Compositions and Methods for Inhibiting Gene Expression of Hif2alpha

Abstract
RNA interference (RNAi) triggers and RNAi trigger conjugates for inhibiting the expression of Hif2α (EPAS1) gene are described. Pharmaceutical compositions comprising one or more Hif2α RNAi triggers optionally with one or more additional therapeutics are also described. Delivery of the described Hif2α RNAi triggers to tumor cells in vivo provides for inhibition of Hif2α gene expression and treatment of cancer.
Description
BACKGROUND

EPAS1 is a member of the HIF (hypoxia inducible factor) gene family. Also known as Hif2alpha or Hif2α, EPAS1 encodes half of a transcription factor involved in the induction of genes regulated by oxygen, and which is induced as oxygen levels fall (a condition known as hypoxia).


Certain variants of this gene provide protection for people living at high altitude. However, at low altitude, over-expression of wild-type (WT) EPAS1 is associated with increased hypertension and stroke, and with symptoms similar to mountain sickness. Mutations in this gene are associated with erythrocytosis familial type 4 and pulmonary hypertension. EPAS1 can cause excessive production of red blood cells, leading to inhibited reproductive abilities or even death.


EPAS1 has been shown to be required for expression of, or enhance the expression of, various genes involved in an assortment of diseases, including tumor progression. For example, EPAS1 may play a role in the progression of uveal melanomas, possibly by promoting the autocrine loop VEGF-pVEGFR2/KDR, and by enhancing the expression of LDHA, thus conferring a growth advantage.


EPAS1 has also been shown to be involved in, or upregulates expression of, other factors, including: cMyc (which favors cell proliferation, transformation, neoplasia and tumorigenesis, and which is highly expressed in most cancers); Interleukin 8 (a pro-inflammatory mediator, e.g., in gingivitis and psoriasis); SP-1 (a transcription factor involved in IL-8 regulation and a coactivator of cMyc); LDH5 (which is linked with tumor necrosis and increased tumor size); and LANA (Latency Associated Nuclear Antigen, which is associated with Kaposi's sarcoma-associated Herpesvirus). In addition, HIF (hypoxia induced factor) activity may play a role in angiogenesis required for cancer tumor growth. EPAS 1 may also be involved in several other diseases, including inflammation, chronic inflammation, neovascular diseases, rheumatoid arthritis, renal cancer, clear cell renal cell carcinoma (and metastases of this and other cancers), melanoma, uveal melanoma, chondrosarcoma, and multiple myeloma.


Mutations in EPAS1 gene have been correlated to early onset of neuroendocrine tumors such as paragangliomas, somatostatinomas and/or pheochromocytomas. The mutations are commonly somatic missense mutations located in the primary hydroxylation site of HIF-2a.


These mutations are believed to disrupt the protein hydroxylation/degradation mechanism and lead to protein stabilization and pseudohypoxic signaling. In addition, neuroendocrine tumors release erythropoietin (EPO) into circulating blood, and lead to polycythemia.


SUMMARY

Described herein are Hif2α (also termed EPAS, or Hif2alpha) gene-specific RNA interference (RNAi) trigger molecules (also termed RNAi agent, RNAi trigger, or trigger) able to selectively and efficiently decrease expression of Hif2α. Each RNAi trigger includes at least a sense strand and an antisense strand. The sense strand and the antisense strand can be partially, substantially, or fully complementary to each other. The length of the RNAi trigger sense and antisense strands described herein each can be 16 to 30 nucleotides in length. In some embodiments, the sense and antisense strands are independently 17 to 26 nucleotides in length. The sense and antisense strands can be either the same length or different lengths. The RNAi triggers described herein, upon delivery to a cell expressing the Hif2α gene, inhibit the expression of the Hif2α gene in vitro or in vivo. Examples of Hif2α RNAi trigger sense strands and antisense strands that can be used in a Hif2α RNAi trigger are provided in Tables 1-2 and 5.


A sense strand of an Hif2α RNAi trigger contains a nucleotide sequence having at least 90% identity over a core stretch of at least 16 consecutive nucleotides to a sequence in an Hif2α mRNA. In some embodiments, the nucleotide sequence having at least 90% identity to a sequence in the Hif2α mRNA is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. An antisense strand of an Hif2α RNAi trigger contains a nucleotide sequence having at least 90% complementary over a core stretch of at least 16 consecutive nucleotides to a sequence in the Hif2α mRNA and the corresponding sense strand. In some embodiments, the nucleotide sequence having at least 90% complementarity to a sequence in the Hif2α mRNA or the corresponding sense strand is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.


In some embodiments, one or more Hif2α RNAi triggers are delivered to target cells or tissues using any oligonucleotide delivery technology known in the art. Nucleic acid delivery methods include, but are not limited to, by encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, proteinaceous vectors or Dynamic Polyconjugates™ (DPCs). In some embodiments, an Hif2α RNAi trigger is conjugated to a targeting group, such as a integrin-binding compound. In some embodiments, an Hif2 RNAi trigger is conjugated to a delivery polymer or vehicle. The delivery polymer can be a reversibly modified membrane active polyamine. The delivery polymer can also be an integrin-targeted reversibly modified membrane active polyamine.


An integrin-targeted reversibly modified membrane active polyamine comprises a membrane active polyamine conjugated to one or more integrin-binding compounds via reversible physiologically labile covalent linkages. In some embodiments, the integrin targeted reversibly modified membrane active polyamine further comprises the membrane active polyamine conjugated to one or more steric stabilizers via reversible physiologically labile covalent linkages. Integrin-binding compounds can be, but are not limited to, RGD peptides and RGD mimics. Reversible physiologically labile covalent linkages include, but are not limited to, dipeptide amidobenzyl carbamate linkages, tetrapeptide linkages, and disubstituted maleamate linkages.


The Hif2α RNAi triggers are optionally combined with one or more additional (i.e., second, third, etc.) therapeutics. A second therapeutic can be another Hif2α RNAi trigger (e.g., a Hif2α RNAi trigger which targets a different sequence within the Hif2α target). An additional therapeutic can also be a small molecule drug, antibody, antibody fragment, and/or vaccine. The Hif2α RNAi triggers, with or without the one or more additional therapeutics, can be combined with one or more excipients to form pharmaceutical compositions.


The present disclosure also encompasses methods of treating a human subject having a pathological state mediated at least in part by Hif2α expression, the methods comprising the step(s) of administering to the subject a therapeutically effective amount of an Hif2α RNAi trigger or Hif2α RNAi trigger-containing composition. The method of treating a subject with an Hif2α RNAi trigger or Hif2α RNAi trigger-containing composition can optionally be combined with one or more steps of administering one or more additional (i.e., second) therapeutics or treatments. The Hif2α RNAi trigger and additional therapeutics can be administered in a single composition or they made be administered separately. Non-limited examples of additional therapeutics include, but are not limited to, VEGFR inhibitors (such as SUTENT®, NEXAVAR®, VOTRIENT®, AVASTIN®, INLYTA®, CABOZANTINIB®), Cytokines (such as IL-2, IFN-α), mTor inhibitors (such as EVEROLIMUS®, TEMSIROLIMUS®), anti-PD1 drugs (such as OPDIVO® and KEYTRUDA®), anti-CTLA4 (such as YERVOY®), drugs targeting signal transduction pathway components in cancer cells (such as VEGF, PI-3-kinase, MEK, JAK, Akt, MYC, Met, Src-family kinases, Abl, Axl, Mer), anti-PD-L1, anti-PD-L2, anti-TIM3, anti-LAG3, anti-CD28, anti-OX40, anti-OX-40L, anti-CD39. anti-CD40, anti-CD80, anti-CD86, anti-CD137, anti-41BBL, anti-TIGIT, anti-GITR, anti-GIRTL, anti-CD155, anti-Fas, anti-FasL, anti-TRAIL/TRAIL-L, IDO-1 inhibitor, and TDO-2 inhibitor.


The pharmaceutical compositions 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, transdermal, oral or parenteral. Parenteral administration includes, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal (e.g., via an implanted device), intracranial, intraparenchymal, intrathecal, and intraventricular, administration.


The described Hif2α RNAi triggers and/or compositions can be used in methods for therapeutic treatment of diseases, including but not limited to: cancer, renal cancer, clear cell renal cell carcinoma, non-small cell lung cancer, astrocytoma (brain cancer), bladder cancer, breast cancer, chondrosarcoma, colorectal carcinoma, gastric carcinoma, glioblastoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, neuroblastoma, melanoma, multiple myeloma, ovarian cancer, rectal cancer, metastases, gingivitis, psoriasis, Kaposi's sarcoma-associated herpesvirus, preemclampsia, inflammation, chronic inflammation, neovascular diseases, and rheumatoid arthritis. Such methods comprise administration of an Hif2α RNAi trigger as described herein to a subject, e.g., a human or animal subject.


Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.


Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.





BRIEF DESCRIPTION OF THE FIGURES


FIG. 1. Chemical structures representing PEGn-FCitFP-TFP modifying agents.



FIG. 2. Chemical structures representing RGD-PEGn-FCitFP-TFP modifying agents.



FIG. 3. Chemical structures representing RGD-PEGn-ACit-PABC-PNP modifying agents.



FIG. 4. Chemical structures representing aldehyde-PEGn-FCit-PABC-PNP modifying agents.



FIG. 5. Chemical structures representing aldehyde-PEGn-ACit-PABC-PNP modifying agents.



FIG. 6. Chemical structures representing SPDP-PEGn-FCit-PABC-PNP modifying agents.



FIG. 7. Chemical structures representing PEGn-ACit-PABC-PNP and PEGn-FCit-PABC-PNP modifying agents.



FIG. 8. Graph illustrating serum SEAP levels during treatment in mice. Fold-changed in serum SEAP levels relative to day (−1) pre-dose levels. For G1 and G2, n=4. For G3, n=3.



