCUGCGAUGGCUGGACCUGUC
1670

AD-76217
A-152250
gsgscaagCfuGfGfAfguaccugcuaL96
1577
A-152251
VPusAfsgcaGfgUfAfcuccAfgCfuugccscsa
1624
UGGGCAAGCUGGAGUACCUGCUG
1671

Example 10—IGF-1 Transcripts, siRNA Design, and siRNA Screening

Bioinformatics

A set of siRNAs targeting the human IGF1, (“insulin like growth factor 1”, NCBI refseqID: NM_000618; NCBI GeneID: 3479 (SEQ ID NO:13), as well as toxicology-species IGF1 orthologs (cynomolgus monkey: XM_005572039) were designed using custom R and Python scripts. The human NM_000618 REFSEQ mRNA, version 3, has a length 7321 of bases.

The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 19mer siRNA from position 10 through the end was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct siRNA designs targeting a large number of vertebrate genes. Subsets of the IGF1 siRNAs were designed with perfect or near-perfect matches between human and cynomolgus monkey. For each strand of the siRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the siRNA and all potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed region, defined here as positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the siRNA, defined here as positions 10-11 of the antisense oligonucleotide. The relative weight of the mismatches was 2.8; 1.2:1 for seed mismatches, cleavage site, and other positions up through antisense position 19. Mismatches in the first position were ignored. A specificity score was calculated for each strand by summing the value of each weighted mismatch. Preference was given to siRNAs whose antisense score in human was >=2.2 and predicted efficacy was >=50% knockdown of the transcript.

In Vitro Dual-Glo® Screening

Cell Culture and Transfections

Cos7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂in DMEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. Three human IGF-1 Dual-Glo® Luciferase constructs were generated using the psiCHECK2 vector. Construct one contained sequence based on NM_001111285 (SEQ ID NO:1672), while constructs two and three contained sequence based on NM_000618 (SEQ ID NOs: 1673 and 1674). Dual-luciferase plasmids were co-transfected with siRNA into 5000 cells using Lipofectamine RNAiMax (Invitrogen, Carlsbad Calif. cat #13778-150). For each well of a 384 well plate, 0.1 μl of Lipofectamine was added to 5 ng of plasmid vector and siRNA in 15 μl of Opti-MEM and allowed to complex at room temperature for 15 minutes. The mixture was then added to the cells resuspended in 35 ul of fresh complete media. Cells were incubated for 48 hours before luciferase was measured. Screen was performed at 10 nM and 0.1 nM final duplex concentration.

Dual-Glo® Luciferase Assay

48 hours after the siRNAs were transfected, Firefly (transfection control) and Renilla (fused to IGF1 target sequence in 3′ UTR) luciferase were measured. First, media was removed from cells. Then Firefly luciferase activity was measured by adding 20 ul of Dual-Glo® Luciferase Reagent mixed with 20 ul of complete media to each well. The mixture was incubated at room temperature for 30 minutes before luminescense (500 nm) was measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 20 ul of room temperature of Dual-Glo® Stop & Glo® Reagent to each well and the plates were incubated for 20 minutes before luminescence was again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent quenched the firefly luciferase signal and sustained luminescence for the Renilla luciferase reaction. siRNA activity was determined by normalizing the Renilla (IGF1) signal to the Firefly (control) signal within each well. The magnitude of siRNA activity was then assessed relative to cells that were transfected with the same vector but were not treated with siRNA or were treated with a non-targeting siRNA. All transfections were done in quadruplicates.