FIG. 9. Tumor gross morphology after 3 weekly treatments in mice. G1, vehicle; G2 400 μg Hif2α-ITG-DPC; G3 280 μg Hif2α-ITG-DPC. Both kidneys from each animal are shown. Tumor was implanted into the kidney shown on the right. Tumor from treatment groups were all significantly smaller and showed some discoloration when compared to controls.



FIG. 10. H&E staining of tumor formalin fixed paraffin sections. Thin arrows indicate apoptotic cells. Thick arrows indicate macrophage infiltration. Panel A, G1 vehicle treated. Typical tubular type RCC morphology with rare apoptotic cells (thin arrows). Panel B, G2 400 μg Hif2α-ITG-DPC treated. Massive necrotic center with numerous apoptotic cells in surrounding areas and overall loss of tumor structure. Panel C, G3 280 μg Hif2α-ITG-DPC treated. Destruction of typical tubular tumor structures with macrophage infiltration and numerous apoptotic cells.





DETAILED DESCRIPTION

Described herein are RNAi triggers for inhibiting expression of the Hif2α gene (referred to herein as Hif2α RNAi triggers). Each Hif2α RNAi trigger comprises a sense strand and an antisense strand. The sense strand and the antisense strand are partially, substantially, or fully complementary to each other. In some embodiments, the length of the herein described RNAi trigger sense and antisense strands are independently 16 to 30 nucleotides in length. In some embodiments, the length of the herein described RNAi trigger sense and antisense strands are independently 17 to 26 nucleotides in length. In some embodiments, the herein described RNAi trigger sense and antisense strands are independently 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. The sense and antisense strands can be either the same length or they can be different lengths. In some embodiments, the sense strand is about 19 nucleotides in length while the antisense strand is about 21 nucleotides in length. In some embodiments, the sense strand is about 21 nucleotides in length while the antisense strand is about 23 nucleotides in length. In other embodiments, the sense and antisense strands are independently 17-21 nucleotides in length. In some embodiments, both the sense and antisense strands are each 21-26 nucleotides in length. Examples of nucleotide sequences used in forming Hif2α RNAi trigger molecules are provided in Tables 1-2 and 5.


RNAi triggers include, but are not limited to: short interfering RNAs (siRNAs), double-strand RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), and dicer substrates (U.S. Pat. No. 8,084,599 8,349,809 and 8,513,207). The RNAi triggers described herein, upon delivery to a cell expressing the Hif2α gene, inhibit or knockdown expression of Hif2α gene in vitro or in vivo through the biological process of RNA interference (RNAi).


An Hif2α RNAi trigger comprises a sense strand and an antisense strand each containing a core sequence of 16-23 nucleobases in length. An antisense strand core sequence is 100% (perfectly) complementary or at least 90% (substantially) complementary to a nucleotide sequence (sometimes referred to, e.g. as a target sequence) present in the Hif2α mRNA. A sense strand core sequence is 100% (perfectly) complementary or at least 90% (substantially) complementary to a sequence in the antisense strand and thus the sense strand core sequence is perfectly identical or at least 90% identical to a nucleotide sequence (target sequence) present in the Hif2α mRNA. A sense strand core sequence can be the same length as a corresponding antisense core sequence or it can be a different length. In some embodiments, the antisense strand core sequence is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. In some embodiments, the sense strand core sequence is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.


The Hif2α RNAi trigger sense and antisense strands typically anneal to form a duplex. Within the complementary duplex region, the sense strand core sequence is at least 90% complementary or 100% complementary to the antisense core sequence. In some embodiments, the sense strand core sequence contains a sequence of at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides that is at least 90% or 100% complementary to a corresponding 16, 17, 18, 19, 20, or 21 nucleotide sequence of the antisense strand core sequence (i.e., the sense strand and antisense core sequences of an Hif2α RNAi trigger have a region of at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides that is at least 90% base paired or 100% base paired.)


As used herein, the term “sequence” or “nucleotide sequence” refers to a succession or order of nucleobases, nucleotides, and/or nucleosides, described with a succession of letters using the standard nucleotide nomenclature and the key for modified nucleotides described herein.


As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence (e.g., RNAi trigger sense strand or Hif2α mRNA) in relation to a second nucleotide sequence (e.g., RNAi trigger antisense strand), refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize (form base pair hydrogen bonds) and form a duplex or double helical structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimics as long as the above requirements with respect to their ability to hybridize are fulfilled. “Perfectly complementary” or “fully complementary” means that all (100%) of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence. As used herein, “partial complementary” means that in a hybridized pair of nucleobase sequences, at least 70% of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. As used herein, “substantial complementary” means that in a hybridized pair of nucleobase sequences, at least 85% of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. The terms “complementary”, “fully complementary” and “substantially complementary” as used herein may be used with respect to the base matching between the sense strand and the antisense strand of an RNAi trigger, or between the antisense strand of an RNAi trigger and a sequence of an Hif2α mRNA. Sequence identity or complementarity is independent of modification. For the purposes of determining identity or complementarity, for example, a and Af are complementary to U (or T) and identical to A.


The sense strand and/or the antisense strand may optionally and independently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides (extension) at the 3′ end, the 5′ end, or both the 3′ and 5′ ends of the core sequences. The antisense strand additional nucleotides, if present, may or may not be complementary to the corresponding sequence in the Hif2α mRNA. The sense strand additional nucleotides, if present, may or may not be identical to the corresponding sequence in the Hif2α mRNA. The antisense strand additional nucleotides, if present, may or may not be complementary to the corresponding sense strand's additional nucleotides, if present.


As used herein, an extension comprises 1, 2, 3, 4, 5, or 6 nucleotides at the 5′ and/or 3′ end of the sense strand core sequence and/or antisense strand core sequence. The extension nucleotides on a sense strand may or may not be complementary to nucleotides, either core sequence nucleotides or extension nucleotides, in the corresponding antisense strand. Conversely, the extension nucleotides on an antisense strand may or may not be complementary to nucleotides, either core sequence nucleotides or extension nucleotides, in the corresponding sense strand. In some embodiments, both the sense strand and the antisense strand of an RNAi trigger contain 3′ and 5′ extensions. In some embodiments, one or more of the 3′ extension nucleotides of one strand base pairs with one or more 5′ extension nucleotides of the other strand. In other embodiments, one or more of 3′ extension nucleotides of one strand do not base pair with one or more 5′ extension nucleotides of the other strand. In some embodiments, an Hif2α RNAi trigger has an antisense strand having a 3′ extension and a sense strand having a 5′ extension.


In some embodiments an Hif2α RNAi trigger molecule comprises an antisense strand having a 3′ extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In other embodiments, an Hif2α RNAi trigger molecule comprises an antisense strand having a 3′ extension of 1, 2, or 3 nucleotides in length. In some embodiments, one or more of the antisense strand extension nucleotides comprise uracil or thymidine nucleotides or nucleotides which are complementary to the corresponding Hif2α mRNA sequence. In some embodiments, the antisense strand extension can be, but is not limited to: uAu, uGu, udTsdT, usdTsdT, UfAu, Aua, Afsusa, UAU, uAfu, uau, udAu, uscu, usgu, uscsu, cAu, aUa, aua, u(invdA)u, cag, agu, gcg, caa, usasu, uAMTM, or usTMsAM (each listed 5′ to 3′, notation is the same as for Table 2).


In some embodiments, an Hif2α RNAi trigger molecule comprises an antisense strand having a 5′ extension of 1, 2, 3, 4, or 5 nucleotides in length. In other embodiments, an Hif2α RNAi trigger molecule comprises an antisense strand having a 5′ extension of 1 or 2 nucleotides in length. In some embodiments, one or more of the antisense strand extension nucleotides comprises uracil or thymidine nucleotides or nucleotides which are complementary to the corresponding Hif2α mRNA sequence. In some embodiments, the antisense strand extension includes or consists of dA, dT, pdT, vpdT, or u, wherein dA and dT represent deoxyadenosine and deoxythimidine nucleotides respectively. pdT represents a deoxythimidine nucleotide having a 5′ phosphate, vpdT represents a vinylphosphonate deoxythimidine nucleotide, and u represents a 2′-OMe modified uracil nucleotide. An antisense strand may have any of the 3′ extensions described above in combination with any of the 5′ antisense strand extensions described, if present.


In some embodiments, an Hif2α RNAi trigger molecule comprises a sense strand having a 3′ extension of 1, 2, 3, 4, or 5 nucleotides in length. In some embodiments, one or more of the sense strand extension nucleotides comprises adenosine, uracil, or thymidine nucleotides, AT dinucleotide, or nucleotides which correspond to nucleotides in the Hif2α mRNA sequence. In some embodiments, the 3′ sense strand extension includes or consists of Af, invdA, invdT, A(invdT), Af(invdT), U(invdT), Uf(invdT), AfAbuAu, dTdT, or dTsdT, wherein Af and Uf represent 2′-fluoro adenosine and uracil nucleotides respectively, invdA and invdT represent 3′-3′ linked (inverted) deoxyadenosine and deoxythimidine nucleotides respectively, Ab represents an abasic ribose, u represents a 2′-OMe modified uracil nucleotide, dT represents a deoxythimidine nucleotide, sdT represents a deoxythimidine nucleotide having a 5′ phosphorothioate, and U and A represent uracil and adenosine ribonucleotides.


In some embodiments, an Hif2α RNAi trigger molecule comprises a sense strand having a 5′ extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In some embodiments, one or more of the sense strand extension nucleotides comprise uracil or adenosine nucleotides or nucleotides which correspond to nucleotides in the Hif2α mRNA sequence. In some embodiments, the sense strand 5′ extension can be, but is not limited to: uAuAus, uAuAu, UAUUAGfs, UfaUfaA, uauaA, AUAUU, AfuAfuU, auauU, uaUfau, uAuA(UUNA), uauau, udAudAu, uuAga, uuAuu, uuGAu, uuaga, uAuga, aUaGas, uauaus, uAuaas, udAuau, adTaga, auaga, u(invdA)uau, gacau, ugaau, gcgau, uauga, uugga, or auaga (each listed 5′ to 3′. notation is the same as for Table 2). A sense strand may have a 3′ extension and/or a 5′ extension.