TABLE 15

Unmodified Sense and Antisense Strand Sequences of IGF-1dsRNAs

Sense

Position

Antisense

Position

Duplex
Oligo

in NM_
SEQ ID
Oligo

in NM_
SEQ ID

Name
Name
Sense Sequence
000618.3
NO
Name
Antisense Sequence
000618.3
NO

AD-75740
A-151647
AAUGUAACUAAUUGAAUCAUA
7181-7201
1675
A-151648
UAUGAUUCAAUUAGUUACAUUUU
7179-7201
1768

AD-75741
A-151649
UAAGAAAGUACUUGACUAAAA
4362-4382
1676
A-151650
UUUUAGUCAAGUACUUUCUUAAA
4360-4382
1769

AD-75747
A-151661
CUUUAUAAUUCUUGAAUGAGA
3765-3785
1677
A-151662
UCUCAUUCAAGAAUUAUAAAGCA
3763-3785
1770

AD-75748
A-151663
AGAUAGAAAUGUAUGUUUGAA
5223-5243
1678
A-151664
UUCAAACAUACAUUUCUAUCUAG
5221-5243
1771

AD-75749
A-151665
UUCCACAAUCCUUAGAAUCUA
6512-6532
1679
A-151666
UAGAUUCUAAGGAUUGUGGAAGG
6510-6532
1772

AD-75750
A-151667
UAUCAAAACCUUUCAAAUAUA
6831-6851
1680
A-151668
UAUAUUUGAAAGGUUUUGAUAUU
6829-6851
1773

AD-75751
A-151669
ACAAGUAAACAUUCCAACAUA
824-844
1681
A-151670
UAUGUUGGAAUGUUUACUUGUGU
822-844
1774

AD-75755
A-151677
ACAUAGAAAGUUUCUUCAACA
1410-1430
1682
A-151678
UGUUGAAGAAACUUUCUAUGUUU
1408-1430
1775

AD-75757
A-151681
CAGUCAACAAGUAUUUUAACA
3122-3142
1683
A-151682
UGUUAAAAUACUUGUUGACUGAA
3120-3142
1776

AD-75759
A-151685
CUCAAGCUGUCUACUUACAUA
6769-6789
1684
A-151686
UAUGUAAGUAGACAGCUUGAGGU
6767-6789
1777

AD-75760
A-151687
GAAUUGUUUCCUUAUUUGCAA
1085-1105
1685
A-151688
UUGCAAAUAAGGAAACAAUUCAU
1083-1105
1778

AD-75761
A-151689
AUCUGUCUUAGUUGAAAAGCA
7071-7091
1686
A-151690
UGCUUUUCAACUAAGACAGAUGU
7069-7091
1779

AD-75765
A-151697
AAUUAGCAAUAUAUUAUCCAA
4632-4652
1687
A-151698
UUGGAUAAUAUAUUGCUAAUUUU
4630-4652
1780

AD-75766
A-151699
AAUUGAAUCAUUAUCUUACAA
7190-7210
1688
A-151700
UUGUAAGAUAAUGAUUCAAUUAG
7188-7210
1781

AD-75769
A-151705
UUUAUCAAUAAUGUUCUAUAA
908-928
1689
A-151706
UUAUAGAACAUUAUUGAUAAAAG
906-928
1782

AD-75772
A-151711
GUAAAAGAAACUAUACAUCAA
3213-3233
1690
A-151712
UUGAUGUAUAGUUUCUUUUACAU
3211-3233
1783

AD-75774
A-151715
AAUGAGGAAUAAUAAGUUAAA
5173-5193
1691
A-151716
UUUAACUUAUUAUUCCUCAUUCU
5171-5193
1784

AD-75776
A-151719
CUCUGUGAAUGUGUUUUAUCA
2737-2757
1692
A-151720
UGAUAAAACACAUUCACAGAGAG
2735-2757
1785

AD-75778
A-151723
GUUCCUUCAAAUGAUGAGUUA
1438-1458
1693
A-151724
UAACUCAUCAUUUGAAGGAACUC
1436-1458
1786

AD-75779
A-151725
CAGGAUAAAGAUAUCAAUUUA
5722-5742
1694
A-151726
UAAAUUGAUAUCUUUAUCCUGUA
5720-5742
1787

AD-75787
A-151741
CAUGUCCUCCUCGCAUCUCUA
297-317
1695
A-151742
UAGAGAUGCGAGGAGGACAUGGU
295-317
1788

AD-75787
A-151741
CAUGUCCUCCUCGCAUCUCUA
297-317
1696
A-151742
UAGAGAUGCGAGGAGGACAUGGU
295-317
1789

AD-75788
A-151743
UUCAACAAGCCCACAGGGUAA
436-456
1697
A-151744
UUACCCUGUGGGCUUGUUGAAAU
434-456
1790

AD-75788
A-151743
UUCAACAAGCCCACAGGGUAA
436-456
1698
A-151744
UUACCCUGUGGGCUUGUUGAAAU
434-456
1791

AD-75789
A-151745
UCCUCGCAUCUCUUCUACCUA
304-324
1699
A-151746
UAGGUAGAAGAGAUGCGAGGAGG
302-324
1792

AD-75789
A-151745
UCCUCGCAUCUCUUCUACCUA
304-324
1700
A-151746
UAGGUAGAAGAGAUGCGAGGAGG
302-324
1793

AD-75790
A-151747
GGGGCUUUUAUUUCAACAAGA
425-445
1701
A-151748
UCUUGUUGAAAUAAAAGCCCCUG
423-445
1794

AD-75790
A-151747
GGGGCUUUUAUUUCAACAAGA
425-445
1702
A-151748
UCUUGUUGAAAUAAAAGCCCCUG
423-445
1795

AD-75791
A-151749
GAUGCUCUUCAGUUCGUGUGA
397-417
1703
A-151750
UCACACGAACUGAAGAGCAUCCA
395-417
1796

AD-75791
A-151749
GAUGCUCUUCAGUUCGUGUGA
397-417
1704
A-151750
UCACACGAACUGAAGAGCAUCCA
395-417
1797

AD-75792
A-151751
UGGAUGCUCUUCAGUUCGUGA
395-415
1705
A-151752
UCACGAACUGAAGAGCAUCCACC
393-415
1798

AD-75792
A-151751
UGGAUGCUCUUCAGUUCGUGA
395-415
1706
A-151752
UCACGAACUGAAGAGCAUCCACC
393-415
1799

AD-75793
A-151753
GGUGGAUGCUCUUCAGUUCGA
393-413
1707
A-151754
UCGAACUGAAGAGCAUCCACCAG
391-413
1800

AD-75793
A-151753
GGUGGAUGCUCUUCAGUUCGA
393-413
1708
A-151754
UCGAACUGAAGAGCAUCCACCAG
391-413
1801

AD-75794
A-151755
GCUGGUGGAUGCUCUUCAGUA
390-410
1709
A-151756
UACUGAAGAGCAUCCACCAGCUC
388-410
1802

AD-75794
A-151755
GCUGGUGGAUGCUCUUCAGUA
390-410
1710
A-151756
UACUGAAGAGCAUCCACCAGCUC
388-410
1803

AD-75795
A-151757
CUGGUGGAUGCUCUUCAGUUA
391-411
1711
A-151758
UAACUGAAGAGCAUCCACCAGCU
389-411
1804

AD-75795
A-151757
CUGGUGGAUGCUCUUCAGUUA
391-411
1712
A-151758
UAACUGAAGAGCAUCCACCAGCU
389-411
1805

AD-77120
A-154752
CCAAAAUGCACUGAUGUAAAA
6586-6606
1713
A-154753
UUUUACAUCAGUGCAUUUUGGGC
6584-6606
1806

AD-77121
A-154754
UCCAGUUGCACUAAAUUCCUA
1009-1029
1714
A-154755
UAGGAAUUUAGUGCAACUGGAUC
1007-1029
1807

AD-77122
A-154756
CAUUCUUCAACUAUCUUUGAA
6325-6345
1715
A-154757
UUCAAAGAUAGUUGAAGAAUGAG
6323-6345
1808

AD-77123
A-154758
ACAUUCCAACAUUGUCUUUAA
832-852
1716
A-154759
UUAAAGACAAUGUUGGAAUGUUU
830-852
1809

AD-77124
A-154760
GGCUUAGAAUAAAAGAUGUAA
4280-4300
1717
A-154761
UUACAUCUUUUAUUCUAAGCCUU
4278-4300
1810

AD-77125
A-154762
UUUCAAGAUAUUUGUAAAAGA
3789-3809
1718
A-154763
UCUUUUACAAAUAUCUUGAAAUU
3787-3809
1811

AD-77126
A-154764
AUAAGCAUAUUUUGAAAAUGA
7221-7241
1719
A-154765
UCAUUUUCAAAAUAUGCUUAUUA
7219-7241
1812

AD-77127
A-154766
GUUUUCAAUGCUAGUGUUUAA
5553-5573
1720
A-154767
UUAAACACUAGCAUUGAAAACAA
5551-5573
1813

AD-77128
A-154768
CAAUGAAAUACACAAGUAAAA
813-833
1721
A-154769
UUUUACUUGUGUAUUUCAUUGGG
811-833
1814

AD-77129
A-154770
CAAAAGUCCACUGAUGCAAAA
1764-1784
1722
A-154771
UUUUGCAUCAGUGGACUUUUGUG
1762-1784
1815

AD-77130
A-154772
AACAUAGAAAGUUUCUUCAAA
1409-1429
1723
A-154773
UUUGAAGAAACUUUCUAUGUUUA
1407-1429
1816

AD-77131
A-154774
AACCUAAUUAGUAACUUUCCA
1465-1485
1724
A-154775
UGGAAAGUUACUAAUUAGGUUGC
1463-1485
1817

AD-77132
A-154776
GACUUAUUUCCUUCACUUAAA
3442-3462
1725
A-154777
UUUAAGUGAAGGAAAUAAGUCAU
3440-3462
1818

AD-77133
A-154778
GGCUCUCAAACUUAGCAAAAA
4614-4634
1726
A-154779
UUUUUGCUAAGUUUGAGAGCCAA
4612-4634
1819

AD-77134
A-154780
ACAAAGAUAAGUGAAAAGAGA
2312-2332
1727
A-154781
UCUCUUUUCACUUAUCUUUGUAU
2310-2332
1820

AD-77135
A-154782
GUAGUGUACUGUUCACCAAAA
4525-4545
1728
A-154783
UUUUGGUGAACAGUACACUACUU
4523-4545
1821

AD-77136
A-154784
UCACCAACUCAUAGCAAAGUA
6663-6683
1729
A-154785
UACUUUGCUAUGAGUUGGUGAGU
6661-6683
1822

AD-77137
A-154786
AAAAUAACUCAUUAUACCAAA
3577-3597
1730
A-154787
UUUGGUAUAAUGAGUUAUUUUCA
3575-3597
1823

AD-77138
A-154788
AAGAAAGAAUCUUUCCAUUCA
5344-5364
1731
A-154789
UGAAUGGAAAGAUUCUUUCUUCU
5342-5364
1824

AD-77139
A-154790
AUUUCAAGAUAUUUGUAAAAA
3788-3808
1732
A-154791
UUUUUACAAAUAUCUUGAAAUUG
3786-3808
1825

AD-77140
A-154792
AGCUGAAACUCUAGAAUUAAA
5908-5928
1733
A-154793
UUUAAUUCUAGAGUUUCAGCUUG
5906-5928
1826

AD-77141
A-154794
AAAGAUAAAUCUGAUUUAUGA
5394-5414
1734
A-154795
UCAUAAAUCAGAUUUAUCUUUUG
5392-5414
1827

AD-77142
A-154796
UAUUAAAUUAAUUCUGAUUGA
7099-7119
1735
A-154797
UCAAUCAGAAUUAAUUUAAUAAA
7097-7119
1828

AD-77143
A-154798
AAGAAUGAGCUUUCAACUCAA
3825-3845
1736
A-154799
UUGAGUUGAAAGCUCAUUCUUAC
3823-3845
1829

AD-77144
A-154800
GUUUGAUAACAUUUAAAAGAA
780-800
1737
A-154801
UUCUUUUAAAUGUUAUCAAACUU
778-800
1830

AD-77145
A-154802
UGUUUUAAACAUAGAAAGUUA
1402-1422
1738
A-154803
UAACUUUCUAUGUUUAAAACAUA
1400-1422
1831

AD-77146
A-154804
UAAAUAACCCAUUCAUAGCAA
2110-2130
1739
A-154805
UUGCUAUGAAUGGGUUAUUUAUA
2108-2130
1832

AD-77147
A-154806
CACAUAGACUCUUUAAAACUA
5196-5216
1740
A-154807
UAGUUUUAAAGAGUCUAUGUGGG
5194-5216
1833

AD-77148
A-154808
AAGUCACAAAGUUAUCUUCUA
2568-2588
1741
A-154809
UAGAAGAUAACUUUGUGACUUUU
2566-2588
1834

AD-77149
A-154810
UAAAGAAAGAAUUUAUGAGAA
5011-5031
1742
A-154811
UUCUCAUAAAUUCUUUCUUUAUC
5009-5031
1835

AD-77150
A-154812
GAAAUGUAUGUUUGACUUGUA
5228-5248
1743
A-154813
UACAAGUCAAACAUACAUUUCUA
5226-5248
1836

AD-77151
A-154814
UUUAUAAGACCUUCCUGUUAA
5611-5631
1744
A-154815
UUAACAGGAAGGUCUUAUAAAAU
5609-5631
1837

AD-77152
A-154816
CUGUGAAUGUGUUUUAUCCAA
2739-2759
1745
A-154817
UUGGAUAAAACACAUUCACAGAG
2737-2759
1838

AD-77153
A-154818
UUAAUUCUGAUUGUAUUUGAA
7106-7126
1746
A-154819
UUCAAAUACAAUCAGAAUUAAUU
7104-7126
1839

AD-77154
A-154820
UGUCAUCUACCUACCUCAAAA
1982-2002
1747
A-154821
UUUUGAGGUAGGUAGAUGACAUA
1980-2002
1840

AD-77155
A-154822
CGUGAAAGCAAAACAAUAGGA
1634-1654
1748
A-154823
UCCUAUUGUUUUGCUUUCACGUA
1632-1654
1841

AD-77156
A-154824
AACAACAAUUCUAUAGAUAGA
4438-4458
1749
A-154825
UCUAUCUAUAGAAUUGUUGUUUU
4436-4458
1842

AD-77157
A-154826
UAAAGUAUUAUUUGAACUUUA
3920-3940
1750
A-154827
UAAAGUUCAAAUAAUACUUUAAU
3918-3940
1843

AD-77158
A-154828
AGAAAGAAUUUAUGAGAAAUA
5014-5034
1751
A-154829
UAUUUCUCAUAAAUUCUUUCUUU
5012-5034
1844

AD-77159
A-154830
AAAUGCACUGAUGUAAAGUAA
6589-6609
1752
A-154831
UUACUUUACAUCAGUGCAUUUUG
6587-6609
1845

AD-77160
A-154832
AUUAAUUCUGAUUGUAUUUGA
7105-7125
1753
A-154833
UCAAAUACAAUCAGAAUUAAUUU
7103-7125
1846

AD-77161
A-154834
CAAUAAUGUUCUAUAGAAAAA
913-933
1754
A-154835
UUUUUCUAUAGAACAUUAUUGAU
911-933
1847

AD-77162
A-154836
GAUUUUCAAUUUGAUUUUGAA
2269-2289
1755
A-154837
UUCAAAAUCAAAUUGAAAAUCAA
2267-2289
1848

AD-77163
A-154840
UUGAUUUUGAAUUCUGCAUUA
2279-2299
1756
A-154841
UAAUGCAGAAUUCAAAAUCAAAU
2277-2299
1849

AD-77164
A-154842
UUUCAAUUUGAUUUUGAAUUA
2272-2292
1757
A-154843
UAAUUCAAAAUCAAAUUGAAAAU
2270-2292
1850

AD-77165
A-154844
GUGUCUGUGUAUCAUGAAAAA
6798-6818
1758
A-154845
UUUUUCAUGAUACACAGACACAG
6796-6818
1851

AD-77166
A-154846
GGUUUUAUGAAUACAAAGAUA
2300-2320
1759
A-154847
UAUCUUUGUAUUCAUAAAACCAA
2298-2320
1852

AD-77167
A-154848
GCAGAUCAAGAUUUUCUCAUA
2837-2857
1760
A-154849
UAUGAGAAAAUCUUGAUCUGCAG
2835-2857
1853

AD-77168
A-154850
AACUAAUUGAAUCAUUAUCUA
7186-7206
1761
A-154851
UAGAUAAUGAUUCAAUUAGUUAC
7184-7206
1854

AD-77169
A-154852
UGGUUUAUGAAUUGUUUCCUA
1077-1097
1762
A-154853
UAGGAAACAAUUCAUAAACCACU
1075-1097
1855

AD-77170
A-154854
UAGUAUAAUGGUGCUAUUUUA
1574-1594
1763
A-154855
UAAAAUAGCACCAUUAUACUAAA
1572-1594
1856

AD-77171
A-154856
CUGUUUAAUAAGCAUAUUUUA
7214-7234
1764
A-154857
UAAAAUAUGCUUAUUAAACAGUA
7212-7234
1857

AD-77172
A-154858
AAAUAUCAAAACCUUUCAAAA
6828-6848
1765
A-154859
UUUUGAAAGGUUUUGAUAUUUUG
6826-6848
1858

AD-77173
A-154860
ACAGAUGUAAAAGAAACUAUA
3207-3227
1766
A-154861
UAUAGUUUCUUUUACAUCUGUCC
3205-3227
1859

AD-77174
A-154862
AACUUUGAGGCCAAUCAUUUA
1373-1393
1767
A-154863
UAAAUGAUUGGCCUCAAAGUUGC
1371-1393
1860

TABLE 16

IGF-1 in vitro 10 nM and 0.1 nM screen

10 nM
10 nM
0.1 nM
0.1 nM
Position in

Duplex Name
AVG
STD
AVG
STD
NM_000618.3

AD-75740
2.9
1.1
20.9
1.6
7179-7201

AD-75741
9.2
1.5
55.6
9.3
4360-4382

AD-75747
50.3
6.8
67.7
8.8
3763-3785

AD-75748
8.4
1.1
36
10.1
5221-5243

AD-75749
7.8
1.3
65.9
3
6510-6532

AD-75750
4.4
1.3
48
9.3
6829-6851

AD-75751
5.7
1.4
51.7
9.4
822-844

AD-75755
29.4
7.2
60.9
3.8
1408-1430

AD-75757
32.4
5.7
62.3
7.9
3120-3142

AD-75759
9.8
4.3
86.5
11.1
6767-6789

AD-75760
32.2
7.8
56.2
7.1
1083-1105

AD-75761
3.5
1.3
53.3
4.3
7069-7091

AD-75765
7.6
1.9
56.5
8.6
4630-4652

AD-75766
3.3
2.4
35.4
7.4
7188-7210

AD-75769
12.4
3.1
33.6
3.9
906-928

AD-75772
36.1
5.2
79.9
19.3
3211-3233

AD-75774
14
1.2
51.8
7.8
5171-5193

AD-75776
41.1
11.6
84.2
10.3
2735-2757

AD-75778
42.3
7.4
56.4
6.6
1436-1458

AD-75779
8.7
1.6
53.7
9.1
5720-5742

AD-75787
59.5
5.5
96.5
6.2
295-317

AD-75787
57.5
12.9
99.5
10.4
295-317

AD-75788
38.5
5.2
82.2
9.3
434-456

AD-75788
28.1
2.1
88.3
4.4
434-456

AD-75789
58
11.5
81.6
3
302-324

AD-75789
65.1
16.2
93.6
5.6
302-324

AD-75790
54.7
5.9
90.1
1.7
423-445

AD-75790
64.1
6.1
92.6
4.3
423-445

AD-75791
17.4
5
78.8
3.9
395-417

AD-75791
19.5
2.1
87.5
7.1
395-417

AD-75792
12.7
1.5
63.7
6.2
393-415

AD-75792
13.9
2
77.8
12.9
393-415

AD-75793
23.6
4.1
78.5
4.7
391-413

AD-75793
27.8
4.1
90.9
11.6
391-413

AD-75794
40.6
3.1
89
12.3
388-410

AD-75794
46.7
4.4
91.1
9.6
388-410

AD-75795
32.2
8.2
75.2
6.3
389-411

AD-75795
27.3
5.3
77.7
7.3
389-411

AD-77120
17.7
1.8
83.5
3.7
6584-6606

AD-77121
28.3
3.4
69
5.9
1007-1029

AD-77122
19.3
6.4
80.4
10.5
6323-6345

AD-77123
21.2
4.1
46.9
8
830-852

AD-77124
13.9
1.5
35.3
5.8
4278-4300

AD-77125
44.5
10.3
52.4
6.9
3787-3809

AD-77126
4.5
3
23.6
3.9
7219-7241

AD-77127
8.3
1.5
43.1
4.9
5551-5573

AD-77128
13.6
4.1
46.6
5.3
811-833

AD-77129
68.5
7.6
99.9
8.2
1762-1784

AD-77130
32.1
5.8
41.4
3.4
1407-1429

AD-77131
36
8.5
53.8
7.2
1463-1485

AD-77132
44.3
8.2
71.7
5.1
3440-3462

AD-77133
21.4
6.4
88.9
5.8
4612-4634

AD-77134
37.9
2.4
82.4
8.7
2310-2332

AD-77135
19.6
5
91.8
17
4523-4545

AD-77136
32.3
8
89.5
3.5
6661-6683

AD-77137
36.8
3.3
67.7
7.7
3575-3597

AD-77138
2.6
1.9
71.3
6.3
5342-5364

AD-77139
45.6
2.5
70.4
8.2
3786-3808

AD-77140
17.1
3.2
65.4
6.1
5906-5928

AD-77141
56.3
17.6
112.7
8.2
5392-5414

AD-77142
18.1
3.7
65.7
8.4
7097-7119

AD-77143
45.7
10.1
93.6
11
3823-3845

AD-77144
5.5
2
43.5
7.6
778-800

AD-77145
59.6
11.1
43.8
5.1
1400-1422

AD-77146
27.7
5.8
64.8
7.7
2108-2130

AD-77147
6.6
5.4
75
10.2
5194-5216

AD-77148
37.6
6.7
65.9
8
2566-2588

AD-77149
5.5
0.7
35.8
5.2
5009-5031

AD-77150
1.1
2.3
48.2
5.1
5226-5248

AD-77151
9.1
2.3
69.2
5.9
5609-5631

AD-77152
52.6
5.9
93.7
6.5
2737-2759

AD-77153
10.4
1
37.8
6.1
7104-7126

AD-77154
57.5
14.5
101.7
11.5
1980-2002

AD-77155
63.3
8.5
53
3.9
1632-1654

AD-77156
14.8
3
47.9
8.9
4436-4458

AD-77158
1.3
1
44.3
0.9
5012-5034

AD-77159
20.5
4.9
92.3
17.2
6587-6609

AD-77160
10.8
5.2
39.3
4.9
7103-7125

AD-77161
26.1
1.8
47.3
6.8
911-933

AD-77162
51.6
5.4
93.6
3.1
2267-2289

AD-77163
41.2
4.4
70
3.1
2277-2299

AD-77164
58.5
5.8
95.5
10.9
2270-2292

AD-77165
11.1
2
61.7
7.1
6796-6818

AD-77166
40.3
5.6
74.2
1.7
2298-2320

AD-77167
55
9.2
84.6
18
2835-2857

AD-77168
7.9
0.6
28.4
3.6
7184-7206

AD-77169
36.1
7.5
51.6
5.6
1075-1097

AD-77170
44.4
8.8
66.7
3.6
1572-1594

AD-77171
13.1
1.6
50.1
9.3
7212-7234

AD-77172
38.8
5.4
103.7
12.7
6826-6848

AD-77173
42.4
2.8
79.8
15.2
3205-3227

AD-77174
48.9
3.5
83.3
6.5
1371-1393

Mock
100
6.1
100
5.6

TABLE 17

Modified Sense and Antisense Strand Sequences of IGF-1 dsRNAs

Sense

Antisense

Duplex
Oligo

SEQ ID
Oligo

SEQ ID

SEQ ID

Name
Name
Sense Sequence
NO
Name
Antisense Sequence
NO
mRNA target sequence
NO

AD-75740
A-151647
asasuguaAfcUfAfAfuugaaucauaL96
1861
A-151648
VPusAfsugaUfuCfAfauuaGfuUfacauususu
1954
AAAAUGUAACUAAUUGAAUCAUU
2047

AD-75741
A-151649
usasagaaAfgUfAfCfuugacuaaaaL96
1862
A-151650
VPusUfsuuaGfuCfAfaguaCfuUfucuuasasa
1955
UUUAAGAAAGUACUUGACUAAAA
2048

AD-75747
A-151661
csusuuauAfaUfUfCfuugaaugagaL96
1863
A-151662
VPusCfsucaUfuCfAfagaaUfuAfuaaagscsa
1956
UGCUUUAUAAUUCUUGAAUGAGG
2049

AD-75748
A-151663
asgsauagAfaAfUfGfuauguuugaaL96
1864
A-151664
VPusUfscaaAfcAfUfacauUfuCfuaucusasg
1957
CUAGAUAGAAAUGUAUGUUUGAC
2050

AD-75749
A-151665
ususccacAfaUfCfCfuuagaaucuaL96
1865
A-151666
VPusAfsgauUfcUfAfaggaUfuGfuggaasgsg
1958
CCUUCCACAAUCCUUAGAAUCUG
2051

AD-75750
A-151667
usasucaaAfaCfCfUfuucaaauauaL96
1866
A-151668
VPusAfsuauUfuGfAfaaggUfuUfugauasusu
1959
AAUAUCAAAACCUUUCAAAUAUC
2052

AD-75751
A-151669
ascsaaguAfaAfCfAfuuccaacauaL96
1867
A-151670
VPusAfsuguUfgGfAfauguUfuAfcuugusgsu
1960
ACACAAGUAAACAUUCCAACAUU
2053

AD-75755
A-151677
ascsauagAfaAfGfUfuucuucaacaL96
1868
A-151678
VPusGfsuugAfaGfAfaacuUfuCfuaugususu
1961
AAACAUAGAAAGUUUCUUCAACU
2054

AD-75757
A-151681
csasgucaAfcAfAfGfuauuuuaacaL96
1869
A-151682
VPusGfsuuaAfaAfUfacuuGfuUfgacugsasa
1962
UUCAGUCAACAAGUAUUUUAACU
2055

AD-75759
A-151685
csuscaagCfuGfUfCfuacuuacauaL96
1870
A-151686
VPusAfsuguAfaGfUfagacAfgCfuugagsgsu
1963
ACCUCAAGCUGUCUACUUACAUC
2056

AD-75760
A-151687
gsasauugUfuUfCfCfuuauuugcaaL96
1871
A-151688
VPusUfsgcaAfaUfAfaggaAfaCfaauucsasu
1964
AUGAAUUGUUUCCUUAUUUGCAC
2057

AD-75761
A-151689
asuscuguCfuUfAfGfuugaaaagcaL96
1872
A-151690
VPusGfscuuUfuCfAfacuaAfgAfcagausgsu
1965
ACAUCUGUCUUAGUUGAAAAGCA
2058

AD-75765
A-151697
asasuuagCfaAfUfAfuauuauccaaL96
1873
A-151698
VPusUfsggaUfaAfUfauauUfgCfuaauususu
1966
AAAAUUAGCAAUAUAUUAUCCAA
2059

AD-75766
A-151699
asasuugaAfuCfAfUfuaucuuacaaL96
1874
A-151700
VPusUfsguaAfgAfUfaaugAfuUfcaauusasg
1967
CUAAUUGAAUCAUUAUCUUACAU
2060

AD-75769
A-151705
ususuaucAfaUfAfAfuguucuauaaL96
1875
A-151706
VPusUfsauaGfaAfCfauuaUfuGfauaaasasg
1968
CUUUUAUCAAUAAUGUUCUAUAG
2061

AD-75772
A-151711
gsusaaaaGfaAfAfCfuauacaucaaL96
1876
A-151712
VPusUfsgauGfuAfUfaguuUfcUfuuuacsasu
1969
AUGUAAAAGAAACUAUACAUCAU
2062

AD-75774
A-151715
asasugagGfaAfUfAfauaaguuaaaL96
1877
A-151716
VPusUfsuaaCfuUfAfuuauUfcCfucauuscsu
1970
AGAAUGAGGAAUAAUAAGUUAAA
2063

AD-75776
A-151719
csuscuguGfaAfUfGfuguuuuaucaL96
1878
A-151720
VPusGfsauaAfaAfCfacauUfcAfcagagsasg
1971
CUCUCUGUGAAUGUGUUUUAUCC
2064

AD-75778
A-151723
gsusuccuUfcAfAfAfugaugaguuaL96
1879
A-151724
VPusAfsacuCfaUfCfauuuGfaAfggaacsusc
1972
GAGUUCCUUCAAAUGAUGAGUUA
2065

AD-75779
A-151725
csasggauAfaAfGfAfuaucaauuuaL96
1880
A-151726
VPusAfsaauUfgAfUfaucuUfuAfuccugsusa
1973
UACAGGAUAAAGAUAUCAAUUUA
2066

AD-75787
A-151741
csasugucCfuCfCfUfcgcaucucuaL96
1881
A-151742
VPusAfsgagAfuGfCfgaggAfgGfacaugsgsu
1974
ACCAUGUCCUCCUCGCAUCUCUU
2067

AD-75787
A-151741
csasugucCfuCfCfUfcgcaucucuaL96
1882
A-151742
VPusAfsgagAfuGfCfgaggAfgGfacaugsgsu
1975
ACCAUGUCCUCCUCGCAUCUCUU
2068

AD-75788
A-151743
ususcaacAfaGfCfCfcacaggguaaL96
1883
A-151744
VPusUfsaccCfuGfUfgggcUfuGfuugaasasu
1976
AUUUCAACAAGCCCACAGGGUAU
2069

AD-75788
A-151743
ususcaacAfaGfCfCfcacaggguaaL96
1884
A-151744
VPusUfsaccCfuGfUfgggcUfuGfuugaasasu
1977
AUUUCAACAAGCCCACAGGGUAU
2070

AD-75789
A-151745
uscscucgCfaUfCfUfcuucuaccuaL96
1885
A-151746
VPusAfsgguAfgAfAfgagaUfgCfgaggasgsg
1978
CCUCCUCGCAUCUCUUCUACCUG
2071

AD-75789
A-151745
uscscucgCfaUfCfUfcuucuaccuaL96
1886
A-151746
VPusAfsgguAfgAfAfgagaUfgCfgaggasgsg
1979
CCUCCUCGCAUCUCUUCUACCUG
2072

AD-75790
A-151747
gsgsggcuUfuUfAfUfuucaacaagaL96
1887
A-151748
VPusCfsuugUfuGfAfaauaAfaAfgccccsusg
1980
CAGGGGCUUUUAUUUCAACAAGC
2073

AD-75790
A-151747
gsgsggcuUfuUfAfUfuucaacaagaL96
1888
A-151748
VPusCfsuugUfuGfAfaauaAfaAfgccccsusg
1981
CAGGGGCUUUUAUUUCAACAAGC
2074

AD-75791
A-151749
gsasugcuCfuUfCfAfguucgugugaL96
1889
A-151750
VPusCfsacaCfgAfAfcugaAfgAfgcaucscsa
1982
UGGAUGCUCUUCAGUUCGUGUGU
2075

AD-75791
A-151749
gsasugcuCfuUfCfAfguucgugugaL96
1890
A-151750
VPusCfsacaCfgAfAfcugaAfgAfgcaucscsa
1983
UGGAUGCUCUUCAGUUCGUGUGU
2076

AD-75792
A-151751
usgsgaugCfuCfUfUfcaguucgugaL96
1891
A-151752
VPusCfsacgAfaCfUfgaagAfgCfauccascsc
1984
GGUGGAUGCUCUUCAGUUCGUGU
2077

AD-75792
A-151751
usgsgaugCfuCfUfUfcaguucgugaL96
1892
A-151752
VPusCfsacgAfaCfUfgaagAfgCfauccascsc
1985
GGUGGAUGCUCUUCAGUUCGUGU
2078

AD-75793
A-151753
gsgsuggaUfgCfUfCfuucaguucgaL96
1893
A-151754
VPusCfsgaaCfuGfAfagagCfaUfccaccsasg
1986
CUGGUGGAUGCUCUUCAGUUCGU
2079

AD-75793
A-151753
gsgsuggaUfgCfUfCfuucaguucgaL96
1894
A-151754
VPusCfsgaaCfuGfAfagagCfaUfccaccsasg
1987
CUGGUGGAUGCUCUUCAGUUCGU
2080

AD-75794
A-151755
gscsugguGfgAfUfGfcucuucaguaL96
1895
A-151756
VPusAfscugAfaGfAfgcauCfcAfccagcsusc
1988
GAGCUGGUGGAUGCUCUUCAGUU
2081

AD-75794
A-151755
gscsugguGfgAfUfGfcucuucaguaL96
1896
A-151756
VPusAfscugAfaGfAfgcauCfcAfccagcsusc
1989
GAGCUGGUGGAUGCUCUUCAGUU
2082

AD-75795
A-151757
csusggugGfaUfGfCfucuucaguuaL96
1897
A-151758
VPusAfsacuGfaAfGfagcaUfcCfaccagscsu
1990
AGCUGGUGGAUGCUCUUCAGUUC
2083

AD-75795
A-151757
csusggugGfaUfGfCfucuucaguuaL96
1898
A-151758
VPusAfsacuGfaAfGfagcaUfcCfaccagscsu
1991
AGCUGGUGGAUGCUCUUCAGUUC
2084

AD-77120
A-154752
cscsaaaaUfgCfAfCfugauguaaaaL96
1899
A-154753
VPusUfsuuaCfaUfCfagugCfaUfuuuggsgsc
1992
GCCCAAAAUGCACUGAUGUAAAG
2085

AD-77121
A-154754
uscscaguUfgCfAfCfuaaauuccuaL96
1900
A-154755
VPusAfsggaAfuUfUfagugCfaAfcuggasusc
1993
GAUCCAGUUGCACUAAAUUCCUC
2086

AD-77122
A-154756
csasuucuUfcAfAfCfuaucuuugaaL96
1901
A-154757
VPusUfscaaAfgAfUfaguuGfaAfgaaugsasg
1994
CUCAUUCUUCAACUAUCUUUGAU
2087

AD-77123
A-154758
ascsauucCfaAfCfAfuugucuuuaaL96
1902
A-154759
VPusUfsaaaGfaCfAfauguUfgGfaaugususu
1995
AAACAUUCCAACAUUGUCUUUAG
2088

AD-77124
A-154760
gsgscuuaGfaAfUfAfaaagauguaaL96
1903
A-154761
VPusUfsacaUfcUfUfuuauUfcUfaagccsusu
1996
AAGGCUUAGAAUAAAAGAUGUAG
2089

AD-77125
A-154762
ususucaaGfaUfAfUfuuguaaaagaL96
1904
A-154763
VPusCfsuuuUfaCfAfaauaUfcUfugaaasusu
1997
AAUUUCAAGAUAUUUGUAAAAGA
2090

AD-77126
A-154764
asusaagcAfuAfUfUfuugaaaaugaL96
1905
A-154765
VPusCfsauuUfuCfAfaaauAfuGfcuuaususa
1998
UAAUAAGCAUAUUUUGAAAAUGU
2091

AD-77127
A-154766
gsusuuucAfaUfGfCfuaguguuuaaL96
1906
A-154767
VPusUfsaaaCfaCfUfagcaUfuGfaaaacsasa
1999
UUGUUUUCAAUGCUAGUGUUUAA
2092

AD-77128
A-154768
csasaugaAfaUfAfCfacaaguaaaaL96
1907
A-154769
VPusUfsuuaCfuUfGfuguaUfuUfcauugsgsg
2000
CCCAAUGAAAUACACAAGUAAAC
2093

AD-77129
A-154770
csasaaagUfcCfAfCfugaugcaaaaL96
1908
A-154771
VPusUfsuugCfaUfCfagugGfaCfuuuugsusg
2001
CACAAAAGUCCACUGAUGCAAAU
2094

AD-77130
A-154772
asascauaGfaAfAfGfuuucuucaaaL96
1909
A-154773
VPusUfsugaAfgAfAfacuuUfcUfauguususa
2002
UAAACAUAGAAAGUUUCUUCAAC
2095

AD-77131
A-154774
asasccuaAfuUfAfGfuaacuuuccaL96
1910
A-154775
VPusGfsgaaAfgUfUfacuaAfuUfagguusgsc
2003
GCAACCUAAUUAGUAACUUUCCU
2096

AD-77132
A-154776
gsascuuaUfuUfCfCfuucacuuaaaL96
1911
A-154777
VPusUfsuaaGfuGfAfaggaAfaUfaagucsasu
2004
AUGACUUAUUUCCUUCACUUAAU
2097

AD-77133
A-154778
gsgscucuCfaAfAfCfuuagcaaaaaL96
1912
A-154779
VPusUfsuuuGfcUfAfaguuUfgAfgagccsasa
2005
UUGGCUCUCAAACUUAGCAAAAU
2098

AD-77134
A-154780
ascsaaagAfuAfAfGfugaaaagagaL96
1913
A-154781
VPusCfsucuUfuUfCfacuuAfuCfuuugusasu
2006
AUACAAAGAUAAGUGAAAAGAGA
2099

AD-77135
A-154782
gsusagugUfaCfUfGfuucaccaaaaL96
1914
A-154783
VPusUfsuugGfuGfAfacagUfaCfacuacsusu
2007
AAGUAGUGUACUGUUCACCAAAU
2100

AD-77136
A-154784
uscsaccaAfcUfCfAfuagcaaaguaL96
1915
A-154785
VPusAfscuuUfgCfUfaugaGfuUfggugasgsu
2008
ACUCACCAACUCAUAGCAAAGUC
2101

AD-77137
A-154786
asasaauaAfcUfCfAfuuauaccaaaL96
1916
A-154787
VPusUfsuggUfaUfAfaugaGfuUfauuuuscsa
2009
UGAAAAUAACUCAUUAUACCAAU
2102

AD-77138
A-154788
asasgaaaGfaAfUfCfuuuccauucaL96
1917
A-154789
VPusGfsaauGfgAfAfagauUfcUfuucuuscsu
2010
AGAAGAAAGAAUCUUUCCAUUCA
2103

AD-77139
A-154790
asusuucaAfgAfUfAfuuuguaaaaaL96
1918
A-154791
VPusUfsuuuAfcAfAfauauCfuUfgaaaususg
2011
CAAUUUCAAGAUAUUUGUAAAAG
2104

AD-77140
A-154792
asgscugaAfaCfUfCfuagaauuaaaL96
1919
A-154793
VPusUfsuaaUfuCfUfagagUfuUfcagcususg
2012
CAAGCUGAAACUCUAGAAUUAAA
2105

AD-77141
A-154794
asasagauAfaAfUfCfugauuuaugaL96
1920
A-154795
VPusCfsauaAfaUfCfagauUfuAfucuuususg
2013
CAAAAGAUAAAUCUGAUUUAUGC
2106

AD-77142
A-154796
usasuuaaAfuUfAfAfuucugauugaL96
1921
A-154797
VPusCfsaauCfaGfAfauuaAfuUfuaauasasa
2014
UUUAUUAAAUUAAUUCUGAUUGU
2107

AD-77143
A-154798
asasgaauGfaGfCfUfuucaacucaaL96
1922
A-154799
VPusUfsgagUfuGfAfaagcUfcAfuucuusasc
2015
GUAAGAAUGAGCUUUCAACUCAU
2108

AD-77144
A-154800
gsusuugaUfaAfCfAfuuuaaaagaaL96
1923
A-154801
VPusUfscuuUfuAfAfauguUfaUfcaaacsusu
2016
AAGUUUGAUAACAUUUAAAAGAU
2109

AD-77145
A-154802
usgsuuuuAfaAfCfAfuagaaaguuaL96
1924
A-154803
VPusAfsacuUfuCfUfauguUfuAfaaacasusa
2017
UAUGUUUUAAACAUAGAAAGUUU
2110

AD-77146
A-154804
usasaauaAfcCfCfAfuucauagcaaL96
1925
A-154805
VPusUfsgcuAfuGfAfauggGfuUfauuuasusa
2018
UAUAAAUAACCCAUUCAUAGCAU
2111

AD-77147
A-154806
csascauaGfaCfUfCfuuuaaaacuaL96
1926
A-154807
VPusAfsguuUfuAfAfagagUfcUfaugugsgsg
2019
CCCACAUAGACUCUUUAAAACUA
2112

AD-77148
A-154808
asasgucaCfaAfAfGfuuaucuucuaL96
1927
A-154809
VPusAfsgaaGfaUfAfacuuUfgUfgacuususu
2020
AAAAGUCACAAAGUUAUCUUCUU
2113

AD-77149
A-154810
usasaagaAfaGfAfAfuuuaugagaaL96
1928
A-154811
VPusUfscucAfuAfAfauucUfuUfcuuuasusc
2021
GAUAAAGAAAGAAUUUAUGAGAA
2114