Unmodified Hif2α RNAi trigger sense strand and antisense strand sequences are provided in Tables 1 and Table 5. In forming Hif2α RNAi triggers, each of the nucleotides in each of the sequences listed in Tables 1 and 5 may be a modified nucleotide.









TABLE 1







Unmodified Hif2α RNAi trigger antisense strand


and sense strand sequences.










SEQ

SEQ



ID
Antisense Strand
ID
Sense Strand


NO.
Sequence 5′ → 3′
NO.
Sequence 5′ → 3′





 1
AGUAAAACAAUUGUGUACUUU
57
AGUACACAAUUGUUUUACUT





 2
AGUAAAACAAUUGUGUACUUU
44
AAAGUACACAAUUGUUUUAC



AA

T





 3
AUUCAUGAAAUCGUUACGUTG
52
ACGUAACGAUUUCAUGAATT





 4
AUUCAUGAAAUCGUUACGUUG
52
ACGUAACGAUUUCAUGAATT





 4
AUUCAUGAAAUCGUUACGUUG
53
ACGUAACGAUUUCAUGAAU





 4
AUUCAUGAAAUCGUUACGUUG
54
ACGUAACGAUUUCAUGAAUT





 4
AUUCAUGAAAUCGUUACGUUG
73
UAUACGUAACGAUUUCAUGA





AUT





 4
AUUCAUGAAAUCGUUACGUUG
74
UAUACGUAACGAUUUCAUGA





AUTT





 5
AUUCAUGAAAUCGUUACGUUG
47
AACGUAACGAUUUCAUGAAU



AT

T





 6
AUUCAUGAAAUCGUUACGUUG
77
UAUCAACGUAACGAUUUCAU



GC

GAAUTT





 7
TAAAUCGUUACGUUGACAGTT
67
CUGUCAACGUAACGAUUUAT





 8
TAACCACAUACGUUGGAGUTT
55
ACUCCAACGUAUGUGGUUAT





 9
TAAGUUAAGCUCCCAUACATT
81
UGUAUGGGAGCUUAACUUAT





10
TAAUCGUUACGUUGACAGGTT
63
CCUGUCAACGUAACGAUUAT





11
TACGUUGACAGGUAGGGUUTT
45
AACCCUACCUGUCAACGUAT





12
TAGAGGAGCUUGUGUGUUCTT
68
GAACACACAAGCUCCUCUAT





13
TAGCUUGUGUGUUCGCAGGTT
62
CCUGCGAACACACAAGCUAT





14
TAGGAGCUUGUGUGUUCGCTT
69
GCGAACACACAAGCUCCUAT





15
TAUCGUUACGUUGACAGGUTT
49
ACCUGUCAACGUAACGUAAT





15
TAUCGUUACGUUGACAGGUTT
71
UAUACCUGUCAACGUAACGU





AAT





16
TCAUGAAAUCGUUACGUUGTT
60
CAACGUAACGAUUUCAUGAT





17
TCGUUACGUUGACAGGUAGTT
65
CUACCUGUCAACGUAACGAT





18
TCUAGCAACAAAACCUUAATT
82
UUAAGGUUUUGUUGCUAGAT





19
TGAGCUUGUGUGUUCGCAGTT
66
CUGCGAACACACAAGCUCAT





19
TGAGCUUGUGUGUUCGCAGTT
68
GAACACACAAGCUCCUCUAT





20
TGAGGAGCUUGUGUGUUCGTT
64
CGAACACACAAGCUCCUCAT





21
TGGAGCUUGUGUGUUCGCATT
79
UGCGAACACACAAGCUCCAT





22
TGGUACUGGGUGGCGUAGCTT
70
GCUACGCCACCCAGUACCAT





23
TGUAAAACAAUUGUGUACUTT
56
AGUACACAAUUGUUUUACAT





24
TUACGUUGACAGGUAGGGUTT
48
ACCCUACCUGUCAAGGUAAT





25
TUCGUUACGUUGACAGGUATT
78
UCACUGUCAACGUAACGAAT





26
TUGAUAAACACUUAACCCATT
80
UGGGUUAAGUGUUUAUCAAT





27
TUGUCACGAUGCGGUGGUUTT
61
CAGUGCAACGCCACCCAGAT





28
TUUCAUGAAAUCGUUACGUCG
76
UAUAUCGACGUAACGAUUUC



GCUAU

AUGAAA





29
TUUCAUGAAAUCGUUACGUCG
76
UAUAUCGACGUAACGAUUUC



GCUGU

AUGAAA





30
TUUCAUGAAAUCGUUACGUTT
50
ACGUAACGAUUUCAUGAAA





30
TUUCAUGAAAUCGUUACGUTT
51
ACGUAACGAUUUCAUGAAAT





30
TUUCAUGAAAUCGUUACGUTT
72
UAUACGUAACGAUUUCAUGA





AAT





31
TUUCAUGAAAUCGUUACGUUG
59
CAACGUAACGAUUUCAUGAA



GC

A





32
TUUCAUGAAAUCGUUACGUUG
75
UAUAUCAACGUAACGAUUUC



GCUAU

AUGAAA





33
TUUCAUGAAAUCGUUACGUUG
75
UAUAUCAACGUAACGAUUUC



GCUGU

AUGAAA





34
TUUCAUGAAAUCGUUACGUUG
75
UAUAUCAACGUAACGAUUUC



GCUTT

AUGAAA





35
UCAUGAAAUCGUUACGUUGTT
58
CAACGUAACGAUUUCAUGA





35
UCAUGAAAUCGUUACGUUGTT
58
CAACGUAACGAUUUCAUGA





36
UCUAGCAACAAAACCUUAATT
82
UUAAGGUUUUGUUGCUAGAT





37
UGUAAAACAAUUGUGUACUTT
56
AGUACACAAUUGUUUUACAT





38
UGUAAAACAAUUGUGUACUUU
56
AGUACACAAUUGUUUUACAT





39
UGUAAAACAAUUGUGUACUUU
43
AAAGUACACAAUUGUUUUAC



AA

A





40
UUUCAUGAAAUCGUUACGUTT
51
ACGUAACGAUUUCAUGAAAT





40
UUUCAUGAAAUCGUUACGUTT
72
UAUACGUAACGAUUUCAUGA





AAT





41
UUUCAUGAAAUCGUUACGUUG
50
ACGUAACGAUUUCAUGAAA





41
UUUCAUGAAAUCGUUACGUUG
51
ACGUAACGAUUUCAUGAAAT





41
UUUCAUGAAAUCGUUACGUUG
72
UAUACGUAACGAUUUCAUGA





AAT





42
UUUCAUGAAAUCGUUACGUUG
46
AACGUAACGAUUUCAUGAAA



AT

T









The Hif2α RNAi triggers described herein are formed by annealing an antisense strand with a sense strand. In some embodiments, an Hif2α RNAi trigger antisense strand comprises a nucleotide sequence of any of the sequences in Tables 1 and 5. In some embodiments, an Hif2α RNAi trigger antisense strand comprises the sequence of nucleotides 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23, 1-24, 2-24, 1-25, 2-25, 1-26, or 2-26 of any of the sequences in Tables 1 and 5. In some embodiments, an Hif2α RNAi trigger sense strand comprises the nucleotide sequence of any of the sequences in Tables 1 and 5. In some embodiments, an Hif2α RNAi trigger sense strand comprises the sequence of nucleotides 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23, 1-24, 2-24, 1-25, 2-25, 1-26, or 2-26 of any of the sequences in Tables 1 and 5.


In some embodiments, the sense and antisense strands of the RNAi triggers described herein contain the same number of nucleotides. In some embodiments the sense and antisense strands of the RNAi triggers described herein contain different numbers of nucleotides. In some embodiments, the sense strand 5′ end and the antisense strand 3′ end of an RNAi trigger form a blunt end. In some embodiments, the sense strand 3′ end and the antisense strand 5′ end of an RNAi trigger form a blunt end. In some embodiments, both ends of an RNAi trigger form a blunt end. In some embodiments, neither end of an RNAi trigger is blunt-ended. As used herein a blunt end refers to an end of a double stranded trigger molecule in which the terminal nucleotides of the two annealed strands are complementary (form a complementary base-pair).


In some embodiments, the sense strand 5′ end and the antisense strand 3′ end of an RNAi trigger form a frayed end. In some embodiments, the sense strand 3′ end and the antisense strand 5′ end of an RNAi trigger form a frayed end. In some embodiments, both ends of an RNAi trigger form a frayed end. In some embodiments, neither end of an RNAi trigger is a frayed end. As used herein a frayed end refers to an end of a double stranded trigger molecule in which the terminal nucleotides of the two annealed strands from a pair (i.e. do not form an overhang) but are not complementary (i.e. form a non-complementary pair). As used herein, an overhang is a stretch of one or more unpaired nucleotides at the end of one strand of a double stranded RNAi trigger molecule. The unpaired nucleotides may be on the sense strand or the antisense strand, creating either 3′ or 5′ overhangs. In some embodiments the RNAi trigger molecule contains: a blunt end and a frayed end, a blunt end and 5′ overhang end, a blunt end and a 3′ overhang end, a frayed end and a 5′ overhand end, a frayed end and a 3′ overhang end, two 5′ overhang ends, two 3′ overhang ends, a 5′ overhang end and a 3′ overhand end, two frayed ends, or two blunt ends.