AD-77150
A-154812
gsasaaugUfaUfGfUfuugacuuguaL96
1929
A-154813
VPusAfscaaGfuCfAfaacaUfaCfauuucsusa
2022
UAGAAAUGUAUGUUUGACUUGUU
2115

AD-77151
A-154814
ususuauaAfgAfCfCfuuccuguuaaL96
1930
A-154815
VPusUfsaacAfgGfAfagguCfuUfauaaasasu
2023
AUUUUAUAAGACCUUCCUGUUAG
2116

AD-77152
A-154816
csusgugaAfuGfUfGfuuuuauccaaL96
1931
A-154817
VPusUfsggaUfaAfAfacacAfuUfcacagsasg
2024
CUCUGUGAAUGUGUUUUAUCCAU
2117

AD-77153
A-154818
ususaauuCfuGfAfUfuguauuugaaL96
1932
A-154819
VPusUfscaaAfuAfCfaaucAfgAfauuaasusu
2025
AAUUAAUUCUGAUUGUAUUUGAA
2118

AD-77154
A-154820
usgsucauCfuAfCfCfuaccucaaaaL96
1933
A-154821
VPusUfsuugAfgGfUfagguAfgAfugacasusa
2026
UAUGUCAUCUACCUACCUCAAAG
2119

AD-77155
A-154822
csgsugaaAfgCfAfAfaacaauaggaL96
1934
A-154823
VPusCfscuaUfuGfUfuuugCfuUfucacgsusa
2027
UACGUGAAAGCAAAACAAUAGGG
2120

AD-77156
A-154824
asascaacAfaUfUfCfuauagauagaL96
1935
A-154825
VPusCfsuauCfuAfUfagaaUfuGfuuguususu
2028
AAAACAACAAUUCUAUAGAUAGA
2121

AD-77157
A-154826
usasaaguAfuUfAfUfuugaacuuuaL96
1936
A-154827
VPusAfsaagUfuCfAfaauaAfuAfcuuuasasu
2029
AUUAAAGUAUUAUUUGAACUUUU
2122

AD-77158
A-154828
asgsaaagAfaUfUfUfaugagaaauaL96
1937
A-154829
VPusAfsuuuCfuCfAfuaaaUfuCfuuucususu
2030
AAAGAAAGAAUUUAUGAGAAAUU
2123

AD-77159
A-154830
asasaugcAfcUfGfAfuguaaaguaaL96
1938
A-154831
VPusUfsacuUfuAfCfaucaGfuGfcauuususg
2031
CAAAAUGCACUGAUGUAAAGUAG
2124

AD-77160
A-154832
asusuaauUfcUfGfAfuuguauuugaL96
1939
A-154833
VPusCfsaaaUfaCfAfaucaGfaAfuuaaususu
2032
AAAUUAAUUCUGAUUGUAUUUGA
2125

AD-77161
A-154834
csasauaaUfgUfUfCfuauagaaaaaL96
1940
A-154835
VPusUfsuuuCfuAfUfagaaCfaUfuauugsasu
2033
AUCAAUAAUGUUCUAUAGAAAAG
2126

AD-77162
A-154836
gsasuuuuCfaAfUfUfugauuuugaaL96
1941
A-154837
VPusUfscaaAfaUfCfaaauUfgAfaaaucsasa
2034
UUGAUUUUCAAUUUGAUUUUGAA
2127

AD-77163
A-154840
ususgauuUfuGfAfAfuucugcauuaL96
1942
A-154841
VPusAfsaugCfaGfAfauucAfaAfaucaasasu
2035
AUUUGAUUUUGAAUUCUGCAUUU
2128

AD-77164
A-154842
ususucaaUfuUfGfAfuuuugaauuaL96
1943
A-154843
VPusAfsauuCfaAfAfaucaAfaUfugaaasasu
2036
AUUUUCAAUUUGAUUUUGAAUUC
2129

AD-77165
A-154844
gsusgucuGfuGfUfAfucaugaaaaaL96
1944
A-154845
VPusUfsuuuCfaUfGfauacAfcAfgacacsasg
2037
CUGUGUCUGUGUAUCAUGAAAAU
2130

AD-77166
A-154846
gsgsuuuuAfuGfAfAfuacaaagauaL96
1945
A-154847
VPusAfsucuUfuGfUfauucAfuAfaaaccsasa
2038
UUGGUUUUAUGAAUACAAAGAUA
2131

AD-77167
A-154848
gscsagauCfaAfGfAfuuuucucauaL96
1946
A-154849
VPusAfsugaGfaAfAfaucuUfgAfucugcsasg
2039
CUGCAGAUCAAGAUUUUCUCAUU
2132

AD-77168
A-154850
asascuaaUfuGfAfAfucauuaucuaL96
1947
A-154851
VPusAfsgauAfaUfGfauucAfaUfuaguusasc
2040
GUAACUAAUUGAAUCAUUAUCUU
2133

AD-77169
A-154852
usgsguuuAfuGfAfAfuuguuuccuaL96
1948
A-154853
VPusAfsggaAfaCfAfauucAfuAfaaccascsu
2041
AGUGGUUUAUGAAUUGUUUCCUU
2134

AD-77170
A-154854
usasguauAfaUfGfGfugcuauuuuaL96
1949
A-154855
VPusAfsaaaUfaGfCfaccaUfuAfuacuasasa
2042
UUUAGUAUAAUGGUGCUAUUUUG
2135

AD-77171
A-154856
csusguuuAfaUfAfAfgcauauuuuaL96
1950
A-154857
VPusAfsaaaUfaUfGfcuuaUfuAfaacagsusa
2043
UACUGUUUAAUAAGCAUAUUUUG
2136

AD-77172
A-154858
asasauauCfaAfAfAfccuuucaaaaL96
1951
A-154859
VPusUfsuugAfaAfGfguuuUfgAfuauuususg
2044
CAAAAUAUCAAAACCUUUCAAAU
2137

AD-77173
A-154860
ascsagauGfuAfAfAfagaaacuauaL96
1952
A-154861
VPusAfsuagUfuUfCfuuuuAfcAfucuguscsc
2045
GGACAGAUGUAAAAGAAACUAUA
2138

AD-77174
A-154862
asascuuuGfaGfGfCfcaaucauuuaL96
1953
A-154863
VPusAfsaauGfaUfUfggccUfcAfaaguusgsc
2046
GCAACUUUGAGGCCAAUCAUUUU
2139

Example 11—Further IGF-1 Transcripts, siRNA Design, and siRNA Screening Bioinformatics

A set of siRNAs targeting human IGF1 (human insulin like growth factor 1, NCBI refseqID: NM_000618; NCBI GeneID: 3479) were designed using custom R and Python scripts. The human IGF1 REFSEQ mRNA has a length of 7366 bases.

In Vitro Dual-Glo® Screening
Cell Culture and Transfections

Cos7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂in DMEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. Three human IGF-1 Dual-Glo® Luciferase constructs were generated using the psiCHECK2 vector. Construct one contained sequence based on NM_001111285, while constructs two and three contained sequence based on NM_000618 as provided in the prior Example. Dual-luciferase plasmids were co-transfected with siRNA into 5000 cells using Lipofectamine RNAiMax (Invitrogen, Carlsbad Calif. cat #13778-150). For each well of a 384 well plate, 0.1 μl of Lipofectamine was added to 15 ng of plasmid vector and siRNA in 15 μl of Opti-MEM and allowed to complex at room temperature for 15 minutes. The mixture was then added to the cells resuspended in 35 ul of fresh complete media. Cells were incubated for 48 hours before luciferase was measured. Single dose experiments were performed at 10 nM final duplex concentration.

Dual-Glo® Luciferase Assay

Forty-eight hours after the siRNAs were transfected, Firefly (transfection control) and Renilla (fused to IGF1 target sequence in 3′ UTR) luciferase were measured. First, media was removed from cells. Then Firefly luciferase activity was measured by adding 20 ul of Dual-Glo® Luciferase Reagent mixed with 20 ul of complete media to each well. The mixture was incubated at room temperature for 30 minutes before luminescense (500 nm) was measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 20 ul of room temperature of Dual-Glo® Stop & Glo® Reagent to each well and the plates were incubated for 20 minutes before luminescence was again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent quenched the firefly luciferase signal and sustained luminescence for the Renilla luciferase reaction. siRNA activity was determined by normalizing the Renilla (IGF1) signal to the Firefly (control) signal within each well. The magnitude of siRNA activity was then assessed relative to cells that were transfected with the same vector but were not treated with siRNA or were treated with a non-targeting siRNA. All transfections were done in quadruplicates.