A nucleotide base (or nucleobase) is a heterocyclic pyrimidine or purine compound which is a constituent of all nucleic acids and includes adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). As used herein, “G”, “g”, “C”, “c”, “A”, “a”, “U”, “u”, and “T”, each generally stand for a nucleobase, nucleoside, nucleotide or nucleotide mimic that contains guanine, cytosine, adenine, uracil and thymidine as a base. Also as used herein, the term “nucleotide” can include a modified nucleotide or nucleotide mimic, abasic site, or a surrogate replacement moiety.


As used herein, a “modified nucleotide” is a nucleotide other than a ribonucleotide (2′-hydroxyl nucleotide). In some embodiments, an Hif2α RNAi trigger contains one or more modified nucleotides. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the nucleotides are modified. Modified nucleotides include, but are not limited to, deoxynucleotides, nucleotide mimics, abasic nucleotides (represented herein as X or Ab), 2′-modified nucleotides, 3′ to 3′ linkages (inverted)nucleotides (represented herein as invdN, invN, invn, invX), non-natural base-comprising nucleotides, bridged nucleotides, peptide nucleic acids, 2′,3′-seco nucleotide mimics (unlocked nucleobase analogues, represented herein as NUNA or NUNA), locked nucleotides (represented herein as NLNA or NLNA), 3′-O-Methoxy (2′ internucleotide linked) nucleotides (represented herein as 3′-OMen), 2′-F-Arabino nucleotides (represented herein as NfANA or NfANA), morpholino nucleotides, vinyl phosphonate deoxyribonucleotides (represented herein as vpdN), and vinyl phosphonate nucleotides. 2′-modified nucleotides (i.e. a nucleotide with a group other than a hydroxyl group at the 2′ position of the five-membered sugar ring) include, but are not limited to, 2′-O-methyl nucleotides (represented herein as a lower case letter ‘n’ in a nucleotide sequence), 2′-deoxy-2′-fluoro nucleotides (represented herein as Nf, also represented herein as 2′-fluoro nucleotide), 2′-deoxy nucleotides (represented herein as dN), 2′-methoxyethyl (2′-O-2-methoxylethyl)nucleotides (represented herein as NM or 2′-MOE), 2′-amino nucleotides, 2′-alkyl nucleotides. It is not necessary for all positions in a given compound to be uniformly modified. Conversely, more than one modification may be incorporated in a single Hif2M RNAi trigger or even in a single nucleotide thereof. The Hif2α RNAi trigger sense strands and antisense strands may be synthesized and/or modified by methods known in the art. Modification at one nucleotide is independent of modification of another nucleotide.


Modified nucleotides also include nucleotides having modified nucleobases. Modified nucleobases include, but are not limited to, synthetic and natural nucleobases, 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 (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 and 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-deazaadenine and 3-deazaguanine and 3-deazaadenine.


In some embodiments 20% or fewer of the modified nucleotides are 2′-fluoro modified nucleotides. In some embodiments, an Hif2α RNAi trigger sense strand contains a 2′-F nucleotide at position 11 from the 3′ end. In some embodiments, an Hif2α RNAi trigger sense strand contains a 2′-F nucleotide at position 12 from the 3′ end. In some embodiments, an Hif2α RNAi trigger sense strand contains a 2′-F nucleotide at position 13 from the 3′ end. In some embodiments, an Hif2α RNAi trigger sense strand contains at least two 2′-F nucleotides at positions 11, 12, and 13 from the 3′ end. In some embodiments, an Hif2α RNAi trigger sense strand contains 2′-F nucleotides at positions 11 and 12, positions 11 and 13, or positions 12 and 13 from the 3′ end. In some embodiments, an Hif2α RNAi trigger sense strand contains 2′-F nucleotides at positions 11, 12, and 13 from the 3′ end.


In some embodiments, an Hif2α RNAi trigger antisense strand contains a 2′-F nucleotide at position 2 from the 5′ end. In some embodiments, an Hif2α RNAi trigger antisense strand contains a 2′-F nucleotide at position 14 from the 5′ end. In some embodiments, an Hif2α RNAi trigger antisense strand contains 2′-F nucleotides at positions 2 and 14 from the 5′ end. In some embodiments, an Hif2α RNAi trigger contains at least two 2′-F nucleotides at positions 11, 12, and 13 from the 3′ end of the sense strand and at positions 2 and 14 from the 5′ end of the antisense strand.


In some embodiments, an Hif2α RNAi trigger antisense strand contains a 2′-F nucleotide at position 4 from the 5′ end. In some embodiments, an Hif2α RNAi trigger antisense strand contains a 2′-F nucleotide at position 6 from the 5′ end. In some embodiments, an Hif2α RNAi trigger antisense strand contains a 2′-F nucleotide at position 8 from the 5′ end. In some embodiments, an Hif2α RNAi trigger antisense strand contains a 2′-F nucleotide at position 10 from the 5′ end. In some embodiments, an Hif2α RNAi trigger antisense strand contains a 2′-F nucleotide at position 12 from the 5′ end. In some embodiments, an Hif2α RNAi trigger antisense strand contains at least two 2′-F nucleotides at positions 4, 6, 8, 10, and 12 from the 5′ end. In some embodiments, an Hif2α RNAi trigger antisense strand contains 2′-F nucleotides at positions 4 and 6, positions 4 and 8, positions 4 and 10, positions 4 and 12, positions 6 and 8, positions 6 and 10, positions 6 and 12, positions 8 and 10, positions 8 and 12, or positions 10 and 12 from the 5′ end. In some embodiments, an Hif2α RNAi trigger antisense strand contains at three 2′-F nucleotides at positions 4, 6, 8, 10, and 12 from the 5′ end. In some embodiments, an Hif2α RNAi trigger antisense strand contains at least four 2′-F nucleotides at positions 4, 6, 8, 10, and 12 from the 5′ end. In some embodiments, an Hif2α RNAi trigger antisense strand contains 2′-F nucleotides at positions 4, 6, 8, and 10, positions 4, 6, 8, and 12, positions 4, 6, 10, and 12, positions 4, 8, 10, and 12 or positions 6, 8, 10, and 12 from the 5′ end.


In some embodiments, an Hif2α RNAi trigger antisense strand contains a 2′-F nucleotide at position 2 and/or position 14 and one, two, or three 2′-F nucleotides at positions 11, 12, and 13 from the 5′ end. In some embodiments, an Hif2α RNAi trigger contains a 2′-F nucleotide at position 2 and/or position 14 and one, two, or three 2′-F nucleotides at positions 11, 12, and 13 from the 5′ end of the antisense strand, and at least two 2′-F nucleotides at positions 11, 12, and 13 from the 3′ end of the sense strand.


In some embodiments, one or more nucleotides of an Hif2α RNAi trigger are linked by non-standard linkages or backbones (i.e. modified internucleoside linkages or modified backbones). In some embodiments, a modified internucleoside linkage is a non-phosphate-containing covalent internucleoside linkage. Modified internucleoside linkages or backbones include, but are not limited to, phosphorothioates, 5′-phosphorothioate group (represented herein as a lower case ‘s’ before a nucleotide, as in sN, sn, sNf, or sdN), chiral phosphorothioates, thiophosphate, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, 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′. In other embodiments, a modified internucleoside linkage or backbone lacks a phosphorus atom. Modified internucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages. In some embodiments, modified internucleoside backbones include, but are not limited to, 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 CH2 component parts.


In some embodiments, an Hif2α RNAi trigger contains one or more modified nucleotides and one or more modified internucleoside linkages. In some embodiments, a 2′-modified nucleotide is combined with modified internucleoside linkage. For example, in some embodiments, a sense strand of an Hif2α RNAi trigger can contain 1, 2, 3, 4 phosphorothioate linkages, an antisense strand of a Hif2α RNAi trigger can contain 1, 2, 3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, or 4 phosphorothioate linkages.


In some embodiments, an Hif2α RNAi trigger sense strand contains two phosphorothioate internucleoside linkages. In some embodiments, the two phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3 from the 3′ end of the sense strand. In some embodiments, the two phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3, 2-4, 3-5, 4-6, 4-5, or 6-8 from the 5′ end of the sense strand. In some embodiments, an Hif2α RNAi trigger antisense strand contains four phosphorothioate internucleoside linkages. In some embodiments, the four phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3 from the 5′ end of the sense strand and between the nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5′ end. In some embodiments, an Hif2α RNAi trigger contains two phosphorothioate internucleoside linkages in the sense strand and four phosphorothioate internucleoside linkages in the antisense strand.


In some embodiments, an Hif2α RNAi trigger is prepared or provided as a salt, mixed salt, or a free-acid.


Examples of antisense strands containing modified nucleotides are provided in Table 2A and Table 5B. Examples of sense strands containing modified nucleotides are provided in Table 2B and Table 5B. In Tables 2A, 2B and 5B, the following notations are used to indicate modified nucleotides:

    • N=2′-OH(unmodified)ribonucleotide (capital letter without or d indication)
    • n=2′-OMe modified nucleotide
    • Nf=2′-fluoro modified nucleotide
    • A dN=2′-deoxynucleotides
    • NUNA=2′,3′-seco nucleotide mimics (unlocked nucleobase analogs)
    • NM=2′-methoxyethyl nucleotide
    • (invdN)=inverted deoxyribonucleotide (3′-3′ linked nucleotide)
    • (invAb)=inverted abasic nucleotide
    • s=phosphorothioate linked nucleotide
    • p=phosphate
    • vpdN=vinyl phosphonate deoxyribonucleotide









TABLE 2A







Hif2α RNAi trigger antisense strands having


modified nucleotides.













Un-





mod.




SEQ 
SEQ


Antisense
Antisense Strand Sequence
ID
ID


Strand ID
(5′ → 3′)
NO.
NO.





AM00159-AS
dTGfgAfgCfuUfgUfgUfgUfuCfgCfad
112
21



TsdT







AM00160-AS
dTGfgUfaCfuGfgGfuGfgCfgUfaGfcd
113
22



TsdT







AM00161-AS
dTCfgUfuAfcGfuUfgAfcAfgGfuAfgd
106
17



TsdT







AM00162-AS
dTAfgCfuUfgUfgUfgUfuCfgCfaGfgd
103
13



TsdT







AM00163-AS
dTGfaGfcUfuGfuGfuGfuUfcGfcAfgd
110
19



TsdT







AM00164-AS
dTAfgGfaGfcUfuGfuGfuGfuUfcGfcd
104
14



TsdT







AM00165-AS
dTGfaGfgAfgCfuUfgUfgUfgUfuCfgd
111
20



TsdT







AM00166-AS
dTAfgAfgGfaGfcUfuGfuGfuGfuUfcd
102
12



TsdT







AM00167-AS
dTUfgUfcAfcGfaUfgCfgGfuGfgUfud
137
27



TsdT







AM00168-AS
dTAfcGfuUfgAfcAfgGfuAfgGfgUfud
101
11



TsdT







AM00169-AS
dTUfaCfgUfuGfaCfaGfgUfaGfgGfud
135
24



TsdT







AM00170-AS
dTUfcGfuUfaCfgUfuGfaCfaGfgUfad
136
25



TsdT







AM00171-AS
dTAfuCfgUfuAfcGfuUfgAfcAfgGfud
105
15



TsdT







AM00172-AS
dTAfaUfcGfuUfaCfgUfuGfaCfaGfgd
100
10



TsdT







AM01770-AS
dTGfuAfaAfaCfaAfuUfgUfgUfaCfud
114
23



TsdT







AM01772-AS
dTGfuAfaAUNAaCfaAfuUfgUfgUfaCf
116
23



udTsdT







AM01773-AS
dTGfuAfaAfAUNACfaAfuUfgUfgUfaC
115
23



fudTsdT







AM01775-AS
dTCfuAfgCfaAfcAfaAfaCfcUfuAfad
107
18



TsdT







AM01777-AS
dTCfuAfgCUNAaAfcAfaAfaCfcUfuAf
109
18



adTsdT







AM01778-AS
dTCfuAfgCfAUNAAfcAfaAfaCfcUfuA
108
18



fadTsdT







AM01780-AS
dTAfaAfuCfgUfuAfcGfuUfgAfcAfgd
 94
 7



TsdT







AM01782-AS
dTAfaAfuCUNAgUfuAfcGfuUfgAfcAf
 96
 7



sgdTdT







AM01783-AS
dTAfaAfuCfGUNAUfuAfcGfuUfgAfcA
 95
 7



fgdTsdT







AM01784-AS
dTUfuCfaUfgAfaAfuCfgUfuAfcGfud
138
30



TsdT







AM01786-AS
dTUfuCfaUUNAgAfaAfuCfgUfuAfcGf
145
30



udTsdT







AM01787-AS
dTUfuCfaUfGUNAAfaAfuCfgUfuAfcG
144
30



fudTsdT







AM01789-AS
dTAfaGfuUfaAfgCfuCfcCfaUfaCfad
 97
 9



TsdT







AM01791-AS
dTAfaGfuUUNAaAfgCfuCfcCfaUfaCf
 99
 9



adTsdT







AM01792-AS
dTAfaGfuUfAUNAAfgCfuCfcCfaUfaC
 98
 9



fadTsdT







AM02090-AS
dTUfuCfaUfgAUNAaAfuCfgUfuAfcGf
143
30



udTsdT







AM02091-AS
dTUfuCfaUfgAfAUNAAfuCfgUfuAfcG
142
30



fudTsdT







AM02092-AS
dTUfuCfaUfgAfaAfuCfgUfUUNAAfcG
139
30



fudTsdT







AM02133-AS
dTUfuCfaUfgAfaAfucgUfuAfcGfudT
141
30



sdT







AM02140-AS
dTsUfsuCfaUfgAfaAfucgUfuAfcGfu
128
31



Ufgsgsc







AM02145-AS
dTsUfsuCfaUfgAfaAfucgUfuAfcGfu
127
33



UfggscsuGu







AM02146-AS
dTsUfsuCfaUfgAfaAfucgUfuAfcGfu
123
29



CfggscsuGu







AM02147-AS
dTsUfsuCfaUfgAfaAfucgUfuAfcGfu
126
32



UfggscsuAu







AM02150-AS
dTsUfsuCfaUfgAfaAfucgUfuAfcGfu
125
34



UfggcusdTsdT







AM02327-AS
pdTUfuCfaUfgAfaAfuCfgUfuAfcGfu
147
30



dTsdT







AM02341-AS
dTsUfsuCfaUfgAUNAaAfuCfgUfuAfc
130
28



GfuCfggscsuAu







AM02342-AS
dTsUfuCfaUfgAUNAaAfuCfgUfuAfcG
134
28



fuCfggcsuAu







AM02345-AS
dTsUfsuCfaUfgAfAUNAAfuCfgUfuAf
129
28



cGfuCfggscsuAu







AM02346-AS
dTsUfuCfaUfgAfAUNAAfuCfgUfuAfc
133
28



GfuCfggcsuAu







AM02508-AS
dTsUfsuCfaUfgAfaAfuCfgUfuAfcGf
120
30



udTsdT







AM02509-AS
dTUfuCfaUfgAfaAfucGfUfuAfcGfud
140
30



TsdT







AM02523-AS
dTsAfsaCfcAfcAfuAfcGfuUfgGfaGf
117
 8



udTsdT







AM02525-AS
dTsUfsgAfuAfaAfcAfcUfuAfaCfcCf
119
26



adTsdT







AM02527-AS
dTsCfsaUfgAfaAfuCfgUfuAfcGfuUf
118
16



gdTsdT







AM02529-AS
usCfsaUfgAfaAfuCfgUfuAfcGfuUfg
148
35



dTsdT







AM02604-AS
dTsUfsuCfaUfgAfaAfucGfUfuAfcGf
122
30



udTsdT







AM02605-AS
dTsUfsuCfaUfgAfaAfucgUfuAfcGfu
124
30



dTsdT







AM02848-AS
dTsUfuCfaUfgAfaAfucGfUfuAfcGfu
132
30



dTsdT







AM02849-AS
dTsUfuCfaUfgAfaAfuCfgUfUfacGfu
131
30



dTsdT







AM02850-AS
dTsUfsuCfaUfgAfaAfuCfgUfUfacGf
121
30



udTsdT







AM02998-AS
usUfsuCfaUfgAfaAfucgUfuAfcGfud
160
40



TsdT







AM03000-AS
usCfsuAfgCfaAfcAfaaAfCfcUfuAfa
151
36



dTsdT







AM03001-AS
usCfsuAfgCfaaCfAfaaAfCfcUfuAfa
149
36



dTsdT







AM03002-AS
usCfsuAfgCfaAfcAfaAfaCfCfuuAfa
152
36



dTsdT







AM03003-AS
usCfsuAfgCfaaCfAfaAfaCfCfuuAfa
150
36



dTsdT







AM03008-AS
usGfsuAfaAfaCfaAfuuGfUfgUfaCfu
154
37



dTsdT







AM03009-AS
usGfsuAfaAfaCfaAfuUfgUfGfuaCfu
153
37



dTsdT







AM03059-AS
pdTsUfsuCfaUfgAfaAfucgUfuAfcGf
146
30



udTsdT







AM03465-AS
usUfsuCfaUfgAfaAfucgUfuAfcGfus
161
41



usg







AM03513-AS
usUfsuCfaUfgAfaAfucguuacgususg
162
41





AM03514-AS
usUfsuCfaUfgAfaaucguuacgususg
165
41





AM03517-AS
asUfsuCfaUfgAfaAfucgUfuAfcGfus
 90
 4



usg







AM03685-AS
usUfsuCfaUfgAfaAfucgUfuacgusus
159
41



g







AM03688-AS
usUfsUfCfaugAfaAfucgUfuacgusus
167
41



g







AM03689-AS
usUfsuCfaUfgAfaaucgUfuacgususg
163
41





AM03690-AS
usUfsUfcaugAfaAfucgUfuacgususg
166
41





AM04001-AS
usGfsuAfaAfaCfaAfuugUfgUfaCfus
155
38



usu







AM04004-AS
asGfsuAfaAfaCfaAfuugUfgUfaCfus
 84
 1



usu







AM04007-AS
usGfsuAfaAfaCfaAfuugUfgUfaCfuu
156
39



usasa







AM04010-AS
asGfsuAfaAfaCfaAfuugUfgUfaCfuu
 85
 2



usasa







AM04015-AS
asGfsuAfaAfaCfaAfuugUfguaCfuuu
 83
 2



sasa







AM04018-AS
asGfsuAfaAfaCfaauugUfguacuuusa
 86
 2



sa







AM04040-AS
vpusUfsuCfaUfgAfaAfucgUfuAfcGf
168
41



ususg







AM04101-AS
asUfsuCfaUfgAfaaucgUfuacgususg
 91
 4





AM04102-AS
usUfsuCfaUfgAfaaucgUfuacguugsa
164
42



s(invdT)







AM04103-AS
asUfsuCfaUfgAfaaucgUfuacguugsa
 92
 5



s(invdT)







AM04104-AS
usUfsucaugAfaAfucgUfuacgususg
157
41





AM04105-AS
asUfsucaugAfaAfucgUfuacgususg
 88
 4





AM04106-AS
asUfsucaugAfaAfucgUfuacgusTMsG
 87
 3



M







AM04244-AS
usUfsuCfaUfgaAfaucgUfuacgususg
158
41





AM04452-AS
asUfsuCfaUfgAfaaucgUfuAfcguugs
 93
 6



gsc







AM04455-AS
asUfsuCfaUfgaAfaucgUfuAfcguugs
 89
 6



gsc
















TABLE 2B







Hif2α RNAi trigger sense strands having modified


nucleotides.













Unmod.


Sense

SEQ
SEQ


Strand

ID
ID


ID
SS Sequence 5′ → 3′
NO.
NO.