TABLE 18

Unmodified Sense and Antisense Strand Sequences of IGF-1 dsRNAs

Sense

Position

Antisense

Position

Duplex
Oligo

in NM_
SEQ ID
Oligo

in NM_
SEQ ID

Name
Name
Sense Sequence
000618.3
NO
Name
Antisense Sequence
000618.3
NO

AD-74963
A-150432
UAGAUAAAUGUGAGGAUUU
6-24
2140
A-150433
AAAUCCUCACAUUUAUCUA
6-24
2420

AD-74964
A-150434
UUCUCUAAAUCCCUCUUCU
24-42
2141
A-150435
AGAAGAGGGAUUUAGAGAA
24-42
2421

AD-74965
A-150436
CUGUUUGCUAAAUCUCACU
41-59
2142
A-150437
AGUGAGAUUUAGCAAACAG
41-59
2422

AD-74966
A-150438
CUCACUGUCACUGCUAAAU
54-72
2143
A-150439
AUUUAGCAGUGACAGUGAG
54-72
2423

AD-74967
A-150440
UUCAGAGCAGAUAGAGCCU
72-90
2144
A-150441
AGGCUCUAUCUGCUCUGAA
72-90
2424

AD-74968
A-150442
CAUUGCUCUCAACAUCUCA
127-145
2145
A-150443
UGAGAUGUUGAGAGCAAUG
127-145
2425

AD-74969
A-150444
ACCAAUUCAUUUUCAGACU
185-203
2146
A-150445
AGUCUGAAAAUGAAUUGGU
185-203
2426

AD-74970
A-150446
UUUGUACUUCAGAAGCAAU
203-221
2147
A-150447
AUUGCUUCUGAAGUACAAA
203-221
2427

AD-74971
A-150448
AUGGGAAAAAUCAGCAGUA
220-238
2148
A-150449
UACUGCUGAUUUUUCCCAU
220-238
2428

AD-74972
A-150450
CAAUUAUUUAAGUGCUGCU
247-265
2149
A-150451
AGCAGCACUUAAAUAAUUG
247-265
2429

AD-74973
A-150452
UUGAAGGUGAAGAUGCACA
277-295
2150
A-150453
UGUGCAUCUUCACCUUCAA
277-295
2430

AD-74974
A-150454
UUUUAUUUCAACAAGCCCA
430-448
2151
A-150455
UGGGCUUGUUGAAAUAAAA
430-448
2431

AD-74975
A-150456
CACAGGGUAUGGCUCCAGA
447-465
2152
A-150457
UCUGGAGCCAUACCCUGUG
447-465
2432

AD-74976
A-150458
CAGCAGUCGGAGGGCGCCU
462-480
2153
A-150459
AGGCGCCCUCCGACUGCUG
462-480
2433

AD-74977
A-150460
UUGCGCACCCCUCAAGCCU
543-561
2154
A-150461
AGGCUUGAGGGGUGCGCAA
543-561
2434

AD-74978
A-150462
UGCAGGAAACAAGAACUAA
654-672
2155
A-150463
UUAGUUCUUGUUUCCUGCA
654-672
2435

AD-74979
A-150464
CAGGAUGUAGGAAGACCCU
672-690
2156
A-150465
AGGGUCUUCCUACAUCCUG
672-690
2436

AD-74980
A-150466
UUAAACUUUGGAACACCUA
750-768
2157
A-150467
UAGGUGUUCCAAAGUUUAA
750-768
2437

AD-74981
A-150468
AAAUAAGUUUGAUAACAUU
774-792
2158
A-150469
AAUGUUAUCAAACUUAUUU
774-792
2438

AD-74982
A-150470
UUAAAAGAUGGGCGUUUCA
792-810
2159
A-150471
UGAAACGCCCAUCUUUUAA
792-810
2439

AD-74983
A-150472
AAAUACACAAGUAAACAUU
818-836
2160
A-150473
AAUGUUUACUUGUGUAUUU
818-836
2440

AD-74984
A-150474
UUCCAACAUUGUCUUUAGA
835-853
2161
A-150475
UCUAAAGACAAUGUUGGAA
835-853
2441

AD-74985
A-150476
GGAGUGAUUUGCACCUUGA
852-870
2162
A-150477
UCAAGGUGCAAAUCACUCC
852-870
2442

AD-74986
A-150478
AUUGCUGUUGAUCUUUUAU
894-912
2163
A-150479
AUAAAAGAUCAACAGCAAU
894-912
2443

AD-74987
A-150480
UCAAUAAUGUUCUAUAGAA
912-930
2164
A-150481
UUCUAUAGAACAUUAUUGA
912-930
2444

AD-74988
A-150482
AAAGAAAAAAAAAAUAUAU
930-948
2165
A-150483
AUAUAUUUUUUUUUUCUUU
930-948
2445

AD-74989
A-150484
AUAUAUAUAUAUAUCUUAA
947-965
2166
A-150485
UUAAGAUAUAUAUAUAUAU
947-965
2446

AD-74990
A-150486
UUUCCUUAUUUGCACUUCU
1091-1109
2167
A-150487
AGAAGUGCAAAUAAGGAAA
1091-1109
2447

AD-74991
A-150488
CUUUCUACACAACUCGGGA
1108-1126
2168
A-150489
UCCCGAGUUGUGUAGAAAG
1108-1126
2448

AD-74992
A-150490
GCUGUUUGUUUUACAGUGU
1125-1143
2169
A-150491
ACACUGUAAAACAAACAGC
1125-1143
2449

AD-74993
A-150492
UUACAGUGUCUGAUAAUCU
1135-1153
2170
A-150493
AGAUUAUCAGACACUGUAA
1135-1153
2450

AD-74994
A-150494
CUGAUAAUCUUGUUAGUCU
1144-1162
2171
A-150495
AGACUAACAAGAUUAUCAG
1144-1162
2451

AD-74995
A-150496
UAUACCCACCACCUCCCUU
1162-1180
2172
A-150497
AAGGGAGGUGGUGGGUAUA
1162-1180
2452

AD-74996
A-150498
UUGCCGAAUUUGGCCUCCU
1195-1213
2173
A-150499
AGGAGGCCAAAUUCGGCAA
1195-1213
2453

AD-74997
A-150500
GCCGAAUUUGGCCUCCUCA
1197-1215
2174
A-150501
UGAGGAGGCCAAAUUCGGC
1197-1215
2454

AD-74998
A-150502
AAAAGCAGCAGCAAGUCGU
1215-1233
2175
A-150503
ACGACUUGCUGCUGCUUUU
1215-1233
2455

AD-74999
A-150504
GUCAAGAAGCACACCAAUU
1232-1250
2176
A-150505
AAUUGGUGUGCUUCUUGAC
1232-1250
2456

AD-75000
A-150506
AGUUGGAUGCAUUUUAUUU
1293-1311
2177
A-150507
AAAUAAAAUGCAUCCAACU
1293-1311
2457

AD-75001
A-150508
UUAGACACAAAGCUUUAUU
1311-1329
2178
A-150509
AAUAAAGCUUUGUGUCUAA
1311-1329
2458

AD-75002
A-150510
CACAUCAUGCUUACAAAAA
1334-1352
2179
A-150511
UUUUUGUAAGCAUGAUGUG
1334-1352
2459

AD-75003
A-150512
AAGAAUAAUGCAAAUAGUU
1352-1370
2180
A-150513
AACUAUUUGCAUUAUUCUU
1352-1370
2460

AD-75004
A-150514
UGCAACUUUGAGGCCAAUA
1370-1388
2181
A-150515
UAUUGGCCUCAAAGUUGCA
1370-1388
2461

AD-75005
A-150516
CAUUUUUAGGCAUAUGUUU
1388-1406
2182
A-150517
AAACAUAUGCCUAAAAAUG
1388-1406
2462

AD-75006
A-150518
UUAAACAUAGAAAGUUUCU
1406-1424
2183
A-150519
AGAAACUUUCUAUGUUUAA
1406-1424
2463

AD-75007
A-150520
CUUCAACUCAAAAGAGUUA
1423-1441
2184
A-150521
UAACUCUUUUGAGUUGAAG
1423-1441
2464

AD-75008
A-150522
UCCUUCAAAUGAUGAGUUA
1440-1458
2185
A-150523
UAACUCAUCAUUUGAAGGA
1440-1458
2465

AD-75009
A-150524
UUAGUAACUUUCCUCUUUU
1472-1490
2186
A-150525
AAAAGAGGAAAGUUACUAA
1472-1490
2466

AD-75010
A-150526
UUUUUCCAUAUAGAGCACU
1494-1512
2187
A-150527
AGUGCUCUAUAUGGAAAAA
1494-1512
2467

AD-75011
A-150528
CUAUGUAAAUUUAGCAUAU
1511-1529
2188
A-150529
AUAUGCUAAAUUUACAUAG
1511-1529
2468

AD-75012
A-150530
AUCAAUUAUACAGGAUAUA
1528-1546
2189
A-150531
UAUAUCCUGUAUAAUUGAU
1528-1546
2469

AD-75013
A-150532
UUUAGUAUAAUGGUGCUAU
1572-1590
2190
A-150533
AUAGCACCAUUAUACUAAA
1572-1590
2470

AD-75014
A-150534
UUGUUAUAUGAAAGAGUCU
1599-1617
2191
A-150535
AGACUCUUUCAUAUAACAA
1599-1617
2471

AD-75015
A-150536
ACGGUAAUACGUGAAAGCA
1625-1643
2192
A-150537
UGCUUUCACGUAUUACCGU
1625-1643
2472

AD-75016
A-150538
AAAACAAUAGGGGAAGCCU
1643-1661
2193
A-150539
AGGCUUCCCCUAUUGUUUU
1643-1661
2473

AD-75017
A-150540
UACUGAAAACACCAUCCAU
1690-1708
2194
A-150541
AUGGAUGGUGUUUUCAGUA
1690-1708
2474

AD-75018
A-150542
UUGGGAAAGAAGGCAAAGU
1709-1727
2195
A-150543
ACUUUGCCUUCUUUCCCAA
1709-1727
2475

AD-75019
A-150544
UCAGACACAAAAGUCCACU
1757-1775
2196
A-150545
AGUGGACUUUUGUGUCUGA
1757-1775
2476

AD-75020
A-150546
CGAGUCCAGAGAGGAAACU
1793-1811
2197
A-150547
AGUUUCCUCUCUGGACUCG
1793-1811
2477

AD-75021
A-150548
AAACUGUGGAAUGGAAAAA
1807-1825
2198
A-150549
UUUUUCCAUUCCACAGUUU
1807-1825
2478

AD-75022
A-150550
AGCAGAAGGCUAGGAAUUU
1825-1843
2199
A-150551
AAAUUCCUAGCCUUCUGCU
1825-1843
2479

AD-75023
A-150552
UUAGCAGUCCUGGUUUCUU
1843-1861
2200
A-150553
AAGAAACCAGGACUGCUAA
1843-1861
2480

AD-75024
A-150554
CAAAAUGGGGGCAAUAUGU
1966-1984
2201
A-150555
ACAUAUUGCCCCCAUUUUG
1966-1984
2481

AD-75025
A-150556
UUUAAAAAGAUAAAGAUUA
2016-2034
2202
A-150557
UAAUCUUUAUCUUUUUAAA
2016-2034
2482

AD-75026
A-150558
UCAGAUUUUUUUUACCCUA
2033-2051
2203
A-150559
UAGGGUAAAAAAAAUCUGA
2033-2051
2483

AD-75027
A-150560
UUUUUUACCCUGGGUUGCU
2040-2058
2204
A-150561
AGCAACCCAGGGUAAAAAA
2040-2058
2484

AD-75028
A-150562
CUGUAAGGGUGCAACAUCA
2057-2075
2205
A-150563
UGAUGUUGCACCCUUACAG
2057-2075
2485

AD-75029
A-150564
CUGAGAUGCAAGGAAUUCU
2090-2108
2206
A-150565
AGAAUUCCUUGCAUCUCAG
2090-2108
2486

AD-75030
A-150566
UUGGUGAAUUGAAUGCUCA
2140-2158
2207
A-150567
UGAGCAUUCAAUUCACCAA
2140-2158
2487

AD-75031
A-150568
UUCUUGUCAGUGAAGCUAU
2170-2188
2208
A-150569
AUAGCUUCACUGACAAGAA
2170-2188
2488

AD-75032
A-150570
AAUAACUGGCCAACUAGUU
2192-2210
2209
A-150571
AACUAGUUGGCCAGUUAUU
2192-2210
2489

AD-75033
A-150572
UGUUAAAAGCUAACAGCUA
2210-2228
2210
A-150573
UAGCUGUUAGCUUUUAACA
2210-2228
2490

AD-75034
A-150574
CAAUCUCUUAAAACACUUU
2228-2246
2211
A-150575
AAAGUGUUUUAAGAGAUUG
2228-2246
2491

AD-75035
A-150576
AAAAUAUGUGGGAAGCAUU
2249-2267
2212
A-150577
AAUGCUUCCCACAUAUUUU
2249-2267
2492

AD-75036
A-150578
UUUGAUUUUCAAUUUGAUU
2266-2284
2213
A-150579
AAUCAAAUUGAAAAUCAAA
2266-2284
2493

AD-75037
A-150580
UUGAAUUCUGCAUUUGGUU
2285-2303
2214
A-150581
AACCAAAUGCAGAAUUCAA
2285-2303
2494

AD-75038
A-150582
UUUAUGAAUACAAAGAUAA
2303-2321
2215
A-150583
UUAUCUUUGUAUUCAUAAA
2303-2321
2495

AD-75039
A-150584
GUGAAAAGAGAGAAAGGAA
2322-2340
2216
A-150585
UUCCUUUCUCUCUUUUCAC
2322-2340
2496

AD-75040
A-150586
AAAGAAAAAGGAGAAAAAC
2340-2358
2217
A-150587
GUUUUUCUCCUUUUUCUUU
2340-2358
2497

AD-75041
A-150588
ACAAAGAGAUUUCUACCAA
2357-2375
2218
A-150589
UUGGUAGAAAUCUCUUUGU
2357-2375
2498

AD-75042
A-150590
UUGUUAGCACUCACUGACU
2398-2416
2219
A-150591
AGUCAGUGAGUGCUAACAA
2398-2416
2499

AD-75043
A-150592
UACAUAUCUAGUAAAACCU
2432-2450
2220
A-150593
AGGUUUUACUAGAUAUGUA
2432-2450
2500

AD-75044
A-150594
CUCGUUUAAUACUAUAAAU
2449-2467
2221
A-150595
AUUUAUAGUAUUAAACGAG
2449-2467
2501

AD-75045
A-150596
UUUAAUACUAUAAAUAAUA
2453-2471
2222
A-150597
UAUUAUUUAUAGUAUUAAA
2453-2471
2502

AD-75046
A-150598
UAUUCUAUUCAUUUUGAAA
2470-2488
2223
A-150599
UUUCAAAAUGAAUAGAAUA
2470-2488
2503

AD-75047
A-150600
UUUGAAAAACACAAUGAUU
2482-2500
2224
A-150601
AAUCAUUGUGUUUUUCAAA
2482-2500
2504

AD-75048
A-150602
AAGGAAAGUGAUCCAAAAU
2521-2539
2225
A-150603
AUUUUGGAUCACUUUCCUU
2521-2539
2505

AD-75049
A-150604
UUUGAAAUAUUAAAAUAAU
2539-2557
2226
A-150605
AUUAUUUUAAUAUUUCAAA
2539-2557
2506

AD-75050
A-150606
UUAAAAUAAUAUCUAAUAA
2548-2566
2227
A-150607
UUAUUAGAUAUUAUUUUAA
2548-2566
2507

AD-75051
A-150608
AAAAGUCACAAAGUUAUCU
2566-2584
2228
A-150609
AGAUAACUUUGUGACUUUU
2566-2584
2508

AD-75052
A-150610
UUCUUUAACAAACUUUACU
2584-2602
2229
A-150611
AGUAAAGUUUGUUAAAGAA
2584-2602
2509

AD-75053
A-150612
CUCUUAUUCUUAGCUGUAU
2601-2619
2230
A-150613
AUACAGCUAAGAAUAAGAG
2601-2619
2510

AD-75054
A-150614
AUAUACAUUUUUUUAAAAG
2618-2636
2231
A-150615
CUUUUAAAAAAAUGUAUAU
2618-2636
2511

AD-75055
A-150616
CAUUUUUUUAAAAGUUUGU
2623-2641
2232
A-150617
ACAAACUUUUAAAAAAAUG
2623-2641
2512

AD-75056
A-150618
GUUAAAAUAUGCUUGACUA
2640-2658
2233
A-150619
UAGUCAAGCAUAUUUUAAC
2640-2658
2513

AD-75057
A-150620
AUGCUUGACUAGAGUUUCA
2648-2666
2234
A-150621
UGAAACUCUAGUCAAGCAU
2648-2666
2514

AD-75058
A-150622
CAGUUGAAAGGCAAAAACU
2666-2684
2235
A-150623
AGUUUUUGCCUUUCAACUG
2666-2684
2515

AD-75059
A-150624
UUCCAUCACAACAAGAAAU
2684-2702
2236
A-150625
AUUUCUUGUUGUGAUGGAA
2684-2702
2516

AD-75060
A-150626
UUGGUAUCAAGAAAGUCCA
2771-2789
2237
A-150627
UGGACUUUCUUGAUACCAA
2771-2789
2517

AD-75061
A-150628
GUUAGUGUACUAGUCCAUA
2793-2811
2238
A-150629
UAUGGACUAGUACACUAAC
2793-2811
2518

AD-75062
A-150630
CAUAGCCUAGAAAAUGAUA
2811-2829
2239
A-150631
UAUCAUUUUCUAGGCUAUG
2811-2829
2519

AD-75063
A-150632
UCCCUAUCUGCAGAUCAAA
2828-2846
2240
A-150633
UUUGAUCUGCAGAUAGGGA
2828-2846
2520

AD-75064
A-150634
UUAUCCAGCAUUCAGAUCU
2869-2887
2241
A-150635
AGAUCUGAAUGCUGGAUAA
2869-2887
2521

AD-75065
A-150636
UUUUUGGUUAAAAGUACCA
2906-2924
2242
A-150637
UGGUACUUUUAACCAAAAA
2906-2924
2522

AD-75066
A-150638
UACCCAGGCUUGAUUAUUU
2920-2938
2243
A-150639
AAAUAAUCAAGCCUGGGUA
2920-2938
2523

AD-75067
A-150640
UCAUGCAAAUUCUAUAUUU
2938-2956
2244
A-150641
AAAUAUAGAAUUUGCAUGA
2938-2956
2524

AD-75068
A-150642
UUACAUUCUUGGAAAGUCU
2956-2974
2245
A-150643
AGACUUUCCAAGAAUGUAA
2956-2974
2525

AD-75069
A-150644
UCUUGGAAAGUCUAUAUGA
2962-2980
2246
A-150645
UCAUAUAGACUUUCCAAGA
2962-2980
2526

AD-75070
A-150646
AAAAACAAAAAUAACAUCU
2980-2998
2247
A-150647
AGAUGUUAUUUUUGUUUUU
2980-2998
2527

AD-75071
A-150648
UUCUCCCACUGGGUCACCU
3006-3024
2248
A-150649
AGGUGACCCAGUGGGAGAA
3006-3024
2528

AD-75072
A-150650
CAAGGAUCAGAGGCCAGGA
3025-3043
2249
A-150651
UCCUGGCCUCUGAUCCUUG
3025-3043
2529

AD-75073
A-150652
AAAAAAAAAAAAAAGACUA
3043-3061
2250
A-150653
UAGUCUUUUUUUUUUUUUU
3043-3061
2530

AD-75074
A-150654
UCCCUGGAUCUCUGAAUAU
3060-3078
2251
A-150655
AUAUUCAGAGAUCCAGGGA
3060-3078
2531

AD-75075
A-150656
AUAUGCAAAAAGAAGGCCA
3077-3095
2252
A-150657
UGGCCUUCUUUUUGCAUAU
3077-3095
2532

AD-75076
A-150658
UAGUGGAGCCAGCAAUCCU
3100-3118
2253
A-150659
AGGAUUGCUGGCUCCACUA
3100-3118
2533

AD-75077
A-150660
UUAACUCUCAGUCCAACAU
3137-3155
2254
A-150661
AUGUUGGACUGAGAGUUAA
3137-3155
2534

AD-75078
A-150662
UUAUUUGAAUUGAGCACCU
3155-3173
2255
A-150663
AGGUGCUCAAUUCAAAUAA
3155-3173
2535

AD-75079
A-150664
CAGAUGUAAAAGAAACUAU
3208-3226
2256
A-150665
AUAGUUUCUUUUACAUCUG
3208-3226
2536

AD-75080
A-150666
AUACAUCAUUUUUGCCCUA
3225-3243
2257
A-150667
UAGGGCAAAAAUGAUGUAU
3225-3243
2537

AD-75081
A-150668
UUUUGCCCUCUGCCUGUUU
3234-3252
2258
A-150669
AAACAGGCAGAGGGCAAAA
3234-3252
2538

AD-75082
A-150670
UUCCAGACAUACAGGUUCU
3252-3270
2259
A-150671
AGAACCUGUAUGUCUGGAA
3252-3270
2539

AD-75083
A-150672
CUGUGGAAUAAGAUACUGA
3269-3287
2260
A-150673
UCAGUAUCUUAUUCCACAG
3269-3287
2540

AD-75084
A-150674
UAAGAUACUGGACUCCUCU
3277-3295
2261
A-150675
AGAGGAGUCCAGUAUCUUA
3277-3295
2541

AD-75085
A-150676
CUUCCCAAGAUGGCACUUA
3294-3312
2262
A-150677
UAAGUGCCAUCUUGGGAAG
3294-3312
2542

AD-75086
A-150678
GUGUACCUUUUAAAAUUAU
3334-3352
2263
A-150679
AUAAUUUUAAAAGGUACAC
3334-3352
2543

AD-75087
A-150680
UUCCCUCUCAACAAAACUU
3352-3370
2264
A-150681
AAGUUUUGUUGAGAGGGAA
3352-3370
2544

AD-75088
A-150682
UUUAUAGGCAGUCUUCUGA
3369-3387
2265
A-150683
UCAGAAGACUGCCUAUAAA
3369-3387
2545

AD-75089
A-150684
UUUUCUGUCAUAGUUAGAU
3400-3418
2266
A-150685
AUCUAACUAUGACAGAAAA
3400-3418
2546

AD-75090
A-150686
AUGUGAUAAUUCUAAGAGU
3417-3435
2267
A-150687
ACUCUUAGAAUUAUCACAU
3417-3435
2547

AD-75091
A-150688
UUCCUUCACUUAAUUCUAU
3449-3467
2268
A-150689
AUAGAAUUAAGUGAAGGAA
3449-3467
2548

AD-75092
A-150690
AUUAUCUUUCUUAACUUUU
3517-3535
2269
A-150691
AAAAGUUAAGAAAGAUAAU
3517-3535
2549

AD-75093
A-150692
UUCCAACACAUAAUCCUCU
3535-3553
2270
A-150693
AGAGGAUUAUGUGUUGGAA
3535-3553
2550

AD-75094
A-150694
AAAUAAAUUGAAAAUAACU
3567-3585
2271
A-150695
AGUUAUUUUCAAUUUAUUU
3567-3585
2551

AD-75095
A-150696
UCAUUAUACCAAUUCACUA
3585-3603
2272
A-150697
UAGUGAAUUGGUAUAAUGA
3585-3603
2552

AD-75096
A-150698
AUUUUAUUUUUUAAUGAAU
3603-3621
2273
A-150699
AUUCAUUAAAAAAUAAAAU
3603-3621
2553

AD-75097
A-150700
UUAAAACUAGAAAACAAAU
3621-3639
2274
A-150701
AUUUGUUUUCUAGUUUUAA
3621-3639
2554

AD-75098
A-150702
UUGAUUACUAUAUACUACA
3662-3680
2275
A-150703
UGUAGUAUAUAGUAAUCAA
3662-3680
2555

AD-75099
A-150704
AUGACUCAGAUUUCAUAGA
3686-3704
2276
A-150705
UCUAUGAAAUCUGAGUCAU
3686-3704
2556

AD-75100
A-150706
AAAGGAGCAACCAAAAUGU
3704-3722
2277
A-150707
ACAUUUUGGUUGCUCCUUU
3704-3722
2557

AD-75101
A-150708
GUCACAACCCAAAACUUUA
3721-3739
2278
A-150709
UAAAGUUUUGGGUUGUGAC
3721-3739
2558

AD-75102
A-150710
AAACUUUACAAGCUUUGCU
3732-3750
2279
A-150711
AGCAAAGCUUGUAAAGUUU
3732-3750
2559

AD-75103
A-150712
UUCAGAAUUAGAUUGCUUU
3750-3768
2280
A-150713
AAAGCAAUCUAAUUCUGAA
3750-3768
2560

AD-75104
A-150714
UUAUAAUUCUUGAAUGAGA
3767-3785
2281
A-150715
UCUCAUUCAAGAAUUAUAA
3767-3785
2561

AD-75105
A-150716
UAAUUCUUGAAUGAGGCAA
3770-3788
2282
A-150717
UUGCCUCAUUCAAGAAUUA
3770-3788
2562

AD-75106
A-150718
AUUUCAAGAUAUUUGUAAA
3788-3806
2283
A-150719
UUUACAAAUAUCUUGAAAU
3788-3806
2563

AD-75107
A-150720
AAGAACAGUAAACAUUGGU
3806-3824
2284
A-150721
ACCAAUGUUUACUGUUCUU
3806-3824
2564

AD-75108
A-150722
UUUCAACUCAUAGGCUUAU
3835-3853
2285
A-150723
AUAAGCCUAUGAGUUGAAA
3835-3853
2565

AD-75109
A-150724
UUGACCAUACUGGAUACUU
3865-3883
2286
A-150725
AAGUAUCCAGUAUGGUCAA
3865-3883
2566

AD-75110
A-150726
UUUAAGAUGAGGCAGUUCA
3939-3957
2287
A-150727
UGAACUGCCUCAUCUUAAA
3939-3957
2567

AD-75111
A-150728
CAUCAGAAUCCACUCUUCU
3982-4000
2288
A-150729
AGAAGAGUGGAUUCUGAUG
3982-4000
2568

AD-75112
A-150730
UAGGGAUAUGAAAAUCUCU
4000-4018
2289
A-150731
AGAGAUUUUCAUAUCCCUA
4000-4018
2569

AD-75113
A-150732
UUCACCCUAAGGAUCCAAU
4081-4099
2290
A-150733
AUUGGAUCCUUAGGGUGAA
4081-4099
2570

AD-75114
A-150734
AUGGAAUACUGAAAAGAAA
4098-4116
2291
A-150735
UUUCUUUUCAGUAUUCCAU
4098-4116
2571

AD-75115
A-150736
GAAUACUGAAAAGAAAUCA
4101-4119
2292
A-150737
UGAUUUCUUUUCAGUAUUC
4101-4119
2572

AD-75116
A-150738
ACUUCCUUGAAAAUUUUAU
4119-4137
2293
A-150739
AUAAAAUUUUCAAGGAAGU
4119-4137
2573

AD-75117
A-150740
UUAAAAAACAAACAAACAA
4137-4155
2294
A-150741
UUGUUUGUUUGUUUUUUAA
4137-4155
2574

AD-75118
A-150742
AAACAAAAAGCCUGUCCAA
4154-4172
2295
A-150743
UUGGACAGGCUUUUUGUUU
4154-4172
2575

AD-75119
A-150744
UUUGUGUAGAUGAAACCAU
4208-4226
2296
A-150745
AUGGUUUCAUCUACACAAA
4208-4226
2576

AD-75120
A-150746
UUGGGAGAAGGCUUAGAAU
4271-4289
2297
A-150747
AUUCUAAGCCUUCUCCCAA
4271-4289
2577

AD-75121
A-150748
UAAAAGAUGUAGCACAUUU
4289-4307
2298
A-150749
AAAUGUGCUACAUCUUUUA
4289-4307
2578

AD-75122
A-150750
UUAUUGUUUGGCCAGCUAU
4319-4337
2299
A-150751
AUAGCUGGCCAAACAAUAA
4319-4337
2579

AD-75123
A-150752
AUGCCAAUGUGGUGCUAUU
4336-4354
2300
A-150753
AAUAGCACCACAUUGGCAU
4336-4354
2580

AD-75124
A-150754
AAUGUGGUGCUAUUGUUUA
4341-4359
2301
A-150755
UAAACAAUAGCACCACAUU
4341-4359
2581

AD-75125
A-150756
CUUUAAGAAAGUACUUGAA
4359-4377
2302
A-150757
UUCAAGUACUUUCUUAAAG
4359-4377
2582

AD-75126
A-150758
CUAAAAAAAAAAGAAAAAA
4377-4395
2303
A-150759
UUUUUUCUUUUUUUUUUAG
4377-4395
2583

AD-75127
A-150760
AAGAAAAAAAAGAAAGCAU
4395-4413
2304
A-150761
AUGCUUUCUUUUUUUUCUU
4395-4413
2584

AD-75128
A-150762
AUAGACAUAUUUUUUUAAA
4412-4430
2305
A-150763
UUUAAAAAAAUAUGUCUAU
4412-4430
2585

AD-75129
A-150764
UUAAAGUAUAAAAACAACA
4426-4444
2306
A-150765
UGUUGUUUUUAUACUUUAA
4426-4444
2586

AD-75130
A-150766
CAAUUCUAUAGAUAGAUGA
4443-4461
2307
A-150767
UCAUCUAUCUAUAGAAUUG
4443-4461
2587

AD-75131
A-150768
GGCUUAAUAAAAUAGCAUU
4460-4478
2308
A-150769
AAUGCUAUUUUAUUAAGCC
4460-4478
2588

AD-75132
A-150770
UAAUAAAAUAGCAUUAGGU
4464-4482
2309
A-150771
ACCUAAUGCUAUUUUAUUA
4464-4482
2589

AD-75133
A-150772
UAUCUAGCCACCACCACCU
4484-4502
2310
A-150773
AGGUGGUGGUGGCUAGAUA
4484-4502
2590

AD-75134
A-150774
UUUAUCACUCACAAGUAGU
4511-4529
2311
A-150775
ACUACUUGUGAGUGAUAAA
4511-4529
2591

AD-75135
A-150776
GGCAGGAGUUGGAAAUUUU
4566-4584
2312
A-150777
AAAAUUUCCAACUCCUGCC
4566-4584
2592

AD-75136
A-150778
UUUAAAGUUAGAAGGCUCA
4584-4602
2313
A-150779
UGAGCCUUCUAACUUUAAA
4584-4602
2593

AD-75137
A-150780
CCAUUGUUUUGUUGGCUCU
4601-4619
2314
A-150781
AGAGCCAACAAAACAAUGG
4601-4619
2594

AD-75138
A-150782
UUAGCAAAAUUAGCAAUAU
4625-4643
2315
A-150783
AUAUUGCUAAUUUUGCUAA
4625-4643
2595

AD-75139
A-150784
AUAUUAUCCAAUCUUCUGA
4642-4660
2316
A-150785
UCAGAAGAUUGGAUAAUAU
4642-4660
2596

AD-75140
A-150786
UUAUCCAAUCUUCUGAACU
4645-4663
2317
A-150787
AGUUCAGAAGAUUGGAUAA
4645-4663
2597

AD-75141
A-150788
AAGAGCAUGGAGAAUAAAC
4669-4687
2318
A-150789
GUUUAUUCUCCAUGCUCUU
4669-4687
2598

AD-75142
A-150790
ACGCGGGAAAAAAGAUCUU
4686-4704
2319
A-150791
AAGAUCUUUUUUCCCGCGU
4686-4704
2599

AD-75143
A-150792
GAUCUUAUAGGCAAAUAGA
4699-4717
2320
A-150793
UCUAUUUGCCUAUAAGAUC
4699-4717
2600

AD-75144
A-150794
AAGAAUUUAAAAGAUAAGU
4717-4735
2321
A-150795
ACUUAUCUUUUAAAUUCUU
4717-4735
2601

AD-75145
A-150796
GUAAGUUCCUUAUUGAUUU
4734-4752
2322
A-150797
AAAUCAAUAAGGAACUUAC
4734-4752
2602

AD-75146
A-150798
UUUUGUGCACUCUGCUCUA
4751-4769
2323
A-150799
UAGAGCAGAGUGCACAAAA
4751-4769
2603

AD-75147
A-150800
AAACAGAUAUUCAGCAAGU
4770-4788
2324
A-150801
ACUUGCUGAAUAUCUGUUU
4770-4788
2604

AD-75148
A-150802
UCAGCAAGUGGAGAAAAUA
4780-4798
2325
A-150803
UAUUUUCUCCACUUGCUGA
4780-4798
2605

AD-75149
A-150804
AAGAACAAAGAGAAAAAAU
4798-4816
2326
A-150805
AUUUUUUCUCUUUGUUCUU
4798-4816
2606

AD-75150
A-150806
AUACAUAGAUUUACCUGCA
4815-4833
2327
A-150807
UGCAGGUAAAUCUAUGUAU
4815-4833
2607

AD-75151
A-150808
UUACCUGCAAAAAAUAGCU
4825-4843
2328
A-150809
AGCUAUUUUUUGCAGGUAA
4825-4843
2608

AD-75152
A-150810
UUUAUAGAAGACAUUCUCA
4884-4902
2329
A-150811
UGAGAAUGUCUUCUAUAAA
4884-4902
2609

AD-75153
A-150812
AGACAUCUCAAAGAGCAGU
4911-4929
2330
A-150813
ACUGCUCUUUGAGAUGUCU
4911-4929
2610

AD-75154
A-150814
UAUGAGAUGGGGGUUAUCU
4987-5005
2331
A-150815
AGAUAACCCCCAUCUCAUA
4987-5005
2611

AD-75155
A-150816
CUACUGAUAAAGAAAGAAU
5004-5022
2332
A-150817
AUUCUUUCUUUAUCAGUAG
5004-5022
2612

AD-75156
A-150818
AAAGAAUUUAUGAGAAAUU
5016-5034
2333
A-150819
AAUUUCUCAUAAAUUCUUU
5016-5034
2613

AD-75157
A-150820
UAACAAUCUGUGAAGAUUU
5050-5068
2334
A-150821
AAAUCUUCACAGAUUGUUA
5050-5068
2614

AD-75158
A-150822
UUUUACUUUAUACAGUCUU
5098-5116
2335
A-150823
AAGACUGUAUAAAGUAAAA
5098-5116
2615

AD-75159
A-150824
UUUAUGAAUUUCUUAAUGU
5115-5133
2336
A-150825
ACAUUAAGAAAUUCAUAAA
5115-5133
2616

AD-75160
A-150826
UUAAUGUUCAAAAUGACUU
5127-5145
2337
A-150827
AAGUCAUUUUGAACAUUAA
5127-5145
2617

AD-75161
A-150828
UUCUUCUUUUUUUAUAUCA
5153-5171
2338
A-150829
UGAUAUAAAAAAAGAAGAA
5153-5171
2618

AD-75162
A-150830
AGAAUGAGGAAUAAUAAGU
5171-5189
2339
A-150831
ACUUAUUAUUCCUCAUUCU
5171-5189
2619

AD-75163
A-150832
UUAAACCCACAUAGACUCU
5189-5207
2340
A-150833
AGAGUCUAUGUGGGUUUAA
5189-5207
2620

AD-75164
A-150834
CUUUAAAACUAUAGGCUAA
5206-5224
2341
A-150835
UUAGCCUAUAGUUUUAAAG
5206-5224
2621

AD-75165
A-150836
AGAUAGAAAUGUAUGUUUA
5223-5241
2342
A-150837
UAAACAUACAUUUCUAUCU
5223-5241
2622

AD-75166
A-150838
UUUGACUUGUUGAAGCUAU
5238-5256
2343
A-150839
AUAGCUUCAACAAGUCAAA
5238-5256
2623

AD-75167
A-150840
UUUUUAAUCUUAAAAGAUU
5284-5302
2344
A-150841
AAUCUUUUAAGAUUAAAAA
5284-5302
2624

AD-75168
A-150842
UUGUGCUAAUUUAUUAGAA
5301-5319
2345
A-150843
UUCUAAUAAAUUAGCACAA
5301-5319
2625

AD-75169
A-150844
UUAUUAGAGCAGAACCUGU
5311-5329
2346
A-150845
ACAGGUUCUGCUCUAAUAA
5311-5329
2626

AD-75170
A-150846
GUUUGGCUCUCCUCAGAAA
5328-5346
2347
A-150847
UUUCUGAGGAGAGCCAAAC
5328-5346
2627

AD-75171
A-150848
CAAUAUUUUCAAAAGAUAA
5383-5401
2348
A-150849
UUAUCUUUUGAAAAUAUUG
5383-5401
2628

AD-75172
A-150850
UCAAAAGAUAAAUCUGAUU
5391-5409
2349
A-150851
AAUCAGAUUUAUCUUUUGA
5391-5409
2629

AD-75173
A-150852
UUAUGCAAUGGCAUCAUUU
5409-5427
2350
A-150853
AAAUGAUGCCAUUGCAUAA
5409-5427
2630

AD-75174
A-150854
UGCAAUGGCAUCAUUUAUU
5412-5430
2351
A-150855
AAUAAAUGAUGCCAUUGCA
5412-5430
2631

AD-75175
A-150856
UUUAAAACAGAAGAAUUGU
5430-5448
2352
A-150857
ACAAUUCUUCUGUUUUAAA
5430-5448
2632

AD-75176
A-150858
AACAACAAAAGGAAAAUGU
5505-5523
2353
A-150859
ACAUUUUCCUUUUGUUGUU
5505-5523
2633

AD-75177
A-150860
UUAAUCCUGUAGUACAUAU
5570-5588
2354
A-150861
AUAUGUACUACAGGAUUAA
5570-5588
2634

AD-75178
A-150862
UUUAAUAUUUUAUAAGACA
5603-5621
2355
A-150863
UGUCUUAUAAAAUAUUAAA
5603-5621
2635

AD-75179
A-150864
CCUUCCUGUUAGGUAUUAA
5620-5638
2356
A-150865
UUAAUACCUAACAGGAAGG
5620-5638
2636

AD-75180
A-150866
UUAGGUAUUAGAAAGUGAU
5628-5646
2357
A-150867
AUCACUUUCUAAUACCUAA
5628-5646
2637

AD-75181
A-150868
AUACAUAGAUAUCUUUUUU
5645-5663
2358
A-150869
AAAAAAGAUAUCUAUGUAU
5645-5663
2638

AD-75182
A-150870
UUUUUGUGUAAUUUCUAUU
5659-5677
2359
A-150871
AAUAGAAAUUACACAAAAA
5659-5677
2639

AD-75183
A-150872
UUAAAAAAGAGAGAAGACU
5677-5695
2360
A-150873
AGUCUUCUCUCUUUUUUAA
5677-5695
2640

AD-75184
A-150874
CUGUCAGAAGCUUUAAGUA
5694-5712
2361
A-150875
UACUUAAAGCUUCUGACAG
5694-5712
2641

AD-75185
A-150876
UAUGGUACAGGAUAAAGAU
5715-5733
2362
A-150877
AUCUUUAUCCUGUACCAUA
5715-5733
2642

AD-75186
A-150878
UUAAAUAACCAAUUCCUAU
5740-5758
2363
A-150879
AUAGGAAUUGGUUAUUUAA
5740-5758
2643

AD-75187
A-150880
UUGUUUUUUAAAGAAACCU
5773-5791
2364
A-150881
AGGUUUCUUUAAAAAACAA
5773-5791
2644

AD-75188
A-150882
CUCUCACAGAUAAGACAGA
5790-5808
2365
A-150883
UCUGUCUUAUCUGUGAGAG
5790-5808
2645

AD-75189
A-150884
CAGAAUUUUAUAGAGGGCU
5884-5902
2366
A-150885
AGCCCUCUAUAAAAUUCUG
5884-5902
2646

AD-75190
A-150886
UCUAGAAUUAAAGGAACCU
5917-5935
2367
A-150887
AGGUUCCUUUAAUUCUAGA
5917-5935
2647

AD-75191
A-150888
CUCACUGAAAACAUAUAUU
5934-5952
2368
A-150889
AAUAUAUGUUUUCAGUGAG
5934-5952
2648

AD-75192
A-150890
AAACAUAUAUUUCACGUGU
5942-5960
2369
A-150891
ACACGUGAAAUAUAUGUUU
5942-5960
2649

AD-75193
A-150892
GUUCCCUCUUUUUUUUUUU
5959-5977
2370
A-150893
AAAAAAAAAAAGAGGGAAC
5959-5977
2650

AD-75194
A-150894
UUAAGCGAUUCUCCUGCCU
6066-6084
2371
A-150895
AGGCAGGAGAAUCGCUUAA
6066-6084
2651

AD-75195
A-150896
CGGCUAAUUUUUUGGAUUU
6129-6147
2372
A-150897
AAAUCCAAAAAAUUAGCCG
6129-6147
2652

AD-75196
A-150898
UUUAAUAGAGACGGGGUUU
6147-6165
2373
A-150899
AAACCCCGUCUCUAUUAAA
6147-6165
2653

AD-75197
A-150900
UUUACCAUGUUGGCCAGGU
6164-6182
2374
A-150901
ACCUGGCCAACAUGGUAAA
6164-6182
2654

AD-75198
A-150902
UUGCUGGGAUUACAGGCAU
6231-6249
2375
A-150903
AUGCCUGUAAUCCCAGCAA
6231-6249
2655

AD-75199
A-150904
UUAAACAUGAUCCUUCUCU
6303-6321
2376
A-150905
AGAGAAGGAUCAUGUUUAA
6303-6321
2656

AD-75200
A-150906
GGGGUCUUUCAAGGGGAAA
6346-6364
2377
A-150907
UUUCCCCUUGAAAGACCCC
6346-6364
2657

AD-75201
A-150908
AAAAAUCCAAGCUUUUUUA
6364-6382
2378
A-150909
UAAAAAAGCUUGGAUUUUU
6364-6382
2658

AD-75202
A-150910
AAAGUAAAAAAAAAAAAAG
6382-6400
2379
A-150911
CUUUUUUUUUUUUUACUUU
6382-6400
2659

AD-75203
A-150912
AGAGAGGACACAAAACCAA
6399-6417
2380
A-150913
UUGGUUUUGUGUCCUCUCU
6399-6417
2660

AD-75204
A-150914
UUAAGAUGGAGACAGAGUU
6444-6462
2381
A-150915
AACUCUGUCUCCAUCUUAA
6444-6462
2661

AD-75205
A-150916
UUUCUCCUAAUAACCGGAA
6461-6479
2382
A-150917
UUCCGGUUAUUAGGAGAAA
6461-6479
2662

AD-75206
A-150918
GCUGAAUUACCUUUCACUU
6479-6497
2383
A-150919
AAGUGAAAGGUAAUUCAGC
6479-6497
2663

AD-75207
A-150920
UUCAAAAACAUGACCUUCA
6497-6515
2384
A-150921
UGAAGGUCAUGUUUUUGAA
6497-6515
2664

AD-75208
A-150922
CAAUCCUUAGAAUCUGCCU
6517-6535
2385
A-150923
AGGCAGAUUCUAAGGAUUG
6517-6535
2665

AD-75209
A-150924
UUUUAUAUUACUGAGGCCU
6538-6556
2386
A-150925
AGGCCUCAGUAAUAUAAAA
6538-6556
2666

AD-75210
A-150926
AAAAGUAAACAUUACUCAU
6557-6575
2387
A-150927
AUGAGUAAUGUUUACUUUU
6557-6575
2667

AD-75211
A-150928
UUUAUUUUGCCCAAAAUGA
6576-6594
2388
A-150929
UCAUUUUGGGCAAAAUAAA
6576-6594
2668

AD-75212
A-150930
CACUGAUGUAAAGUAGGAA
6594-6612
2389
A-150931
UUCCUACUUUACAUCAGUG
6594-6612
2669

AD-75213
A-150932
AAAAUAAAAACAGAGCUCU
6612-6630
2390
A-150933
AGAGCUCUGUUUUUAUUUU
6612-6630
2670

AD-75214
A-150934
CUAAAAUCCCUUUCAAGCA
6629-6647
2391
A-150935
UGCUUGAAAGGGAUUUUAG
6629-6647
2671

AD-75215
A-150936
UUGACCCCACUCACCAACU
6653-6671
2392
A-150937
AGUUGGUGAGUGGGGUCAA
6653-6671
2672

AD-75216
A-150938
UUAUCUUGUACCCGCUGCU
6722-6740
2393
A-150939
AGCAGCGGGUACAAGAUAA
6722-6740
2673

AD-75217
A-150940
CUGAAACCUCAAGCUGUCU
6762-6780
2394
A-150941
AGACAGCUUGAGGUUUCAG
6762-6780
2674

AD-75218
A-150942
GUAUCAUGAAAAUGUCUAU
6806-6824
2395
A-150943
AUAGACAUUUUCAUGAUAC
6806-6824
2675

AD-75219
A-150944
UUCAAAAUAUCAAAACCUU
6824-6842
2396
A-150945
AAGGUUUUGAUAUUUUGAA
6824-6842
2676

AD-75220
A-150946
UUUCAAAUAUCACGCAGCU
6841-6859
2397
A-150947
AGCUGCGUGAUAUUUGAAA
6841-6859
2677

AD-75221
A-150948
CUUAUAUUCAGUUUACAUA
6858-6876
2398
A-150949
UAUGUAAACUGAAUAUAAG
6858-6876
2678

AD-75222
A-150950
UUACAUAAAGGCCCCAAAU
6870-6888
2399
A-150951
AUUUGGGGCCUUUAUGUAA
6870-6888
2679

AD-75223
A-150952
AUACCAUGUCAGAUCUUUU
6887-6905
2400
A-150953
AAAAGAUCUGACAUGGUAU
6887-6905
2680

AD-75224
A-150954
AAAAGAGUUAAUGAACUAU
6910-6928
2401
A-150955
AUAGUUCAUUAACUCUUUU
6910-6928
2681

AD-75225
A-150956
AUGAGAAUUGGGAUUACAU
6927-6945
2402
A-150957
AUGUAAUCCCAAUUCUCAU
6927-6945
2682

AD-75226
A-150958
AUCAUGUAUUUUGCCUCAU
6944-6962
2403
A-150959
AUGAGGCAAAAUACAUGAU
6944-6962
2683

AD-75227
A-150960
UUAUCACACUUAUAGGCCA
6969-6987
2404
A-150961
UGGCCUAUAAGUGUGAUAA
6969-6987
2684

AD-75228
A-150962
CAAGUGUGAUAAAUAAACU
6986-7004
2405
A-150963
AGUUUAUUUAUCACACUUG
6986-7004
2685

AD-75229
A-150964
UUACAGACACUGAAUUAAU
7004-7022
2406
A-150965
AUUAAUUCAGUGUCUGUAA
7004-7022
2686

AD-75230
A-150966
UUUGAAACCAGAAAAUAAU
7035-7053
2407
A-150967
AUUAUUUUCUGGUUUCAAA
7035-7053
2687

AD-75231
A-150968
AUGACUGGCCAUUCGUUAA
7052-7070
2408
A-150969
UUAACGAAUGGCCAGUCAU
7052-7070
2688

AD-75232
A-150970
UUAGUUGAAAAGCAUAUUU
7078-7096
2409
A-150971
AAAUAUGCUUUUCAACUAA
7078-7096
2689

AD-75233
A-150972
UUUUUAUUAAAUUAAUUCU
7095-7113
2410
A-150973
AGAAUUAAUUUAAUAAAAA
7095-7113
2690

AD-75234
A-150974
CUGAUUGUAUUUGAAAUUA
7112-7130
2411
A-150975
UAAUUUCAAAUACAAUCAG
7112-7130
2691

AD-75235
A-150976
UUUGAAAUUAUUAUUCAAU
7121-7139
2412
A-150977
AUUGAAUAAUAAUUUCAAA
7121-7139
2692

AD-75236
A-150978
UUAUGGCAGAGGAAUAUCA
7144-7162
2413
A-150979
UGAUAUUCCUCUGCCAUAA
7144-7162
2693

AD-75237
A-150980
UCUAAAAAUGUAACUAAUU
7175-7193
2414
A-150981
AAUUAGUUACAUUUUUAGA
7175-7193
2694

AD-75238
A-150982
UUUACUGUUUAAUAAGCAU
7210-7228
2415
A-150983
AUGCUUAUUAAACAGUAAA
7210-7228
2695

AD-75239
A-150984
UGUCAUAAUAAAAUGGUAU
7252-7270
2416
A-150985