AM00158-SS
CfuGfuCfaAfcGfuAfaCfgAfuUfuAf(invdT)
277
67





AM00188-SS
(NH2-C6)uAuUfgCfgAfaCfaCfaCfaAfgCfuCfcAf(invdT)
265
79





AM00189-SS
(NH2-C6)uAuGfcUfaCfgCfcAfcCfcAfgUfaCfcAf(invdT)
264
70





AM00190-SS
(NH2-C6)uAuCfuAfcCfuGfuCfaAfcGfuAfaCfgAf(invdT)
263
65





AM00191-SS
(NH2-C6)uAuCfcUfgCfgAfaCfaCfaCfaAfgCfuAf(invdT)
262
62





AM00207-SS
(NH2-C6)CfuGfcGfaAfcAfcAfcAfaGfcUfcAf(invdT)
258
66





AM00208-SS
(NH2-C6)GfcGfaAfcAfcAfcAfaGfcUfcCfuAf(invdT)
260
69





AM00209-SS
(NH2-C6)CfgAfaCfaCfaCfaAfgCfuCfcUfcAf(invdT)
256
64





AM00210-SS
(NH2-C6)GfaAfcAfcAfcAfaGfcUfcCfuCfuAf(invdT)
259
68





AM00211-SS
(NH2-C6)CfaGfuGfcAfaCfgCfcAfcCfcAfgAf(invdT)
253
61





AM00212-SS
(NH2-C6)AfaCfcCfuAfcCfuGfuCfaAfcGfuAf(invdT)
247
45





AM00213-SS
(NH2-C6)AfcCfcUfaCfcUfgUfcAfaGfgUfaAf(invdT)
248
48





AM00214-SS
(NH2-C6)UfcAfcUfgUfcAfaCfgUfaAfcGfaAf(invdT)
266
78





AM00219-SS
(NH2-C6)AfcCfuGfuCfaAfcGfuAfaCfgUfaAf(invdT)
249
49





AM00220-SS
(NH2-C6)CfcUfgUfcAfaCfgUfaAfcGfaUfuAf(invdT)
255
63





AM00221-SS
(NH2-C6)UfgCfgAfaCfaCfaCfaAfgCfuCfcAf(invdT)
267
79





AM00222-SS
(NH2-C6)GfcUfaCfgCfcAfcCfcAfgUfaCfcAf(invdT)
261
70





AM00223-SS
(NH2-C6)CfuAfcCfuGfuCfaAfcGfuAfaCfgAf(invdT)
257
65





AM00224-SS
(NH2-C6)CfcUfgCfgAfaCfaCfaCfaAfgCfuAf(invdT)
254
62





AM00366-SS
(Alk-SS-C6)CfuGfcGfaAfcAfcAfcAfaGfcUfcAf(invdT)
220
66





AM00367-SS
(Alk-SS-C6)GfcGfaAfcAfcAfcAfaGfcUfcCfuAf(invdT)
223
69





AM00369-SS
(Alk-SS-C6)GfaAfcAfcAfcAfaGfcUfcCfuCfuAf(invdT)
222
68





AM00530-SS
(Alk-SS-C6)AfcCfcUfaCfcUfgUfcAfaGfgUfaAf(invdT)
212
48





AM00531-SS
(Alk-SS-C6)UfcAfcUfgUfcAfaCfgUfaAfcGfaAf(invdT)
224
78





AM00543-SS
(Alk-SS-C6)AfaCfcCfuAfcCfuGfuCfaAfcGfuAf(invdT)
211
45





AM00544-SS
(Alk-SS-C6)AfcCfuGfuCfaAfcGfuAfaCfgUfaAf(invdT)
213
49





AM00545-SS
(Alk-SS-C6)CfuAfcCfuGfuCfaAfcGfuAfaCfgAf(invdT)
219
65





AM01771-SS
AfgUfaCfaCfaAfuUfgUfuUfuAfcAf(invdT)
271
56





AM01776-SS
UfuAfaGfgUfuUfuGfuUfgCfuAfgAf(invdT)
284
82





AM01785-SS
AfcGfuAfaCfgAfuUfuCfaUfgAfaAf(invdT)
269
51





AM01790-SS
UfgUfaUfgGfgAfgCfuUfaAfcUfuAf(invdT)
283
81





AM01859-SS
(Alk-SS-C6)AfgUfaCfaCfaAfuUfgUfuUfuAfcAf(invdT)
218
56





AM01860-SS
(Alk-SS-C6)UfuAfaGfgUfuUfuGfuUfgCfuAfgAf(invdT)
226
82





AM01861-SS
(Alk-SS-C6)CfuGfuCfaAfcGfuAfaCfgAfuUfuAf(invdT)
221
67





AM01862-SS
(Alk-SS-C6)AfcGfuAfaCfgAfuUfuCfaUfgAfaAf(invdT)
215
51





AM01863-SS
(Alk-SS-C6)UfgUfaUfgGfgAfgCfuUfaAfcUfuAf(invdT)
225
81





AM01994-SS
(Alk-C6)uAuAfcCfuGfuCfaAfcGfuAfaCfgUfaAf(invdT)
169
71





AM02043-SS
(Me-Alk-SS-C6)AfcGfuAfaCfgAfuUfuCfaUfgAfaAf(invdT)
231
51





AM02093-SS
(DBCO-TEG)uAuAfcGfuAfaCfgAfuUfuCfaUfgAfaAf(invdT)
228
72





AM02135-SS
(Alk-SS-C6)AfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(invdT)
216
51





AM02137-SS
(Alk-SS-C6)AfcGfuAfAfCfgAfuUfuCfaUfgAfaAf(invdT)
217
51





AM02139-SS
(Alk-SS-C6)AfcGfuaaCfgAfuUfuCfaUfgAfaAf(invdT)
214
51





AM02142-SS
CfsasAfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me)
276
59





AM02144-SS
CUNAsasAfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me)
278
59





AM02149-SS
uAuAusCfsaAfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me)
280
75





AM02163-SS
uAuAusCfsgAfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me)
282
76





AM02363-SS
(DBCO-TEG)uAuAusCfsgAfcGfuAfaCfgAfuUfuCfaUfgAfa(invdA)
230
76





AM02364-SS
uAuAusCfsgAfcGfuAfaCfgAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me)
281
76





AM02365-SS
uAuAusCfgAfcGfuAfaCfgAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me)
279
76





AM02456-SS
(Alk-SMPT-C6)AfcGfuAfaCfgAfuUfuCfaUfgAfaAf(invdT)
193
51





AM02510-SS
(Me-Alk-SS-C6)AfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(invdT)
232
51





AM02512-SS
(Me-Alk-SS-C6)AfcGfuAfacGfAfuUfuCfaUfgAfaAf(invdT)
233
51





AM02522-SS
(NH2-C6)AfcUfcCfaAfcGfuAfuGfuGfgUfuAf(invdT)
250
55





AM02524-SS
(NH2-C6)UfgGfgUfuAfaGfuGfuUfuAfuCfaAf(invdT)
268
80





AM02526-SS
(NH2-C6)CfaAfcGfuAfaCfgAfuUfuCfaUfgAf(invdT)
252
60





AM02528-SS
(NH2-C6)CfaAfcGfuAfaCfgAfuUfuCfaUfg(invdA)
251
58





AM02546-SS
(Me-Alk-SS-C6)AfcUfcCfaAfcGfuAfuGfuGfgUfuAf(invdT)
235
55





AM02547-SS
(Me-Alk-SS-C6)UfgGfgUfuAfaGfuGfuUfuAfuCfaAf(invdT)
242
80





AM02548-SS
(Me-Alk-SS-C6)CfaAfcGfuAfaCfgAfuUfuCfaUfgAf(invdT)
241
60





AM02549-SS
(Me-Alk-SS-C6)CfaAfcGfuAfaCfgAfuUfuCfaUfg(invdA)
240
58





AM02852-SS
AfscGfuAfacGfAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me)
272
50





AM02853-SS
AfscGfUfaaCfgAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me)
273
50





AM02856-SS
(Me-Alk-SS-C6)AfcGfUfaaCfgAfuUfuCfaUfgAfaAf(invdT)
234
51





AM02999-SS
(DBCO-TEG)uAuAfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(invdT)
229
72





AM03004-SS
(Me-Alk-SS-C6)UfuAfaGfguUfUfuGfuUfgCfuAfgAf(invdT)
244
82





AM03005-SS
(Me-Alk-SS-C6)uuAfaGfguUfUfuguUfgCfuAfga(invdT)
246
82





AM03006-SS
(Me-Alk-SS-C6)UfuAfAfggUfuUfuGfuUfgCfuAfgAf(invdT)
243
82





AM03007-SS
(Me-Alk-SS-C6)uuAfAfggUfuUfuguUfgCfuAfga(invdT)
245
82





AM03010-SS
(Me-Alk-SS-C6)AfgUfaCfacAfAfuUfgUfuUfuAfcAf(invdT)
236
56





AM03011-SS
(Me-Alk-SS-C6)agUfaCfacAfAfuUfgUfuuuAfca(invdT)
238
56





AM03012-SS
(Me-Alk-SS-C6)AfgUfAfcaCfaAfuUfgUfuUfuAfcAf(invdT)
237
56





AM03013-SS
(Me-Alk-SS-C6)agUfAfcaCfaAfuUfgUfuuuAfca(invdT)
239
56





AM03058-SS
AfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(invdT)
270
51





AM03307-SS
agUfAfcaCfaAfuUfgUfuuuAfca(invdT)
274
56





AM03308-SS
(Alk-C6-C6)AfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(invdT)
170
51