AUACCAUUUUAUUAUGACA
7252-7270
2696

AD-75240
A-150986
AUAUCUUUCUUUAGUAAUU
7269-7287
2417
A-150987
AAUUACUAAAGAAAGAUAU
7269-7287
2697

AD-75241
A-150988
UUAGUAAUUACAUUAAAAU
7279-7297
2418
A-150989
AUUUUAAUGUAAUUACUAA
7279-7297
2698

AD-75242
A-150990
AUUAGUCAUGUUUGAUUAA
7296-7314
2419
A-150991
UUAAUCAAACAUGACUAAU
7296-7314
2699

TABLE 19

IGF-1 in vitro 10 nM screen

Position in

Duplex Name
10 nM AVG
10 nM STD
NM_000618.3

AD-74963
2.44
2.3
6-24

AD-74964
7.2
5.49
24-42

AD-74965
3.3
3.72
41-59

AD-74966
4.25
1.91
54-72

AD-74967
15.72
4.3
72-90

AD-74968
3.11
0.38
127-145

AD-74969
17.28
0.98
185-203

AD-74970
7.75
1.22
203-221

AD-74971
4.93
4
220-238

AD-74972
21.83
3.83
247-265

AD-74973
14.71
5.65
277-295

AD-74974
38.48
1.73
430-448

AD-74975
8.99
1.93
447-465

AD-74976
22.76
2.57
462-480

AD-74977
34.47
2.7
543-561

AD-74978
10.33
10.14
654-672

AD-74979
4.03
0.6
672-690

AD-74980
22.84
18.43
750-768

AD-74981
10.09
7.19
774-792

AD-74982
5.27
0.53
792-810

AD-74983
7.33
3.35
818-836

AD-74984
25.15
1.05
835-853

AD-74985
9.51
1.12
852-870

AD-74986
13.08
1.24
894-912

AD-74987
15.81
0.07
912-930

AD-74988
74.25
9.8
930-948

AD-74989
53.17
16.23
947-965

AD-74990
34.92
1.88
1091-1109

AD-74991
35.6
2.75
1108-1126

AD-74992
54.21
4.47
1125-1143

AD-74993
51.57
3.65
1135-1153

AD-74994
20.06
0.5
1144-1162

AD-74995
49.73
0.85
1162-1180

AD-74996
54.67
2.95
1195-1213

AD-74997
24.76
9.85
1197-1215

AD-74998
116.31
7.45
1215-1233

AD-74999
37.97
1.63
1232-1250

AD-75000
17.29
1.27
1293-1311

AD-75001
44.75
3.5
1311-1329

AD-75002
33.61
1.4
1334-1352

AD-75003
50.72
3.07
1352-1370

AD-75004
49.47
3.37
1370-1388

AD-75005
33.73
0.68
1388-1406

AD-75006
62.64
3.34
1406-1424

AD-75007
36.36
0.24
1423-1441

AD-75008
37.81
2.85
1440-1458

AD-75009
19.35
1.31
1472-1490

AD-75010
71.78
3.37
1494-1512

AD-75011
25.82
2.55
1511-1529

AD-75012
55.66
5.16
1528-1546

AD-75013
83.97
4.37
1572-1590

AD-75014
29.26
11.24
1599-1617

AD-75015
40.19
0.14
1625-1643

AD-75016
38.98
4.61
1643-1661

AD-75017
32.96
4.54
1690-1708

AD-75018
19.3
1.74
1709-1727

AD-75019
71.36
5.08
1757-1775

AD-75020
20.46
1.94
1793-1811

AD-75021
33.72
1.42
1807-1825

AD-75022
84.23
3.34
1825-1843

AD-75023
24.26
1.03
1843-1861

AD-75024
40.83
2.06
1966-1984

AD-75025
48.65
1.28
2016-2034

AD-75026
70.35
5.83
2033-2051

AD-75027
94.74
14.09
2040-2058

AD-75028
29.93
7.99
2057-2075

AD-75029
31.95
1.57
2090-2108

AD-75030
64.72
8.6
2140-2158

AD-75031
44.8
3.93
2170-2188

AD-75032
32.33
2.6
2192-2210

AD-75033
26.13
0.64
2210-2228

AD-75034
43.7
7.02
2228-2246

AD-75035
70.89
7.82
2249-2267

AD-75036
106.4
4.66
2266-2284

AD-75037
47.27
4.85
2285-2303

AD-75038
48.86
4.15
2303-2321

AD-75039
40.63
4.83
2322-2340

AD-75040
107.1
7.22
2340-2358

AD-75041
35.81
5.21
2357-2375

AD-75042
52.59
6.79
2398-2416

AD-75043
55.9
9.3
2432-2450

AD-75044
46.66
11.14
2449-2467

AD-75045
82.13
13.7
2453-2471

AD-75046
73.55
5.15
2470-2488

AD-75047
71.67
15.96
2482-2500

AD-75048
38.18
30.65
2521-2539

AD-75049
92.97
13.14
2539-2557

AD-75050
86
15.13
2548-2566

AD-75051
59.23
8.69
2566-2584

AD-75052
104.39
19.17
2584-2602

AD-75053
58.66
10.73
2601-2619

AD-75054
89.3
16.07
2618-2636

AD-75055
64.45
10.53
2623-2641

AD-75056
95.7
23.97
2640-2658

AD-75057
38.59
5.32
2648-2666

AD-75058
35.56
5.16
2666-2684

AD-75059
62.95
9.3
2684-2702

AD-75060
50.41
11.52
2771-2789

AD-75061
34.92
9.92
2793-2811

AD-75062
52.01
10.49
2811-2829

AD-75063
49.98
9.93
2828-2846

AD-75064
113.1
26.07
2869-2887

AD-75065
73.65
7.55
2906-2924

AD-75066
43.56
5.6
2920-2938

AD-75067
56.32
9.44
2938-2956

AD-75068
57.61
8.64
2956-2974

AD-75069
34.69
12.05
2962-2980

AD-75070
89.29
14.15
2980-2998

AD-75071
64.02
16.69
3006-3024

AD-75072
125.6
49.27
3025-3043

AD-75073
111.64
19.51
3043-3061

AD-75074
75.61
13.18
3060-3078

AD-75075
111.51
16.74
3077-3095

AD-75076
69.43
9.4
3100-3118

AD-75077
44.04
5
3137-3155

AD-75078
57.27
10.2
3155-3173

AD-75079
28.28
5.64
3208-3226

AD-75080
59.53
7.5
3225-3243

AD-75081
61.41
5.87
3234-3252

AD-75082
54.31
7.14
3252-3270

AD-75083
34.99
5.51
3269-3287

AD-75084
46.86
9.9
3277-3295

AD-75085
56.82
7.96
3294-3312

AD-75086
58.83
11.06
3334-3352

AD-75087
55.26
2.93
3352-3370

AD-75088
34.12
16.28
3369-3387

AD-75089
63.74
11.09
3400-3418

AD-75090
36.81
7.73
3417-3435

AD-75091
31.56
5.56
3449-3467

AD-75092
61.39
11.47
3517-3535

AD-75093
83.2
19.37
3535-3553

AD-75094
80.16
13.64
3567-3585

AD-75095
38.33
8.26
3585-3603

AD-75096
103.57
22.84
3603-3621

AD-75097
69.98
7.03
3621-3639

AD-75098
51.57
14.6
3662-3680

AD-75099
31.97
13.18
3686-3704

AD-75100
94.94
9.26
3704-3722

AD-75101
36.43
11.54
3721-3739

AD-75102
70.66
7.75
3732-3750

AD-75103
57.38
5.65
3750-3768

AD-75104
77.58
15.39
3767-3785

AD-75105
70.13
8.3
3770-3788

AD-75106
50.24
6.25
3788-3806

AD-75107
34.8
6.73
3806-3824

AD-75108
58.82
3.73
3835-3853

AD-75109
65.08
10.73
3865-3883

AD-75110
31.63
14.97
3939-3957

AD-75111
5.82
0.91
3982-4000

AD-75112
11.18
0.76
4000-4018

AD-75113
38.66
8.55
4081-4099

AD-75114
14.58
5.96
4098-4116

AD-75115
12.98
2.49
4101-4119

AD-75116
35.3
6.1
4119-4137

AD-75117
57.1
9.56
4137-4155

AD-75118
23.23
4.43
4154-4172

AD-75119
54.12
7.09
4208-4226

AD-75120
15.15
2.22
4271-4289

AD-75121
19.41
4.81
4289-4307

AD-75122
33.51
8.79
4319-4337

AD-75123
10.61
1.98
4336-4354

AD-75124
22.01
6.31
4341-4359

AD-75125
10.88
0.88
4359-4377

AD-75126
84.91
10.8
4377-4395

AD-75127
71.33
14.6
4395-4413

AD-75128
78.44
5.21
4412-4430

AD-75129
76.77
25.96
4426-4444

AD-75130
15.56
6.25
4443-4461

AD-75131
13.16
4.18
4460-4478

AD-75132
86.74
17.91
4464-4482

AD-75133
36.15
4.26
4484-4502

AD-75134
51.96
9.57
4511-4529

AD-75135
19.14
4.52
4566-4584

AD-75136
43.64
6.37
4584-4602

AD-75137
3.8
0.43
4601-4619

AD-75138
40.31
6.39
4625-4643

AD-75139
20.1
4.15
4642-4660

AD-75140
53.32
1.96
4645-4663

AD-75141
51.87
3.72
4669-4687

AD-75142
32.17
4.32
4686-4704

AD-75143
17.5
6.52
4699-4717

AD-75144
94.26
24.89
4717-4735

AD-75145
8.73
1.94
4734-4752

AD-75146
39.8
9.22
4751-4769

AD-75147
44.81
9.36
4770-4788

AD-75148
21.99
3.67
4780-4798

AD-75149
56.19
8.99
4798-4816

AD-75150
22.7
2.67
4815-4833

AD-75151
33.03
2.82
4825-4843

AD-75152
28.29
11.66
4884-4902

AD-75153
34.14
6.63
4911-4929

AD-75154
43.03
6.81
4987-5005

AD-75155
10.11
2.68
5004-5022

AD-75156
49.77
4.33
5016-5034

AD-75157
10.55
1.54
5050-5068

AD-75158
98.46
15.78
5098-5116

AD-75159
59.88
7.14
5115-5133

AD-75160
67.78
10.53
5127-5145

AD-75161
71.58
12.59
5153-5171

AD-75162
9.5
2.65
5171-5189

AD-75163
99.59
10.77
5189-5207

AD-75164
24.32
6.23
5206-5224

AD-75165
48.65
10.6
5223-5241

AD-75166
20.31
3.83
5238-5256

AD-75167
96.76
8.31
5284-5302

AD-75168
50.33
6.42
5301-5319

AD-75169
35.41
3.61
5311-5329

AD-75170
9.16
1.79
5328-5346

AD-75171
74.55
6.92
5383-5401

AD-75172
61.38
10.44
5391-5409

AD-75173
18.61
2.81
5409-5427

AD-75174
12.46
3.74
5412-5430

AD-75175
64.04
8.99
5430-5448

AD-75176
47.5
11.54
5505-5523

AD-75177
26.63
1.26
5570-5588

AD-75178
93.5
13.15
5603-5621

AD-75179
13.82
1.72
5620-5638

AD-75180
40.37
3.65
5628-5646

AD-75181
68.1
14
5645-5663

AD-75182
91.45
7.76
5659-5677

AD-75183
52.7
9.56
5677-5695

AD-75184
10.64
1.91
5694-5712

AD-75185
22.43
3.75
5715-5733

AD-75186
39.19
4.23
5740-5758

AD-75187
80.34
23.05
5773-5791

AD-75188
18.72
4.87
5790-5808

AD-75189
71.77
15.33
5884-5902

AD-75190
69.3
7.75
5917-5935

AD-75191
13.99
6.28
5934-5952

AD-75192
83.17
9.31
5942-5960

AD-75193
79.66
18.37
5959-5977

AD-75194
64.7
6.93
6066-6084

AD-75195
79.21
5.63
6129-6147

AD-75196
105.5
9.43
6147-6165

AD-75197
128.21
12.85
6164-6182

AD-75198
46.08
7.51
6231-6249

AD-75199
117.2
7.51
6303-6321

AD-75200
50.47
12.5
6346-6364

AD-75201
59.91
14.09
6364-6382

AD-75202
107.53
22.57
6382-6400

AD-75203
25.97
4.96
6399-6417

AD-75204
71.96
3.58
6444-6462

AD-75205
39.19
13.83
6461-6479

AD-75206
11.68
4.11
6479-6497

AD-75207
40.79
10.66
6497-6515

AD-75208
34.43
5.15
6517-6535

AD-75209
107.98
25.75
6538-6556

AD-75210
52.4
6.95
6557-6575

AD-75211
90.01
19.17
6576-6594

AD-75212
22.91
6.75
6594-6612

AD-75213
87.12
9.51
6612-6630

AD-75214
19.83
5.95
6629-6647

AD-75215
50.88
6.82
6653-6671

AD-75216
43.88
4.7
6722-6740

AD-75217
18.19
2.83
6762-6780

AD-75218
9.91
1.3
6806-6824

AD-75219
54.72
5.78
6824-6842

AD-75220
60.73
10.07
6841-6859

AD-75221
23.15
1.19
6858-6876

AD-75222
36.29
4.92
6870-6888

AD-75223
21.88
2.24
6887-6905

AD-75224
32.13
3.75
6910-6928

AD-75225
12.65
3.49
6927-6945

AD-75226
48.19
14.5
6944-6962

AD-75227
58.51
8.21
6969-6987

AD-75228
53.16
6.09
6986-7004

AD-75229
10.94
2.92
7004-7022

AD-75230
31.83
5.18
7035-7053

AD-75231
15.75
2.98
7052-7070

AD-75232
18.71
3.47
7078-7096

AD-75233
106.56
8.46
7095-7113

AD-75234
18.49
4.54
7112-7130

AD-75235
113.68
22.34
7121-7139

AD-75236
20.92
10.52
7144-7162

AD-75237
29.1
4.1
7175-7193

AD-75238
39.59
10.16
7210-7228

AD-75239
23.08
3.81
7252-7270

AD-75240
98.59
16.79
7269-7287

AD-75241
109.49
8.89
7279-7297

AD-75242
92.6
7.78
7296-7314

TABLE 20

Modified Sense and Antisense Strand Sequences of IGF-1 dsRNAs

Sense

Antisense

Duplex
Oligo

SEQ ID
Oligo

SEQ ID

SEQ ID

Name
Name
Sense Sequence
NO
Name
Antisense Sequence
NO
mRNA target sequence
NO

AD-74963
A-150432
UAGAUAAAUGUGAGGAUUUdTdT
2700
A-150433
AAAUCCUCACAUUUAUCUAdTdT
2980
UAGAUAAAUGUGAGGAUUU
3260

AD-74964
A-150434
UUCUCUAAAUCCCUCUUCUdTdT
2701
A-150435
AGAAGAGGGAUUUAGAGAAdTdT
2981
UUCUCUAAAUCCCUCUUCU
3261

AD-74965
A-150436
CUGUUUGCUAAAUCUCACUdTdT
2702
A-150437
AGUGAGAUUUAGCAAACAGdTdT
2982
CUGUUUGCUAAAUCUCACU
3262

AD-74966
A-150438
CUCACUGUCACUGCUAAAUdTdT
2703
A-150439
AUUUAGCAGUGACAGUGAGdTdT
2983
CUCACUGUCACUGCUAAAU
3263

AD-74967
A-150440
UUCAGAGCAGAUAGAGCCUdTdT
2704
A-150441
AGGCUCUAUCUGCUCUGAAdTdT
2984
UUCAGAGCAGAUAGAGCCU
3264

AD-74968
A-150442
CAUUGCUCUCAACAUCUCAdTdT
2705
A-150443
UGAGAUGUUGAGAGCAAUGdTdT
2985
CAUUGCUCUCAACAUCUCC
3265

AD-74969
A-150444
ACCAAUUCAUUUUCAGACUdTdT
2706
A-150445
AGUCUGAAAAUGAAUUGGUdTdT
2986
ACCAAUUCAUUUUCAGACU
3266

AD-74970
A-150446
UUUGUACUUCAGAAGCAAUdTdT
2707
A-150447
AUUGCUUCUGAAGUACAAAdTdT
2987
UUUGUACUUCAGAAGCAAU
3267

AD-74971
A-150448
AUGGGAAAAAUCAGCAGUAdTdT
2708
A-150449
UACUGCUGAUUUUUCCCAUdTdT
2988
AUGGGAAAAAUCAGCAGUC
3268

AD-74972
A-150450
CAAUUAUUUAAGUGCUGCUdTdT
2709
A-150451
AGCAGCACUUAAAUAAUUGdTdT
2989
CAAUUAUUUAAGUGCUGCU
3269

AD-74973
A-150452
UUGAAGGUGAAGAUGCACAdTdT
2710
A-150453
UGUGCAUCUUCACCUUCAAdTdT
2990
UUGAAGGUGAAGAUGCACA
3270

AD-74974
A-150454
UUUUAUUUCAACAAGCCCAdTdT
2711
A-150455
UGGGCUUGUUGAAAUAAAAdTdT
2991
UUUUAUUUCAACAAGCCCA
3271

AD-74975
A-150456
CACAGGGUAUGGCUCCAGAdTdT
2712
A-150457
UCUGGAGCCAUACCCUGUGdTdT
2992
CACAGGGUAUGGCUCCAGC
3272

AD-74976
A-150458
CAGCAGUCGGAGGGCGCCUdTdT
2713
A-150459
AGGCGCCCUCCGACUGCUGdTdT
2993
CAGCAGUCGGAGGGCGCCU
3273

AD-74977
A-150460
UUGCGCACCCCUCAAGCCUdTdT
2714
A-150461
AGGCUUGAGGGGUGCGCAAdTdT
2994
UUGCGCACCCCUCAAGCCU
3274

AD-74978
A-150462
UGCAGGAAACAAGAACUAAdTdT
2715
A-150463
UUAGUUCUUGUUUCCUGCAdTdT
2995
UGCAGGAAACAAGAACUAC
3275

AD-74979
A-150464
CAGGAUGUAGGAAGACCCUdTdT
2716
A-150465
AGGGUCUUCCUACAUCCUGdTdT
2996
CAGGAUGUAGGAAGACCCU
3276

AD-74980
A-150466
UUAAACUUUGGAACACCUAdTdT
2717
A-150467
UAGGUGUUCCAAAGUUUAAdTdT
2997
UUAAACUUUGGAACACCUA
3277

AD-74981
A-150468
AAAUAAGUUUGAUAACAUUdTdT
2718
A-150469
AAUGUUAUCAAACUUAUUUdTdT
2998
AAAUAAGUUUGAUAACAUU
3278

AD-74982
A-150470
UUAAAAGAUGGGCGUUUCAdTdT
2719
A-150471
UGAAACGCCCAUCUUUUAAdTdT
2999
UUAAAAGAUGGGCGUUUCC
3279

AD-74983
A-150472
AAAUACACAAGUAAACAUUdTdT
2720
A-150473
AAUGUUUACUUGUGUAUUUdTdT
3000
AAAUACACAAGUAAACAUU
3280

AD-74984
A-150474
UUCCAACAUUGUCUUUAGAdTdT
2721
A-150475
UCUAAAGACAAUGUUGGAAdTdT
3001
UUCCAACAUUGUCUUUAGG
3281

AD-74985
A-150476
GGAGUGAUUUGCACCUUGAdTdT
2722
A-150477
UCAAGGUGCAAAUCACUCCdTdT
3002
GGAGUGAUUUGCACCUUGC
3282

AD-74986
A-150478
AUUGCUGUUGAUCUUUUAUdTdT
2723
A-150479
AUAAAAGAUCAACAGCAAUdTdT
3003
AUUGCUGUUGAUCUUUUAU
3283

AD-74987
A-150480
UCAAUAAUGUUCUAUAGAAdTdT
2724
A-150481
UUCUAUAGAACAUUAUUGAdTdT
3004
UCAAUAAUGUUCUAUAGAA
3284

AD-74988
A-150482
AAAGAAAAAAAAAAUAUAUdTdT
2725
A-150483
AUAUAUUUUUUUUUUCUUUdTdT
3005
AAAGAAAAAAAAAAUAUAU
3285

AD-74989
A-150484
AUAUAUAUAUAUAUCUUAAdTdT
2726
A-150485
UUAAGAUAUAUAUAUAUAUdTdT
3006
AUAUAUAUAUAUAUCUUAG
3286

AD-74990
A-150486
UUUCCUUAUUUGCACUUCUdTdT
2727
A-150487
AGAAGUGCAAAUAAGGAAAdTdT
3007
UUUCCUUAUUUGCACUUCU
3287

AD-74991
A-150488
CUUUCUACACAACUCGGGAdTdT
2728
A-150489
UCCCGAGUUGUGUAGAAAGdTdT
3008
CUUUCUACACAACUCGGGC
3288

AD-74992
A-150490
GCUGUUUGUUUUACAGUGUdTdT
2729
A-150491
ACACUGUAAAACAAACAGCdTdT
3009
GCUGUUUGUUUUACAGUGU
3289

AD-74993
A-150492
UUACAGUGUCUGAUAAUCUdTdT
2730
A-150493
AGAUUAUCAGACACUGUAAdTdT
3010
UUACAGUGUCUGAUAAUCU
3290

AD-74994
A-150494
CUGAUAAUCUUGUUAGUCUdTdT
2731
A-150495
AGACUAACAAGAUUAUCAGdTdT
3011
CUGAUAAUCUUGUUAGUCU
3291

AD-74995
A-150496
UAUACCCACCACCUCCCUUdTdT
2732
A-150497
AAGGGAGGUGGUGGGUAUAdTdT
3012
UAUACCCACCACCUCCCUU
3292

AD-74996
A-150498
UUGCCGAAUUUGGCCUCCUdTdT
2733
A-150499
AGGAGGCCAAAUUCGGCAAdTdT
3013
UUGCCGAAUUUGGCCUCCU
3293

AD-74997
A-150500
GCCGAAUUUGGCCUCCUCAdTdT
2734
A-150501
UGAGGAGGCCAAAUUCGGCdTdT
3014
GCCGAAUUUGGCCUCCUCA
3294

AD-74998
A-150502
AAAAGCAGCAGCAAGUCGUdTdT
2735
A-150503
ACGACUUGCUGCUGCUUUUdTdT
3015
AAAAGCAGCAGCAAGUCGU
3295

AD-74999
A-150504
GUCAAGAAGCACACCAAUUdTdT
2736
A-150505
AAUUGGUGUGCUUCUUGACdTdT
3016
GUCAAGAAGCACACCAAUU
3296

AD-75000
A-150506
AGUUGGAUGCAUUUUAUUUdTdT
2737
A-150507
AAAUAAAAUGCAUCCAACUdTdT
3017
AGUUGGAUGCAUUUUAUUU
3297

AD-75001
A-150508
UUAGACACAAAGCUUUAUUdTdT
2738
A-150509
AAUAAAGCUUUGUGUCUAAdTdT
3018
UUAGACACAAAGCUUUAUU
3298

AD-75002
A-150510
CACAUCAUGCUUACAAAAAdTdT
2739
A-150511
UUUUUGUAAGCAUGAUGUGdTdT
3019
CACAUCAUGCUUACAAAAA
3299

AD-75003
A-150512
AAGAAUAAUGCAAAUAGUUdTdT
2740
A-150513
AACUAUUUGCAUUAUUCUUdTdT
3020
AAGAAUAAUGCAAAUAGUU
3300

AD-75004
A-150514
UGCAACUUUGAGGCCAAUAdTdT
2741
A-150515
UAUUGGCCUCAAAGUUGCAdTdT
3021
UGCAACUUUGAGGCCAAUC
3301

AD-75005
A-150516
CAUUUUUAGGCAUAUGUUUdTdT
2742
A-150517
AAACAUAUGCCUAAAAAUGdTdT
3022
CAUUUUUAGGCAUAUGUUU
3302

AD-75006
A-150518
UUAAACAUAGAAAGUUUCUdTdT
2743
A-150519
AGAAACUUUCUAUGUUUAAdTdT
3023
UUAAACAUAGAAAGUUUCU
3303

AD-75007
A-150520
CUUCAACUCAAAAGAGUUAdTdT
2744
A-150521
UAACUCUUUUGAGUUGAAGdTdT
3024
CUUCAACUCAAAAGAGUUC
3304

AD-75008
A-150522
UCCUUCAAAUGAUGAGUUAdTdT
2745
A-150523
UAACUCAUCAUUUGAAGGAdTdT
3025
UCCUUCAAAUGAUGAGUUA
3305

AD-75009
A-150524
UUAGUAACUUUCCUCUUUUdTdT
2746
A-150525
AAAAGAGGAAAGUUACUAAdTdT
3026
UUAGUAACUUUCCUCUUUU
3306

AD-75010
A-150526
UUUUUCCAUAUAGAGCACUdTdT
2747
A-150527
AGUGCUCUAUAUGGAAAAAdTdT
3027
UUUUUCCAUAUAGAGCACU
3307

AD-75011
A-150528
CUAUGUAAAUUUAGCAUAUdTdT
2748
A-150529
AUAUGCUAAAUUUACAUAGdTdT
3028
CUAUGUAAAUUUAGCAUAU
3308

AD-75012
A-150530
AUCAAUUAUACAGGAUAUAdTdT
2749
A-150531
UAUAUCCUGUAUAAUUGAUdTdT
3029
AUCAAUUAUACAGGAUAUA
3309

AD-75013
A-150532
UUUAGUAUAAUGGUGCUAUdTdT
2750
A-150533
AUAGCACCAUUAUACUAAAdTdT
3030
UUUAGUAUAAUGGUGCUAU
3310

AD-75014
A-150534
UUGUUAUAUGAAAGAGUCUdTdT
2751
A-150535
AGACUCUUUCAUAUAACAAdTdT
3031
UUGUUAUAUGAAAGAGUCU
3311

AD-75015
A-150536
ACGGUAAUACGUGAAAGCAdTdT
2752
A-150537
UGCUUUCACGUAUUACCGUdTdT
3032
ACGGUAAUACGUGAAAGCA
3312

AD-75016
A-150538
AAAACAAUAGGGGAAGCCUdTdT
2753
A-150539
AGGCUUCCCCUAUUGUUUUdTdT
3033
AAAACAAUAGGGGAAGCCU
3313

AD-75017
A-150540
UACUGAAAACACCAUCCAUdTdT
2754
A-150541
AUGGAUGGUGUUUUCAGUAdTdT
3034
UACUGAAAACACCAUCCAU
3314

AD-75018
A-150542
UUGGGAAAGAAGGCAAAGUdTdT
2755
A-150543
ACUUUGCCUUCUUUCCCAAdTdT
3035
UUGGGAAAGAAGGCAAAGU
3315

AD-75019
A-150544
UCAGACACAAAAGUCCACUdTdT
2756
A-150545
AGUGGACUUUUGUGUCUGAdTdT
3036
UCAGACACAAAAGUCCACU
3316

AD-75020
A-150546
CGAGUCCAGAGAGGAAACUdTdT
2757
A-150547
AGUUUCCUCUCUGGACUCGdTdT
3037
CGAGUCCAGAGAGGAAACU
3317

AD-75021
A-150548
AAACUGUGGAAUGGAAAAAdTdT
2758
A-150549
UUUUUCCAUUCCACAGUUUdTdT
3038
AAACUGUGGAAUGGAAAAA
3318

AD-75022
A-150550
AGCAGAAGGCUAGGAAUUUdTdT
2759
A-150551
AAAUUCCUAGCCUUCUGCUdTdT
3039
AGCAGAAGGCUAGGAAUUU
3319

AD-75023
A-150552
UUAGCAGUCCUGGUUUCUUdTdT
2760
A-150553
AAGAAACCAGGACUGCUAAdTdT
3040
UUAGCAGUCCUGGUUUCUU
3320

AD-75024
A-150554
CAAAAUGGGGGCAAUAUGUdTdT
2761
A-150555
ACAUAUUGCCCCCAUUUUGdTdT
3041
CAAAAUGGGGGCAAUAUGU
3321

AD-75025
A-150556
UUUAAAAAGAUAAAGAUUAdTdT
2762
A-150557
UAAUCUUUAUCUUUUUAAAdTdT
3042
UUUAAAAAGAUAAAGAUUC
3322

AD-75026
A-150558
UCAGAUUUUUUUUACCCUAdTdT
2763
A-150559
UAGGGUAAAAAAAAUCUGAdTdT
3043
UCAGAUUUUUUUUACCCUG
3323

AD-75027
A-150560
UUUUUUACCCUGGGUUGCUdTdT
2764
A-150561
AGCAACCCAGGGUAAAAAAdTdT
3044
UUUUUUACCCUGGGUUGCU
3324

AD-75028
A-150562
CUGUAAGGGUGCAACAUCAdTdT
2765
A-150563
UGAUGUUGCACCCUUACAGdTdT
3045
CUGUAAGGGUGCAACAUCA
3325

AD-75029
A-150564
CUGAGAUGCAAGGAAUUCUdTdT
2766
A-150565
AGAAUUCCUUGCAUCUCAGdTdT
3046
CUGAGAUGCAAGGAAUUCU
3326

AD-75030
A-150566
UUGGUGAAUUGAAUGCUCAdTdT
2767
A-150567
UGAGCAUUCAAUUCACCAAdTdT
3047
UUGGUGAAUUGAAUGCUCC
3327

AD-75031
A-150568
UUCUUGUCAGUGAAGCUAUdTdT
2768
A-150569
AUAGCUUCACUGACAAGAAdTdT
3048
UUCUUGUCAGUGAAGCUAU
3328

AD-75032
A-150570
AAUAACUGGCCAACUAGUUdTdT
2769
A-150571
AACUAGUUGGCCAGUUAUUdTdT
3049
AAUAACUGGCCAACUAGUU
3329

AD-75033
A-150572
UGUUAAAAGCUAACAGCUAdTdT
2770
A-150573
UAGCUGUUAGCUUUUAACAdTdT
3050
UGUUAAAAGCUAACAGCUC
3330

AD-75034
A-150574
CAAUCUCUUAAAACACUUUdTdT
2771
A-150575
AAAGUGUUUUAAGAGAUUGdTdT
3051
CAAUCUCUUAAAACACUUU
3331

AD-75035
A-150576
AAAAUAUGUGGGAAGCAUUdTdT
2772
A-150577
AAUGCUUCCCACAUAUUUUdTdT
3052
AAAAUAUGUGGGAAGCAUU
3332

AD-75036
A-150578
UUUGAUUUUCAAUUUGAUUdTdT
2773
A-150579
AAUCAAAUUGAAAAUCAAAdTdT
3053
UUUGAUUUUCAAUUUGAUU
3333

AD-75037
A-150580
UUGAAUUCUGCAUUUGGUUdTdT
2774
A-150581
AACCAAAUGCAGAAUUCAAdTdT
3054
UUGAAUUCUGCAUUUGGUU
3334

AD-75038
A-150582
UUUAUGAAUACAAAGAUAAdTdT
2775
A-150583
UUAUCUUUGUAUUCAUAAAdTdT
3055
UUUAUGAAUACAAAGAUAA
3335

AD-75039
A-150584
GUGAAAAGAGAGAAAGGAAdTdT
2776
A-150585
UUCCUUUCUCUCUUUUCACdTdT
3056
GUGAAAAGAGAGAAAGGAA
3336

AD-75040
A-150586
AAAGAAAAAGGAGAAAAACdTdT
2777
A-150587
GUUUUUCUCCUUUUUCUUUdTdT
3057
AAAGAAAAAGGAGAAAAAC
3337

AD-75041
A-150588
ACAAAGAGAUUUCUACCAAdTdT
2778
A-150589
UUGGUAGAAAUCUCUUUGUdTdT
3058
ACAAAGAGAUUUCUACCAG
3338

AD-75042
A-150590
UUGUUAGCACUCACUGACUdTdT
2779
A-150591
AGUCAGUGAGUGCUAACAAdTdT
3059
UUGUUAGCACUCACUGACU
3339

AD-75043
A-150592
UACAUAUCUAGUAAAACCUdTdT
2780
A-150593
AGGUUUUACUAGAUAUGUAdTdT
3060
UACAUAUCUAGUAAAACCU
3340

AD-75044
A-150594
CUCGUUUAAUACUAUAAAUdTdT
2781
A-150595
AUUUAUAGUAUUAAACGAGdTdT
3061
CUCGUUUAAUACUAUAAAU
3341

AD-75045
A-150596
UUUAAUACUAUAAAUAAUAdTdT
2782
A-150597
UAUUAUUUAUAGUAUUAAAdTdT
3062
UUUAAUACUAUAAAUAAUA
3342

AD-75046
A-150598
UAUUCUAUUCAUUUUGAAAdTdT
2783
A-150599
UUUCAAAAUGAAUAGAAUAdTdT
3063
UAUUCUAUUCAUUUUGAAA
3343

AD-75047
A-150600
UUUGAAAAACACAAUGAUUdTdT
2784
A-150601
AAUCAUUGUGUUUUUCAAAdTdT
3064
UUUGAAAAACACAAUGAUU
3344

AD-75048
A-150602
AAGGAAAGUGAUCCAAAAUdTdT
2785
A-150603
AUUUUGGAUCACUUUCCUUdTdT
3065
AAGGAAAGUGAUCCAAAAU
3345

AD-75049
A-150604
UUUGAAAUAUUAAAAUAAUdTdT
2786
A-150605
AUUAUUUUAAUAUUUCAAAdTdT
3066
UUUGAAAUAUUAAAAUAAU
3346

AD-75050
A-150606
UUAAAAUAAUAUCUAAUAAdTdT
2787
A-150607
UUAUUAGAUAUUAUUUUAAdTdT
3067
UUAAAAUAAUAUCUAAUAA
3347

AD-75051
A-150608
AAAAGUCACAAAGUUAUCUdTdT
2788
A-150609
AGAUAACUUUGUGACUUUUdTdT
3068
AAAAGUCACAAAGUUAUCU
3348

AD-75052
A-150610
UUCUUUAACAAACUUUACUdTdT
2789
A-150611
AGUAAAGUUUGUUAAAGAAdTdT
3069
UUCUUUAACAAACUUUACU
3349

AD-75053
A-150612
CUCUUAUUCUUAGCUGUAUdTdT
2790
A-150613
AUACAGCUAAGAAUAAGAGdTdT
3070
CUCUUAUUCUUAGCUGUAU
3350

AD-75054
A-150614
AUAUACAUUUUUUUAAAAGdTdT
2791
A-150615
CUUUUAAAAAAAUGUAUAUdTdT
3071
AUAUACAUUUUUUUAAAAG
3351

AD-75055
A-150616
CAUUUUUUUAAAAGUUUGUdTdT
2792
A-150617
ACAAACUUUUAAAAAAAUGdTdT
3072
CAUUUUUUUAAAAGUUUGU
3352

AD-75056
A-150618
GUUAAAAUAUGCUUGACUAdTdT
2793
A-150619
UAGUCAAGCAUAUUUUAACdTdT
3073
GUUAAAAUAUGCUUGACUA
3353

AD-75057
A-150620
AUGCUUGACUAGAGUUUCAdTdT
2794
A-150621
UGAAACUCUAGUCAAGCAUdTdT
3074
AUGCUUGACUAGAGUUUCC
3354

AD-75058
A-150622
CAGUUGAAAGGCAAAAACUdTdT
2795
A-150623
AGUUUUUGCCUUUCAACUGdTdT
3075
CAGUUGAAAGGCAAAAACU
3355

AD-75059
A-150624
UUCCAUCACAACAAGAAAUdTdT
2796
A-150625
AUUUCUUGUUGUGAUGGAAdTdT
3076
UUCCAUCACAACAAGAAAU
3356

AD-75060
A-150626
UUGGUAUCAAGAAAGUCCAdTdT
2797
A-150627
UGGACUUUCUUGAUACCAAdTdT
3077
UUGGUAUCAAGAAAGUCCA
3357

AD-75061
A-150628
GUUAGUGUACUAGUCCAUAdTdT
2798
A-150629
UAUGGACUAGUACACUAACdTdT
3078
GUUAGUGUACUAGUCCAUC
3358

AD-75062
A-150630
CAUAGCCUAGAAAAUGAUAdTdT
2799
A-150631
UAUCAUUUUCUAGGCUAUGdTdT
3079
CAUAGCCUAGAAAAUGAUC
3359

AD-75063
A-150632
UCCCUAUCUGCAGAUCAAAdTdT
2800
A-150633
UUUGAUCUGCAGAUAGGGAdTdT
3080
UCCCUAUCUGCAGAUCAAG
3360

AD-75064
A-150634
UUAUCCAGCAUUCAGAUCUdTdT
2801
A-150635
AGAUCUGAAUGCUGGAUAAdTdT
3081
UUAUCCAGCAUUCAGAUCU
3361

AD-75065
A-150636
UUUUUGGUUAAAAGUACCAdTdT
2802
A-150637
UGGUACUUUUAACCAAAAAdTdT
3082
UUUUUGGUUAAAAGUACCC
3362

AD-75066
A-150638
UACCCAGGCUUGAUUAUUUdTdT
2803
A-150639
AAAUAAUCAAGCCUGGGUAdTdT
3083
UACCCAGGCUUGAUUAUUU
3363

AD-75067
A-150640
UCAUGCAAAUUCUAUAUUUdTdT
2804
A-150641
AAAUAUAGAAUUUGCAUGAdTdT
3084
UCAUGCAAAUUCUAUAUUU
3364

AD-75068
A-150642
UUACAUUCUUGGAAAGUCUdTdT
2805
A-150643
AGACUUUCCAAGAAUGUAAdTdT
3085
UUACAUUCUUGGAAAGUCU
3365

AD-75069
A-150644
UCUUGGAAAGUCUAUAUGAdTdT
2806
A-150645
UCAUAUAGACUUUCCAAGAdTdT
3086
UCUUGGAAAGUCUAUAUGA
3366

AD-75070
A-150646
AAAAACAAAAAUAACAUCUdTdT
2807
A-150647
AGAUGUUAUUUUUGUUUUUdTdT
3087
AAAAACAAAAAUAACAUCU
3367

AD-75071
A-150648
UUCUCCCACUGGGUCACCUdTdT
2808
A-150649
AGGUGACCCAGUGGGAGAAdTdT
3088
UUCUCCCACUGGGUCACCU
3368

AD-75072
A-150650
CAAGGAUCAGAGGCCAGGAdTdT
2809
A-150651
UCCUGGCCUCUGAUCCUUGdTdT
3089
CAAGGAUCAGAGGCCAGGA
3369

AD-75073
A-150652
AAAAAAAAAAAAAAGACUAdTdT
2810
A-150653
UAGUCUUUUUUUUUUUUUUdTdT
3090
AAAAAAAAAAAAAAGACUC
3370

AD-75074
A-150654
UCCCUGGAUCUCUGAAUAUdTdT
2811
A-150655
AUAUUCAGAGAUCCAGGGAdTdT
3091
UCCCUGGAUCUCUGAAUAU
3371

AD-75075
A-150656
AUAUGCAAAAAGAAGGCCAdTdT
2812
A-150657
UGGCCUUCUUUUUGCAUAUdTdT
3092
AUAUGCAAAAAGAAGGCCC
3372

AD-75076
A-150658
UAGUGGAGCCAGCAAUCCUdTdT
2813
A-150659
AGGAUUGCUGGCUCCACUAdTdT
3093
UAGUGGAGCCAGCAAUCCU
3373

AD-75077
A-150660
UUAACUCUCAGUCCAACAUdTdT
2814
A-150661
AUGUUGGACUGAGAGUUAAdTdT
3094
UUAACUCUCAGUCCAACAU
3374

AD-75078
A-150662
UUAUUUGAAUUGAGCACCUdTdT
2815
A-150663
AGGUGCUCAAUUCAAAUAAdTdT
3095
UUAUUUGAAUUGAGCACCU
3375

AD-75079
A-150664
CAGAUGUAAAAGAAACUAUdTdT
2816
A-150665
AUAGUUUCUUUUACAUCUGdTdT
3096
CAGAUGUAAAAGAAACUAU
3376

AD-75080
A-150666
AUACAUCAUUUUUGCCCUAdTdT
2817
A-150667
UAGGGCAAAAAUGAUGUAUdTdT
3097
AUACAUCAUUUUUGCCCUC
3377

AD-75081
A-150668
UUUUGCCCUCUGCCUGUUUdTdT
2818
A-150669
AAACAGGCAGAGGGCAAAAdTdT
3098
UUUUGCCCUCUGCCUGUUU
3378

AD-75082
A-150670
UUCCAGACAUACAGGUUCUdTdT
2819
A-150671
AGAACCUGUAUGUCUGGAAdTdT
3099
UUCCAGACAUACAGGUUCU
3379

AD-75083
A-150672
CUGUGGAAUAAGAUACUGAdTdT
2820
A-150673
UCAGUAUCUUAUUCCACAGdTdT
3100
CUGUGGAAUAAGAUACUGG
3380

AD-75084
A-150674
UAAGAUACUGGACUCCUCUdTdT
2821
A-150675
AGAGGAGUCCAGUAUCUUAdTdT
3101
UAAGAUACUGGACUCCUCU
3381

AD-75085
A-150676
CUUCCCAAGAUGGCACUUAdTdT
2822
A-150677
UAAGUGCCAUCUUGGGAAGdTdT
3102
CUUCCCAAGAUGGCACUUC
3382

AD-75086
A-150678
GUGUACCUUUUAAAAUUAUdTdT
2823
A-150679
AUAAUUUUAAAAGGUACACdTdT
3103
GUGUACCUUUUAAAAUUAU
3383

AD-75087
A-150680
UUCCCUCUCAACAAAACUUdTdT
2824
A-150681
AAGUUUUGUUGAGAGGGAAdTdT
3104
UUCCCUCUCAACAAAACUU
3384

AD-75088
A-150682
UUUAUAGGCAGUCUUCUGAdTdT
2825
A-150683
UCAGAAGACUGCCUAUAAAdTdT
3105
UUUAUAGGCAGUCUUCUGC
3385

AD-75089
A-150684
UUUUCUGUCAUAGUUAGAUdTdT
2826
A-150685
AUCUAACUAUGACAGAAAAdTdT
3106
UUUUCUGUCAUAGUUAGAU
3386

AD-75090
A-150686
AUGUGAUAAUUCUAAGAGUdTdT
2827
A-150687
ACUCUUAGAAUUAUCACAUdTdT
3107
AUGUGAUAAUUCUAAGAGU
3387

AD-75091
A-150688
UUCCUUCACUUAAUUCUAUdTdT
2828
A-150689
AUAGAAUUAAGUGAAGGAAdTdT
3108
UUCCUUCACUUAAUUCUAU
3388

AD-75092
A-150690
AUUAUCUUUCUUAACUUUUdTdT
2829
A-150691
AAAAGUUAAGAAAGAUAAUdTdT
3109
AUUAUCUUUCUUAACUUUU
3389

AD-75093
A-150692
UUCCAACACAUAAUCCUCUdTdT
2830
A-150693
AGAGGAUUAUGUGUUGGAAdTdT
3110
UUCCAACACAUAAUCCUCU
3390

AD-75094
A-150694
AAAUAAAUUGAAAAUAACUdTdT
2831
A-150695
AGUUAUUUUCAAUUUAUUUdTdT
3111
AAAUAAAUUGAAAAUAACU
3391

AD-75095
A-150696
UCAUUAUACCAAUUCACUAdTdT
2832
A-150697
UAGUGAAUUGGUAUAAUGAdTdT
3112
UCAUUAUACCAAUUCACUA
3392

AD-75096
A-150698
AUUUUAUUUUUUAAUGAAUdTdT
2833
A-150699
AUUCAUUAAAAAAUAAAAUdTdT
3113
AUUUUAUUUUUUAAUGAAU
3393

AD-75097
A-150700
UUAAAACUAGAAAACAAAUdTdT
2834
A-150701
AUUUGUUUUCUAGUUUUAAdTdT
3114
UUAAAACUAGAAAACAAAU
3394

AD-75098
A-150702
UUGAUUACUAUAUACUACAdTdT
2835
A-150703
UGUAGUAUAUAGUAAUCAAdTdT
3115
UUGAUUACUAUAUACUACA
3395

AD-75099
A-150704
AUGACUCAGAUUUCAUAGAdTdT
2836
A-150705
UCUAUGAAAUCUGAGUCAUdTdT
3116
AUGACUCAGAUUUCAUAGA
3396

AD-75100
A-150706
AAAGGAGCAACCAAAAUGUdTdT
2837
A-150707
ACAUUUUGGUUGCUCCUUUdTdT
3117
AAAGGAGCAACCAAAAUGU
3397

AD-75101
A-150708
GUCACAACCCAAAACUUUAdTdT
2838
A-150709
UAAAGUUUUGGGUUGUGACdTdT
3118
GUCACAACCCAAAACUUUA
3398

AD-75102
A-150710
AAACUUUACAAGCUUUGCUdTdT
2839
A-150711
AGCAAAGCUUGUAAAGUUUdTdT
3119
AAACUUUACAAGCUUUGCU
3399

AD-75103
A-150712
UUCAGAAUUAGAUUGCUUUdTdT
2840
A-150713
AAAGCAAUCUAAUUCUGAAdTdT
3120
UUCAGAAUUAGAUUGCUUU
3400

AD-75104
A-150714
UUAUAAUUCUUGAAUGAGAdTdT
2841
A-150715
UCUCAUUCAAGAAUUAUAAdTdT
3121
UUAUAAUUCUUGAAUGAGG
3401

AD-75105
A-150716
UAAUUCUUGAAUGAGGCAAdTdT
2842
A-150717
UUGCCUCAUUCAAGAAUUAdTdT
3122
UAAUUCUUGAAUGAGGCAA
3402

AD-75106
A-150718
AUUUCAAGAUAUUUGUAAAdTdT
2843
A-150719
UUUACAAAUAUCUUGAAAUdTdT
3123
AUUUCAAGAUAUUUGUAAA
3403

AD-75107
A-150720
AAGAACAGUAAACAUUGGUdTdT
2844
A-150721
ACCAAUGUUUACUGUUCUUdTdT
3124
AAGAACAGUAAACAUUGGU
3404

AD-75108
A-150722
UUUCAACUCAUAGGCUUAUdTdT
2845
A-150723
AUAAGCCUAUGAGUUGAAAdTdT
3125
UUUCAACUCAUAGGCUUAU
3405

AD-75109
A-150724
UUGACCAUACUGGAUACUUdTdT
2846
A-150725
AAGUAUCCAGUAUGGUCAAdTdT
3126
UUGACCAUACUGGAUACUU
3406

AD-75110
A-150726
UUUAAGAUGAGGCAGUUCAdTdT
2847
A-150727
UGAACUGCCUCAUCUUAAAdTdT
3127
UUUAAGAUGAGGCAGUUCC
3407

AD-75111
A-150728
CAUCAGAAUCCACUCUUCUdTdT
2848
A-150729
AGAAGAGUGGAUUCUGAUGdTdT
3128
CAUCAGAAUCCACUCUUCU
3408

AD-75112
A-150730
UAGGGAUAUGAAAAUCUCUdTdT
2849
A-150731
AGAGAUUUUCAUAUCCCUAdTdT
3129
UAGGGAUAUGAAAAUCUCU
3409

AD-75113
A-150732
UUCACCCUAAGGAUCCAAUdTdT
2850
A-150733
AUUGGAUCCUUAGGGUGAAdTdT
3130
UUCACCCUAAGGAUCCAAU
3410

AD-75114
A-150734
AUGGAAUACUGAAAAGAAAdTdT
2851
A-150735
UUUCUUUUCAGUAUUCCAUdTdT
3131
AUGGAAUACUGAAAAGAAA
3411

AD-75115
A-150736
GAAUACUGAAAAGAAAUCAdTdT
2852
A-150737
UGAUUUCUUUUCAGUAUUCdTdT
3132
GAAUACUGAAAAGAAAUCA
3412

AD-75116
A-150738
ACUUCCUUGAAAAUUUUAUdTdT
2853
A-150739
AUAAAAUUUUCAAGGAAGUdTdT
3133
ACUUCCUUGAAAAUUUUAU
3413

AD-75117
A-150740
UUAAAAAACAAACAAACAAdTdT
2854
A-150741
UUGUUUGUUUGUUUUUUAAdTdT
3134
UUAAAAAACAAACAAACAA
3414

AD-75118
A-150742
AAACAAAAAGCCUGUCCAAdTdT
2855
A-150743
UUGGACAGGCUUUUUGUUUdTdT
3135
AAACAAAAAGCCUGUCCAC
3415

AD-75119
A-150744
UUUGUGUAGAUGAAACCAUdTdT
2856
A-150745
AUGGUUUCAUCUACACAAAdTdT
3136
UUUGUGUAGAUGAAACCAU
3416

AD-75120
A-150746
UUGGGAGAAGGCUUAGAAUdTdT
2857
A-150747
AUUCUAAGCCUUCUCCCAAdTdT
3137
UUGGGAGAAGGCUUAGAAU
3417

AD-75121
A-150748
UAAAAGAUGUAGCACAUUUdTdT
2858
A-150749
AAAUGUGCUACAUCUUUUAdTdT
3138
UAAAAGAUGUAGCACAUUU
3418

AD-75122
A-150750
UUAUUGUUUGGCCAGCUAUdTdT
2859
A-150751
AUAGCUGGCCAAACAAUAAdTdT
3139
UUAUUGUUUGGCCAGCUAU
3419

AD-75123
A-150752
AUGCCAAUGUGGUGCUAUUdTdT
2860
A-150753
AAUAGCACCACAUUGGCAUdTdT
3140
AUGCCAAUGUGGUGCUAUU
3420

AD-75124
A-150754
AAUGUGGUGCUAUUGUUUAdTdT
2861
A-150755
UAAACAAUAGCACCACAUUdTdT
3141
AAUGUGGUGCUAUUGUUUC