AM03467-SS
(Alk-SMPT-C6)AfscGfuAfaCfGfAfuUfuCfaUfgAfaa(invdT)
194
51





AM03469-SS
(Alk-SMPT-C6)ascGfuAfaCfGfAfuuuCfaUfgAfaa(invdT)
201
51





AM03471-SS
(Alk-SMPT-C6)ascguAfaCfgAfuuuCfaUfgAfaa(invdT)
208
51





AM03473-SS
(Alk-SMPT-C6)ascGfuaaCfgAfuuuCfaugAfaa(invdT)
200
51





AM03475-SS
(Alk-SMPT-C6)ascguaaCfgAfuuuCfaugaaa(invdT)
204
51





AM03516-SS
(Alk-SMPT-C6)ascguaaCfgAfuuucaugaaa(invdT)
203
51





AM03519-SS
(Alk-SMPT-C6)ascGfuAfaCfGfAfuuuCfaUfgAfau(invdT)
202
54





AM03687-SS
(Alk-SMPT-C6)ascguaaCfGfAfuuucaugaaa(invdT)
205
51





AM03692-SS
(Alk-PEGS-C6)uAuascguaaCfGfAfuuucaugaaa(invdT)
185
72





AM03694-SS
(Alk-PEGS-C6)uAuascGfuAfaCfGfAfuuuCfaUfgAfau(invdT)
183
73





AM03708-SS
(Alk-PEGS-C6)uAuascGfuAfaCfGfAfuuuCfaUfgAfaa(invdT)
182
72





AM03710-SS
(Alk-PEGS-C6)uAuascguaaCfGfAfuuuCfaugaaa(invdT)
189
72





AM03712-SS
(Alk-PEGS-C6)uAuascguaaCfGfAfuuuCfaugaau(invdT)
190
73





AM03714-SS
(Alk-PEG5-C6)(Alk-PEG5-Ser)-
180
72



uAuascguaaCfGfAfuuuCfaugaaa(invdT)







AM03774-SS
(Alk-C6-SMPT-C6)ascguaaCfgAfuuuCfaugaaa(invdT)
174
51





AM03829-SS
(Alk-PEGS-C6)uAuascguaaCfgAfuuuCfaugaaa(invdT)
184
72





AM03830-SS
(Chol-TEG)uAuascguaaCfgAfuuuCfaugaaa(invdT)
227
72





AM03831-SS
ascguaaCfgAfuuuCfaugaaa(NAG13)
275
50





AM04003-SS
(Alk-SMPT-C6)asgUfaCfaCfAfAfuUfgUfuuuAfca(invdT)
209
56





AM04006-SS
(Alk-SMPT-C6)asgUfaCfaCfAfAfuUfgUfuuuAfcu(invdT)
210
57





AM04009-SS
(Alk-SMPT-C6)asaAfgUfaCfaCfAfAfuUfgUfuuuAfc(invdA)
195
43





AM04012-SS
(Alk-SMPT-C6)asaAfgUfaCfaCfAfAfuUfgUfuuuAfc(invdT)
196
44





AM04014-SS
(Alk-SMPT-C6)asaaguaCfaCfAfAfuuguuuuac(invdT)
198
44





AM04017-SS
(Alk-SMPT-C6)asaagUfaCfaCfAfAfuUfgUfuuuac(invdT)
199
44





AM04020-SS
(Alk-SMPT-C6)asaaguacaCfAfAfuuguuuuac(invdT)
197
44





AM04107-SS
(Alk-C6-SMPT-C6)ascguaaCfGfAfuuucaugaaa(invdT)
175
51





AM04107-SS
(Alk-C6-SMPT-C6)ascguaaCfGfAfuuucaugaaa(invdT)
175
51





AM04109-SS
(Alk-C6-SMPT-C6)ascguaaCfGfAfuuucaugaAMTM(invdT)
176
52





AM04111-SS
(Alk-C6-SMPT-C6)AMsCMguaaCfGfAfuuucaugaAMTM(invdT)
171
52





AM04113-SS
(Alk-C6-SMPT-C6)ascguaaCfGfAfuuucaugaasus(invdT)
178
54





AM04115-SS
(Alk-C6-SMPT-C6)ascguaaCfGfAfuuucaugaasus(invAb)
177
53





AM04117-SS
(Alk-C6-SMPT-C6)ascguaaCfGfAfuuucaugaau(invdT)
179
54





AM04119-SS
(Alk-C6-SMPT-C6)asacguaaCfGfAfuuucaugaau(invdT)
173
47





AM04121-SS
(Alk-C6-SMPT-C6)asacguaaCfGfAfuuucaugaaa(invdT)
172
46





AM04122-SS
(Alk-PEGS-C6)ascguaaCfGfAfuuucaugaaa(invdT)
181
51





AM04241-SS
(Alk-SMPT-C6)ascguaaCfGfAfuuucaugaaAM(invdT)
206
51





AM04243-SS
(Alk-SMPT-C6)ascguaaCfGfAfuuucaugaaAMs(invdT)
207
51





AM04246-SS
(Alk-PEG5-C6)uAuascguaaCfGfAfuuucaugaaAMs(invdT)
186
72





AM04248-SS
(Alk-PEG5-C6)uauascguaaCfGfAfuuucaugaaAMs(invdT)
187
72





AM04451-SS
(Alk-PEG5-C6)uAuascguaaCfGfAfuuucaugaaudTs(invdT)
188
74





AM04454-SS
(Alk-PEG5-C6)uAucsasacguAfaCfGfAfuuucaugAfaudTs(invdT)
192
77





AM04457-SS
(Alk-PEG5-C6)uAucsaacguAfaCfGfAfuuucaugAfaudTs(invdT)
191
77





AM03710-SS
(Alk-PEG5-C6)uAuascguaaCfGfAfuuuCfaugaaa(invdT)
189
72
















TABLE 3







Hif2α RNAi trigger duplexes with Duplex ID numbers.















SEQ
Unmod.

SEQ
Unmod.


Duplex
Antisense
ID
SEQ
Sense Strand
ID
SEQ


ID
Strand ID
NO.
ID NO.
ID
NO.
ID NO.
