3421

AD-75125
A-150756
CUUUAAGAAAGUACUUGAAdTdT
2862
A-150757
UUCAAGUACUUUCUUAAAGdTdT
3142
CUUUAAGAAAGUACUUGAC
3422

AD-75126
A-150758
CUAAAAAAAAAAGAAAAAAdTdT
2863
A-150759
UUUUUUCUUUUUUUUUUAGdTdT
3143
CUAAAAAAAAAAGAAAAAA
3423

AD-75127
A-150760
AAGAAAAAAAAGAAAGCAUdTdT
2864
A-150761
AUGCUUUCUUUUUUUUCUUdTdT
3144
AAGAAAAAAAAGAAAGCAU
3424

AD-75128
A-150762
AUAGACAUAUUUUUUUAAAdTdT
2865
A-150763
UUUAAAAAAAUAUGUCUAUdTdT
3145
AUAGACAUAUUUUUUUAAA
3425

AD-75129
A-150764
UUAAAGUAUAAAAACAACAdTdT
2866
A-150765
UGUUGUUUUUAUACUUUAAdTdT
3146
UUAAAGUAUAAAAACAACA
3426

AD-75130
A-150766
CAAUUCUAUAGAUAGAUGAdTdT
2867
A-150767
UCAUCUAUCUAUAGAAUUGdTdT
3147
CAAUUCUAUAGAUAGAUGG
3427

AD-75131
A-150768
GGCUUAAUAAAAUAGCAUUdTdT
2868
A-150769
AAUGCUAUUUUAUUAAGCCdTdT
3148
GGCUUAAUAAAAUAGCAUU
3428

AD-75132
A-150770
UAAUAAAAUAGCAUUAGGUdTdT
2869
A-150771
ACCUAAUGCUAUUUUAUUAdTdT
3149
UAAUAAAAUAGCAUUAGGU
3429

AD-75133
A-150772
UAUCUAGCCACCACCACCUdTdT
2870
A-150773
AGGUGGUGGUGGCUAGAUAdTdT
3150
UAUCUAGCCACCACCACCU
3430

AD-75134
A-150774
UUUAUCACUCACAAGUAGUdTdT
2871
A-150775
ACUACUUGUGAGUGAUAAAdTdT
3151
UUUAUCACUCACAAGUAGU
3431

AD-75135
A-150776
GGCAGGAGUUGGAAAUUUUdTdT
2872
A-150777
AAAAUUUCCAACUCCUGCCdTdT
3152
GGCAGGAGUUGGAAAUUUU
3432

AD-75136
A-150778
UUUAAAGUUAGAAGGCUCAdTdT
2873
A-150779
UGAGCCUUCUAACUUUAAAdTdT
3153
UUUAAAGUUAGAAGGCUCC
3433

AD-75137
A-150780
CCAUUGUUUUGUUGGCUCUdTdT
2874
A-150781
AGAGCCAACAAAACAAUGGdTdT
3154
CCAUUGUUUUGUUGGCUCU
3434

AD-75138
A-150782
UUAGCAAAAUUAGCAAUAUdTdT
2875
A-150783
AUAUUGCUAAUUUUGCUAAdTdT
3155
UUAGCAAAAUUAGCAAUAU
3435

AD-75139
A-150784
AUAUUAUCCAAUCUUCUGAdTdT
2876
A-150785
UCAGAAGAUUGGAUAAUAUdTdT
3156
AUAUUAUCCAAUCUUCUGA
3436

AD-75140
A-150786
UUAUCCAAUCUUCUGAACUdTdT
2877
A-150787
AGUUCAGAAGAUUGGAUAAdTdT
3157
UUAUCCAAUCUUCUGAACU
3437

AD-75141
A-150788
AAGAGCAUGGAGAAUAAACdTdT
2878
A-150789
GUUUAUUCUCCAUGCUCUUdTdT
3158
AAGAGCAUGGAGAAUAAAC
3438

AD-75142
A-150790
ACGCGGGAAAAAAGAUCUUdTdT
2879
A-150791
AAGAUCUUUUUUCCCGCGUdTdT
3159
ACGCGGGAAAAAAGAUCUU
3439

AD-75143
A-150792
GAUCUUAUAGGCAAAUAGAdTdT
2880
A-150793
UCUAUUUGCCUAUAAGAUCdTdT
3160
GAUCUUAUAGGCAAAUAGA
3440

AD-75144
A-150794
AAGAAUUUAAAAGAUAAGUdTdT
2881
A-150795
ACUUAUCUUUUAAAUUCUUdTdT
3161
AAGAAUUUAAAAGAUAAGU
3441

AD-75145
A-150796
GUAAGUUCCUUAUUGAUUUdTdT
2882
A-150797
AAAUCAAUAAGGAACUUACdTdT
3162
GUAAGUUCCUUAUUGAUUU
3442

AD-75146
A-150798
UUUUGUGCACUCUGCUCUAdTdT
2883
A-150799
UAGAGCAGAGUGCACAAAAdTdT
3163
UUUUGUGCACUCUGCUCUA
3443

AD-75147
A-150800
AAACAGAUAUUCAGCAAGUdTdT
2884
A-150801
ACUUGCUGAAUAUCUGUUUdTdT
3164
AAACAGAUAUUCAGCAAGU
3444

AD-75148
A-150802
UCAGCAAGUGGAGAAAAUAdTdT
2885
A-150803
UAUUUUCUCCACUUGCUGAdTdT
3165
UCAGCAAGUGGAGAAAAUA
3445

AD-75149
A-150804
AAGAACAAAGAGAAAAAAUdTdT
2886
A-150805
AUUUUUUCUCUUUGUUCUUdTdT
3166
AAGAACAAAGAGAAAAAAU
3446

AD-75150
A-150806
AUACAUAGAUUUACCUGCAdTdT
2887
A-150807
UGCAGGUAAAUCUAUGUAUdTdT
3167
AUACAUAGAUUUACCUGCA
3447

AD-75151
A-150808
UUACCUGCAAAAAAUAGCUdTdT
2888
A-150809
AGCUAUUUUUUGCAGGUAAdTdT
3168
UUACCUGCAAAAAAUAGCU
3448

AD-75152
A-150810
UUUAUAGAAGACAUUCUCAdTdT
2889
A-150811
UGAGAAUGUCUUCUAUAAAdTdT
3169
UUUAUAGAAGACAUUCUCC
3449

AD-75153
A-150812
AGACAUCUCAAAGAGCAGUdTdT
2890
A-150813
ACUGCUCUUUGAGAUGUCUdTdT
3170
AGACAUCUCAAAGAGCAGU
3450

AD-75154
A-150814
UAUGAGAUGGGGGUUAUCUdTdT
2891
A-150815
AGAUAACCCCCAUCUCAUAdTdT
3171
UAUGAGAUGGGGGUUAUCU
3451

AD-75155
A-150816
CUACUGAUAAAGAAAGAAUdTdT
2892
A-150817
AUUCUUUCUUUAUCAGUAGdTdT
3172
CUACUGAUAAAGAAAGAAU
3452

AD-75156
A-150818
AAAGAAUUUAUGAGAAAUUdTdT
2893
A-150819
AAUUUCUCAUAAAUUCUUUdTdT
3173
AAAGAAUUUAUGAGAAAUU
3453

AD-75157
A-150820
UAACAAUCUGUGAAGAUUUdTdT
2894
A-150821
AAAUCUUCACAGAUUGUUAdTdT
3174
UAACAAUCUGUGAAGAUUU
3454

AD-75158
A-150822
UUUUACUUUAUACAGUCUUdTdT
2895
A-150823
AAGACUGUAUAAAGUAAAAdTdT
3175
UUUUACUUUAUACAGUCUU
3455

AD-75159
A-150824
UUUAUGAAUUUCUUAAUGUdTdT
2896
A-150825
ACAUUAAGAAAUUCAUAAAdTdT
3176
UUUAUGAAUUUCUUAAUGU
3456

AD-75160
A-150826
UUAAUGUUCAAAAUGACUUdTdT
2897
A-150827
AAGUCAUUUUGAACAUUAAdTdT
3177
UUAAUGUUCAAAAUGACUU
3457

AD-75161
A-150828
UUCUUCUUUUUUUAUAUCAdTdT
2898
A-150829
UGAUAUAAAAAAAGAAGAAdTdT
3178
UUCUUCUUUUUUUAUAUCA
3458

AD-75162
A-150830
AGAAUGAGGAAUAAUAAGUdTdT
2899
A-150831
ACUUAUUAUUCCUCAUUCUdTdT
3179
AGAAUGAGGAAUAAUAAGU
3459

AD-75163
A-150832
UUAAACCCACAUAGACUCUdTdT
2900
A-150833
AGAGUCUAUGUGGGUUUAAdTdT
3180
UUAAACCCACAUAGACUCU
3460

AD-75164
A-150834
CUUUAAAACUAUAGGCUAAdTdT
2901
A-150835
UUAGCCUAUAGUUUUAAAGdTdT
3181
CUUUAAAACUAUAGGCUAG
3461

AD-75165
A-150836
AGAUAGAAAUGUAUGUUUAdTdT
2902
A-150837
UAAACAUACAUUUCUAUCUdTdT
3182
AGAUAGAAAUGUAUGUUUG
3462

AD-75166
A-150838
UUUGACUUGUUGAAGCUAUdTdT
2903
A-150839
AUAGCUUCAACAAGUCAAAdTdT
3183
UUUGACUUGUUGAAGCUAU
3463

AD-75167
A-150840
UUUUUAAUCUUAAAAGAUUdTdT
2904
A-150841
AAUCUUUUAAGAUUAAAAAdTdT
3184
UUUUUAAUCUUAAAAGAUU
3464

AD-75168
A-150842
UUGUGCUAAUUUAUUAGAAdTdT
2905
A-150843
UUCUAAUAAAUUAGCACAAdTdT
3185
UUGUGCUAAUUUAUUAGAG
3465

AD-75169
A-150844
UUAUUAGAGCAGAACCUGUdTdT
2906
A-150845
ACAGGUUCUGCUCUAAUAAdTdT
3186
UUAUUAGAGCAGAACCUGU
3466

AD-75170
A-150846
GUUUGGCUCUCCUCAGAAAdTdT
2907
A-150847
UUUCUGAGGAGAGCCAAACdTdT
3187
GUUUGGCUCUCCUCAGAAG
3467

AD-75171
A-150848
CAAUAUUUUCAAAAGAUAAdTdT
2908
A-150849
UUAUCUUUUGAAAAUAUUGdTdT
3188
CAAUAUUUUCAAAAGAUAA
3468

AD-75172
A-150850
UCAAAAGAUAAAUCUGAUUdTdT
2909
A-150851
AAUCAGAUUUAUCUUUUGAdTdT
3189
UCAAAAGAUAAAUCUGAUU
3469

AD-75173
A-150852
UUAUGCAAUGGCAUCAUUUdTdT
2910
A-150853
AAAUGAUGCCAUUGCAUAAdTdT
3190
UUAUGCAAUGGCAUCAUUU
3470

AD-75174
A-150854
UGCAAUGGCAUCAUUUAUUdTdT
2911
A-150855
AAUAAAUGAUGCCAUUGCAdTdT
3191
UGCAAUGGCAUCAUUUAUU
3471

AD-75175
A-150856
UUUAAAACAGAAGAAUUGUdTdT
2912
A-150857
ACAAUUCUUCUGUUUUAAAdTdT
3192
UUUAAAACAGAAGAAUUGU
3472

AD-75176
A-150858
AACAACAAAAGGAAAAUGUdTdT
2913
A-150859
ACAUUUUCCUUUUGUUGUUdTdT
3193
AACAACAAAAGGAAAAUGU
3473

AD-75177
A-150860
UUAAUCCUGUAGUACAUAUdTdT
2914
A-150861
AUAUGUACUACAGGAUUAAdTdT
3194
UUAAUCCUGUAGUACAUAU
3474

AD-75178
A-150862
UUUAAUAUUUUAUAAGACAdTdT
2915
A-150863
UGUCUUAUAAAAUAUUAAAdTdT
3195
UUUAAUAUUUUAUAAGACC
3475

AD-75179
A-150864
CCUUCCUGUUAGGUAUUAAdTdT
2916
A-150865
UUAAUACCUAACAGGAAGGdTdT
3196
CCUUCCUGUUAGGUAUUAG
3476

AD-75180
A-150866
UUAGGUAUUAGAAAGUGAUdTdT
2917
A-150867
AUCACUUUCUAAUACCUAAdTdT
3197
UUAGGUAUUAGAAAGUGAU
3477

AD-75181
A-150868
AUACAUAGAUAUCUUUUUUdTdT
2918
A-150869
AAAAAAGAUAUCUAUGUAUdTdT
3198
AUACAUAGAUAUCUUUUUU
3478

AD-75182
A-150870
UUUUUGUGUAAUUUCUAUUdTdT
2919
A-150871
AAUAGAAAUUACACAAAAAdTdT
3199
UUUUUGUGUAAUUUCUAUU
3479

AD-75183
A-150872
UUAAAAAAGAGAGAAGACUdTdT
2920
A-150873
AGUCUUCUCUCUUUUUUAAdTdT
3200
UUAAAAAAGAGAGAAGACU
3480

AD-75184
A-150874
CUGUCAGAAGCUUUAAGUAdTdT
2921
A-150875
UACUUAAAGCUUCUGACAGdTdT
3201
CUGUCAGAAGCUUUAAGUG
3481

AD-75185
A-150876
UAUGGUACAGGAUAAAGAUdTdT
2922
A-150877
AUCUUUAUCCUGUACCAUAdTdT
3202
UAUGGUACAGGAUAAAGAU
3482

AD-75186
A-150878
UUAAAUAACCAAUUCCUAUdTdT
2923
A-150879
AUAGGAAUUGGUUAUUUAAdTdT
3203
UUAAAUAACCAAUUCCUAU
3483

AD-75187
A-150880
UUGUUUUUUAAAGAAACCUdTdT
2924
A-150881
AGGUUUCUUUAAAAAACAAdTdT
3204
UUGUUUUUUAAAGAAACCU
3484

AD-75188
A-150882
CUCUCACAGAUAAGACAGAdTdT
2925
A-150883
UCUGUCUUAUCUGUGAGAGdTdT
3205
CUCUCACAGAUAAGACAGA
3485

AD-75189
A-150884
CAGAAUUUUAUAGAGGGCUdTdT
2926
A-150885
AGCCCUCUAUAAAAUUCUGdTdT
3206
CAGAAUUUUAUAGAGGGCU
3486

AD-75190
A-150886
UCUAGAAUUAAAGGAACCUdTdT
2927
A-150887
AGGUUCCUUUAAUUCUAGAdTdT
3207
UCUAGAAUUAAAGGAACCU
3487

AD-75191
A-150888
CUCACUGAAAACAUAUAUUdTdT
2928
A-150889
AAUAUAUGUUUUCAGUGAGdTdT
3208
CUCACUGAAAACAUAUAUU
3488

AD-75192
A-150890
AAACAUAUAUUUCACGUGUdTdT
2929
A-150891
ACACGUGAAAUAUAUGUUUdTdT
3209
AAACAUAUAUUUCACGUGU
3489

AD-75193
A-150892
GUUCCCUCUUUUUUUUUUUdTdT
2930
A-150893
AAAAAAAAAAAGAGGGAACdTdT
3210
GUUCCCUCUUUUUUUUUUU
3490

AD-75194
A-150894
UUAAGCGAUUCUCCUGCCUdTdT
2931
A-150895
AGGCAGGAGAAUCGCUUAAdTdT
3211
UUAAGCGAUUCUCCUGCCU
3491

AD-75195
A-150896
CGGCUAAUUUUUUGGAUUUdTdT
2932
A-150897
AAAUCCAAAAAAUUAGCCGdTdT
3212
CGGCUAAUUUUUUGGAUUU
3492

AD-75196
A-150898
UUUAAUAGAGACGGGGUUUdTdT
2933
A-150899
AAACCCCGUCUCUAUUAAAdTdT
3213
UUUAAUAGAGACGGGGUUU
3493

AD-75197
A-150900
UUUACCAUGUUGGCCAGGUdTdT
2934
A-150901
ACCUGGCCAACAUGGUAAAdTdT
3214
UUUACCAUGUUGGCCAGGU
3494

AD-75198
A-150902
UUGCUGGGAUUACAGGCAUdTdT
2935
A-150903
AUGCCUGUAAUCCCAGCAAdTdT
3215
UUGCUGGGAUUACAGGCAU
3495

AD-75199
A-150904
UUAAACAUGAUCCUUCUCUdTdT
2936
A-150905
AGAGAAGGAUCAUGUUUAAdTdT
3216
UUAAACAUGAUCCUUCUCU
3496

AD-75200
A-150906
GGGGUCUUUCAAGGGGAAAdTdT
2937
A-150907
UUUCCCCUUGAAAGACCCCdTdT
3217
GGGGUCUUUCAAGGGGAAA
3497

AD-75201
A-150908
AAAAAUCCAAGCUUUUUUAdTdT
2938
A-150909
UAAAAAAGCUUGGAUUUUUdTdT
3218
AAAAAUCCAAGCUUUUUUA
3498

AD-75202
A-150910
AAAGUAAAAAAAAAAAAAGdTdT
2939
A-150911
CUUUUUUUUUUUUUACUUUdTdT
3219
AAAGUAAAAAAAAAAAAAG
3499

AD-75203
A-150912
AGAGAGGACACAAAACCAAdTdT
2940
A-150913
UUGGUUUUGUGUCCUCUCUdTdT
3220
AGAGAGGACACAAAACCAA
3500

AD-75204
A-150914
UUAAGAUGGAGACAGAGUUdTdT
2941
A-150915
AACUCUGUCUCCAUCUUAAdTdT
3221
UUAAGAUGGAGACAGAGUU
3501

AD-75205
A-150916
UUUCUCCUAAUAACCGGAAdTdT
2942
A-150917
UUCCGGUUAUUAGGAGAAAdTdT
3222
UUUCUCCUAAUAACCGGAG
3502

AD-75206
A-150918
GCUGAAUUACCUUUCACUUdTdT
2943
A-150919
AAGUGAAAGGUAAUUCAGCdTdT
3223
GCUGAAUUACCUUUCACUU
3503

AD-75207
A-150920
UUCAAAAACAUGACCUUCAdTdT
2944
A-150921
UGAAGGUCAUGUUUUUGAAdTdT
3224
UUCAAAAACAUGACCUUCC
3504

AD-75208
A-150922
CAAUCCUUAGAAUCUGCCUdTdT
2945
A-150923
AGGCAGAUUCUAAGGAUUGdTdT
3225
CAAUCCUUAGAAUCUGCCU
3505

AD-75209
A-150924
UUUUAUAUUACUGAGGCCUdTdT
2946
A-150925
AGGCCUCAGUAAUAUAAAAdTdT
3226
UUUUAUAUUACUGAGGCCU
3506

AD-75210
A-150926
AAAAGUAAACAUUACUCAUdTdT
2947
A-150927
AUGAGUAAUGUUUACUUUUdTdT
3227
AAAAGUAAACAUUACUCAU
3507

AD-75211
A-150928
UUUAUUUUGCCCAAAAUGAdTdT
2948
A-150929
UCAUUUUGGGCAAAAUAAAdTdT
3228
UUUAUUUUGCCCAAAAUGC
3508

AD-75212
A-150930
CACUGAUGUAAAGUAGGAAdTdT
2949
A-150931
UUCCUACUUUACAUCAGUGdTdT
3229
CACUGAUGUAAAGUAGGAA
3509

AD-75213
A-150932
AAAAUAAAAACAGAGCUCUdTdT
2950
A-150933
AGAGCUCUGUUUUUAUUUUdTdT
3230
AAAAUAAAAACAGAGCUCU
3510

AD-75214
A-150934
CUAAAAUCCCUUUCAAGCAdTdT
2951
A-150935
UGCUUGAAAGGGAUUUUAGdTdT
3231
CUAAAAUCCCUUUCAAGCC
3511

AD-75215
A-150936
UUGACCCCACUCACCAACUdTdT
2952
A-150937
AGUUGGUGAGUGGGGUCAAdTdT
3232
UUGACCCCACUCACCAACU
3512

AD-75216
A-150938
UUAUCUUGUACCCGCUGCUdTdT
2953
A-150939
AGCAGCGGGUACAAGAUAAdTdT
3233
UUAUCUUGUACCCGCUGCU
3513

AD-75217
A-150940
CUGAAACCUCAAGCUGUCUdTdT
2954
A-150941
AGACAGCUUGAGGUUUCAGdTdT
3234
CUGAAACCUCAAGCUGUCU
3514

AD-75218
A-150942
GUAUCAUGAAAAUGUCUAUdTdT
2955
A-150943
AUAGACAUUUUCAUGAUACdTdT
3235
GUAUCAUGAAAAUGUCUAU
3515

AD-75219
A-150944
UUCAAAAUAUCAAAACCUUdTdT
2956
A-150945
AAGGUUUUGAUAUUUUGAAdTdT
3236
UUCAAAAUAUCAAAACCUU
3516

AD-75220
A-150946
UUUCAAAUAUCACGCAGCUdTdT
2957
A-150947
AGCUGCGUGAUAUUUGAAAdTdT
3237
UUUCAAAUAUCACGCAGCU
3517

AD-75221
A-150948
CUUAUAUUCAGUUUACAUAdTdT
2958
A-150949
UAUGUAAACUGAAUAUAAGdTdT
3238
CUUAUAUUCAGUUUACAUA
3518

AD-75222
A-150950
UUACAUAAAGGCCCCAAAUdTdT
2959
A-150951
AUUUGGGGCCUUUAUGUAAdTdT
3239
UUACAUAAAGGCCCCAAAU
3519

AD-75223
A-150952
AUACCAUGUCAGAUCUUUUdTdT
2960
A-150953
AAAAGAUCUGACAUGGUAUdTdT
3240
AUACCAUGUCAGAUCUUUU
3520

AD-75224
A-150954
AAAAGAGUUAAUGAACUAUdTdT
2961
A-150955
AUAGUUCAUUAACUCUUUUdTdT
3241
AAAAGAGUUAAUGAACUAU
3521

AD-75225
A-150956
AUGAGAAUUGGGAUUACAUdTdT
2962
A-150957
AUGUAAUCCCAAUUCUCAUdTdT
3242
AUGAGAAUUGGGAUUACAU
3522

AD-75226
A-150958
AUCAUGUAUUUUGCCUCAUdTdT
2963
A-150959
AUGAGGCAAAAUACAUGAUdTdT
3243
AUCAUGUAUUUUGCCUCAU
3523

AD-75227
A-150960
UUAUCACACUUAUAGGCCAdTdT
2964
A-150961
UGGCCUAUAAGUGUGAUAAdTdT
3244
UUAUCACACUUAUAGGCCA
3524

AD-75228
A-150962
CAAGUGUGAUAAAUAAACUdTdT
2965
A-150963
AGUUUAUUUAUCACACUUGdTdT
3245
CAAGUGUGAUAAAUAAACU
3525

AD-75229
A-150964
UUACAGACACUGAAUUAAUdTdT
2966
A-150965
AUUAAUUCAGUGUCUGUAAdTdT
3246
UUACAGACACUGAAUUAAU
3526

AD-75230
A-150966
UUUGAAACCAGAAAAUAAUdTdT
2967
A-150967
AUUAUUUUCUGGUUUCAAAdTdT
3247
UUUGAAACCAGAAAAUAAU
3527

AD-75231
A-150968
AUGACUGGCCAUUCGUUAAdTdT
2968
A-150969
UUAACGAAUGGCCAGUCAUdTdT
3248
AUGACUGGCCAUUCGUUAC
3528

AD-75232
A-150970
UUAGUUGAAAAGCAUAUUUdTdT
2969
A-150971
AAAUAUGCUUUUCAACUAAdTdT
3249
UUAGUUGAAAAGCAUAUUU
3529

AD-75233
A-150972
UUUUUAUUAAAUUAAUUCUdTdT
2970
A-150973
AGAAUUAAUUUAAUAAAAAdTdT
3250
UUUUUAUUAAAUUAAUUCU
3530

AD-75234
A-150974
CUGAUUGUAUUUGAAAUUAdTdT
2971
A-150975
UAAUUUCAAAUACAAUCAGdTdT
3251
CUGAUUGUAUUUGAAAUUA
3531

AD-75235
A-150976
UUUGAAAUUAUUAUUCAAUdTdT
2972
A-150977
AUUGAAUAAUAAUUUCAAAdTdT
3252
UUUGAAAUUAUUAUUCAAU
3532

AD-75236
A-150978
UUAUGGCAGAGGAAUAUCAdTdT
2973
A-150979
UGAUAUUCCUCUGCCAUAAdTdT
3253
UUAUGGCAGAGGAAUAUCA
3533

AD-75237
A-150980
UCUAAAAAUGUAACUAAUUdTdT
2974
A-150981
AAUUAGUUACAUUUUUAGAdTdT
3254
UCUAAAAAUGUAACUAAUU
3534

AD-75238
A-150982
UUUACUGUUUAAUAAGCAUdTdT
2975
A-150983
AUGCUUAUUAAACAGUAAAdTdT
3255
UUUACUGUUUAAUAAGCAU
3535

AD-75239
A-150984
UGUCAUAAUAAAAUGGUAUdTdT
2976
A-150985
AUACCAUUUUAUUAUGACAdTdT
3256
UGUCAUAAUAAAAUGGUAU
3536

AD-75240
A-150986
AUAUCUUUCUUUAGUAAUUdTdT
2977
A-150987
AAUUACUAAAGAAAGAUAUdTdT
3257
AUAUCUUUCUUUAGUAAUU
3537

AD-75241
A-150988
UUAGUAAUUACAUUAAAAUdTdT
2978
A-150989
AUUUUAAUGUAAUUACUAAdTdT
3258
UUAGUAAUUACAUUAAAAU
3538

AD-75242
A-150990
AUUAGUCAUGUUUGAUUAAdTdT
2979
A-150991
UUAAUCAAACAUGACUAAUdTdT
3259
AUUAGUCAUGUUUGAUUAA
3539

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

Number	Date	Country
62316726	Apr 2016	US
62269401	Dec 2015	US
62191008	Jul 2015	US

	Number	Date	Country
Parent	15743349	Jan 2018	US
Child	16601681		US

INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN, ACID LABILE SUBUNIT (IGFALS) AND INSULIN-LIKE GROWTH FACTOR 1 (IGF-1) iRNA COMPOSITIONS AND METHODS OF USE THEREOF

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

RELATED APPLICATION

Provisional Applications (3)

Divisions (1)