AD00086
AM00159-AS
112
21
AM00188-SS
265
79


AD00087
AM00160-AS
113
22
AM00189-SS
264
70


AD00088
AM00161-AS
106
17
AM00190-SS
263
65


AD00089
AM00162-AS
103
13
AM00191-SS
262
62


AD00102
AM00163-AS
110
19
AM00207-SS
258
66


AD00103
AM00164-AS
104
14
AM00208-SS
260
69


AD00104
AM00165-AS
111
20
AM00209-SS
256
64


AD00105
AM00166-AS
102
12
AM00210-SS
259
68


AD00106
AM00167-AS
137
27
AM00211-SS
253
61


AD00107
AM00168-AS
101
11
AM00212-SS
247
45


AD00108
AM00169-AS
135
24
AM00213-SS
248
48


AD00109
AM00170-AS
136
25
AM00214-SS
266
78


AD00110
AM00171-AS
105
15
AM00219-SS
249
49


AD00111
AM00172-AS
100
10
AM00220-SS
255
63


AD00112
AM00159-AS
112
21
AM00221-SS
267
79


AD00113
AM00160-AS
113
22
AM00222-SS
261
70


AD00114
AM00161-AS
106
17
AM00223-SS
257
65


AD00115
AM00162-AS
103
13
AM00224-SS
254
62


AD00215
AM00163-AS
110
19
AM00369-SS
222
68


AD00268
AM00166-AS
102
12
AM00369-SS
222
68


AD00269
AM00169-AS
135
24
AM00530-SS
212
48


AD00270
AM00170-AS
136
25
AM00531-SS
224
78


AD00274
AM00168-AS
101
11
AM00543-SS
211
45


AD00275
AM00171-AS
105
15
AM00544-SS
213
49


AD00276
AM00161-AS
106
17
AM00545-SS
219
65


AD00285
AM00163-AS
110
19
AM00366-SS
220
66


AD00286
AM00164-AS
104
14
AM00367-SS
223
69


AD00373
AM00169-AS
135
24
AM00659-SS
212
48


AD00374
AM00170-AS
136
25
AM00660-SS
224
78


AD00375
AM00168-AS
101
11
AM00679-SS
211
45


AD00376
AM00171-AS
105
15
AM00661-SS
213
49


AD00377
AM00161-AS
106
17
AM00662-SS
219
65


AD00988
AM01772-AS
116
23
AM01771-SS
271
56


AD00989
AM01777-AS
109
18
AM01776-SS
284
82


AD00990
AM01782-AS
96
7
AM00158-SS
277
67


AD00991
AM01786-AS
145
30
AM01785-SS
269
51


AD00992
AM01791-AS
99
9
AM01790-SS
283
81


AD00993
AM01773-AS
115
23
AM01771-SS
271
56


AD00994
AM01778-AS
108
18
AM01776-SS
284
82


AD00995
AM01783-AS
95
7
AM00158-SS
277
67


AD00996
AM01787-AS
144
30
AM01785-SS
269
51


AD00997
AM01792-AS
98
9
AM01790-SS
283
81


AD01020
AM01772-AS
116
23
AM01859-SS
218
56


AD01021
AM01773-AS
115
23
AM01859-SS
218
56


AD01022
AM01770-AS
114
23
AM01859-SS
218
56


AD01023
AM01777-AS
109
18
AM01860-SS
226
82


AD01024
AM01778-AS
108
18
AM01860-SS
226
82


AD01025
AM01775-AS
107
18
AM01860-SS
226
82


AD01026
AM01782-AS
96
7
AM01861-SS
221
67


AD01027
AM01783-AS
95
7
AM01861-SS
221
67


AD01028
AM01780-AS
94
7
AM01861-SS
221
67


AD01029
AM01786-AS
145
30
AM01862-SS
215
51


AD01030
AM01787-AS
144
30
AM01862-SS
215
51


AD01031
AM01784-AS
138
30
AM01862-SS
215
51


AD01032
AM01791-AS
99
9
AM01863-SS
225
81


AD01033
AM01792-AS
98
9
AM01863-SS
225
81


AD01034
AM01789-AS
97
9
AM01863-SS
225
81


AD01180
AM00171-AS
105
15
AM01994-SS
169
71


AD01214
AM01784-AS
138
30
AM02043-SS
231
51


AD01255
AM01784-AS
138
30
AM02093-SS
228
72


AD01256
AM02090-AS
143
30
AM01862-SS
215
51


AD01257
AM02091-AS
142
30
AM01862-SS
215
51


AD01258
AM02092-AS
139
30
AM01862-SS
215
51


AD01288
AM02133-AS
141
30
AM02135-SS
216
51


AD01289
AM01784-AS
138
30
AM02137-SS
217
51


AD01290
AM01784-AS
138
30
AM02139-SS
214
51


AD01291
AM02140-AS
128
31
AM02142-SS
276
59


AD01292
AM02140-AS
128
31
AM02144-SS
278
59


AD01293
AM02145-AS
127
33
AM02149-SS
280
75


AD01294
AM02146-AS
123
29
AM02163-SS
282
76


AD01295
AM02147-AS
126
32
AM02149-SS
280
75


AD01296
AM02150-AS
125
34
AM02149-SS
280
75


AD01391
AM01784-AS
138
30
AM01785-SS
269
51


AD01392
AM02327-AS
147
30
AM01785-SS
269
51


AD01404
AM02341-AS
130
28
AM02364-SS
281
76


AD01405
AM02341-AS
130
28
AM02365-SS
279
76


AD01406
AM02342-AS
134
28
AM02364-SS
281
76


AD01407
AM02342-AS
134
28
AM02365-SS
279
76


AD01408
AM02345-AS
129
28
AM02364-SS
281
76


AD01409
AM02345-AS
129
28
AM02365-SS
279
76


AD01410
AM02346-AS
133
28
AM02364-SS
281
76


AD01411
AM02346-AS
133
28
AM02365-SS
279
76


AD01424
AM02345-AS
129
28
AM02363-SS
230
76


AD01476
AM01784-AS
138
30
AM02456-SS
193
51


AD01522
AM02133-AS
141
30
AM02510-SS
232
51


AD01523
AM02509-AS
140
30
AM02512-SS
233
51


AD01524
AM02508-AS
120
30
AM02043-SS
231
51


AD01525
AM02523-AS
117
8
AM02522-SS
250
55


AD01526
AM02525-AS
119
26
AM02524-SS
268
80


AD01527
AM02527-AS
118
16
AM02526-SS
252
60


AD01528
AM02529-AS
148
35
AM02528-SS
251
58


AD01546
AM02523-AS
117
8
AM02546-SS
235
55


AD01547
AM02525-AS
119
26
AM02547-SS
242
80


AD01548
AM02527-AS
118
16
AM02548-SS
241
60


AD01549
AM02529-AS
148
35
AM02549-SS
240
58


AD01554
AM02604-AS
122
30
AM02512-SS
233
51


AD01555
AM02605-AS
124
30
AM02510-SS
232
51


AD01654
AM02848-AS
132
30
AM02852-SS
272
50


AD01655
AM02848-AS
132
30
AM02512-SS
233
51


AD01656
AM02849-AS
131
30
AM02853-SS
273
50


AD01657
AM02849-AS
131
30
AM02856-SS
234
51


AD01658
AM02850-AS
121
30
AM02853-SS
273
50


AD01659
AM02850-AS
121
30
AM02856-SS
234
51


AD01884
AM02998-AS
160
40
AM02510-SS
232
51


AD01885
AM02998-AS
160
40
AM02999-SS
229
72


AD01886
AM03000-AS
151
36
AM03004-SS
244
82


AD01887
AM03001-AS
149
36
AM03005-SS
246
82


AD01888
AM03002-AS
152
36
AM03006-SS
243
82


AD01889
AM03003-AS
150
36
AM03007-SS
245
82


AD01890
AM03008-AS
154
37
AM03010-SS
236
56


AD01891
AM03008-AS
154
37
AM03011-SS
238
56


AD01892
AM03009-AS
153
37
AM03012-SS
237
56


AD01893
AM03009-AS
153
37
AM03013-SS
239
56


AD01910
AM02605-AS
124
30
AM03058-SS
270
51


AD01911
AM03059-AS
146
30
AM03058-SS
270
51


AD02073
AM03009-AS
153
37
AM03307-SS
274
56


AD02074
AM02605-AS
124
30
AM03308-SS
170
51


AD02691
AM03465-AS
161
41
AM03467-SS
194
51


AD02692
AM03465-AS
161
41
AM03469-SS
201
51


AD02693
AM03465-AS
161
41
AM03471-SS
208
51


AD02694
AM03465-AS
161
41
AM03473-SS
200
51


AD02695
AM03465-AS
161
41
AM03475-SS
204
51


AD02733
AM03513-AS
162
41
AM03516-SS
203
51


AD02734
AM03514-AS
165
41
AM03516-SS
203
51


AD02735
AM03517-AS
90
4
AM03519-SS
202
54


AD02857
AM03685-AS
159
41
AM03687-SS
205
51


AD02858
AM03688-AS
167
41
AM03687-SS
205
51


AD02859
AM03689-AS
163
41
AM03687-SS
205
51


AD02860
AM03690-AS
166
41
AM03687-SS
205
51


AD02861
AM03685-AS
159
41
AM03692-SS
185
72


AD02862
AM03517-AS
90
4
AM03694-SS
183
73


AD02873
AM03465-AS
161
41
AM03708-SS
182
72


AD02874
AM03465-AS
161
41
AM03710-SS
189
72


AD02875
AM03517-AS
90
4
AM03712-SS
190
73


AD02876
AM03465-AS
161
41
AM03714-SS
180
72


AD02949
AM03465-AS
161
41
AM03774-SS
174
51


AD03011
AM03465-AS
161
41
AM03829-SS
184
72


AD03012
AM03465-AS
161
41
AM03830-SS
227
72


AD03013
AM03465-AS
161
41
AM03831-SS
275
50


AD03187
AM04001-AS
155
38
AM04003-SS
209
56


AD03188
AM04004-AS
84
1
AM04006-SS
210
57


AD03189
AM04007-AS
156
39
AM04009-SS
195
43


AD03190
AM04010-AS
85
2
AM04012-SS
196
44


AD03191
AM04010-AS
85
2
AM04014-SS
198
44


AD03192
AM04015-AS
83
2
AM04017-SS
199
44


AD03193
AM04018-AS
86
2
AM04020-SS
197
44


AD03215
AM04040-AS
168
41
AM03469-SS
201
51


AD03216
AM04040-AS
168
41
AM03475-SS
204
51


AD03253
AM03689-AS
163
41
AM04107-SS
175
51


AD03254
AM04101-AS
91
4
AM04117-SS
179
54


AD03255
AM04102-AS
164
42
AM04121-SS
172
46


AD03256
AM04103-AS
92
5
AM04119-SS
173
47


AD03257
AM04104-AS
157
41
AM04107-SS
175
51


AD03258
AM04105-AS
88
4
AM04117-SS
179
54


AD03259
AM04106-AS
87
3
AM04109-SS
176
52


AD03260
AM04105-AS
88
4
AM04109-SS
176
52


AD03261
AM04105-AS
88
4
AM04111-SS
171
52


AD03262
AM04105-AS
88
4
AM04113-SS
178
54


AD03263
AM04105-AS
88
4
AM04115-SS
177
53


AD03264
AM03690-AS
166
41
AM04107-SS
175
51


AD03265
AM03689-AS
163
41
AM03692-SS
185
72


AD03266
AM03689-AS
163
41
AM04122-SS
181
51


AD03345
AM03689-AS
163
41
AM04241-SS
206
51


AD03346
AM03689-AS
163
41
AM04243-SS
207
51


AD03347
AM04244-AS
158
41
AM04243-SS
207
51


AD03348
AM03689-AS
163
41
AM04246-SS
186
72


AD03349
AM03689-AS
163
41
AM04248-SS
187
72


AD03505
AM04101-AS
91
4
AM04451-SS
188
74


AD03506
AM04452-AS
93
6
AM04454-SS
192
77


AD03507
AM04455-AS
89
6
AM04454-SS
192
77


AD03508
AM04452-AS
93
6
AM04457-SS
191
77









A sense strand containing a sequence listed in Table 2B can be hybridized to any antisense strand containing a sequence listed in Table 2A provided the two sequences have a region of at least 90% complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence. Representative Hif2α RNA triggers are represented by the Duplex ID Nos. shown in Table 3. In some embodiments an Hif2α RNAi trigger consists of any of the Duplex ID Nos. presented herein. In some embodiments an Hif2α RNAi trigger comprises of any of the Duplex ID Nos. presented herein. In some embodiments, an Hif2α RNAi trigger comprises the sense strand and antisense strand nucleotide sequences of any of the Duplex ID Nos. presented herein. In some embodiments, an Hif2α RNAi trigger comprises the sense strand and antisense strand nucleotide sequences of any of the Duplex ID Nos. presented herein and a targeting group and/or linking group wherein the targeting group and/or linking group is covalently linked to the sense strand or the antisense strand. In some embodiments, an Hif2α RNAi trigger comprises the sense strand and antisense strand modified nucleotide sequences of any of the Duplex ID Nos. presented herein. In some embodiments, an Hif2α RNAi trigger comprises the sense strand and antisense strand modified nucleotide sequences of any of the Duplex ID Nos. presented herein and a targeting group and/or linking group wherein the targeting group and/or linking group is covalently linked to the sense strand or the antisense strand.


In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising a nucleotide base sequence of nucleotides 2-21 of SEQ ID NO. 4. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising a nucleotide base sequence of nucleotides 2-21 of SEQ ID NO. 4 and a sense strand comprising a nucleotide base sequence of SEQ ID NO. 53.


In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising a nucleotide base sequence of SEQ ID NO. 88, SEQ ID NO. 157, SEQ ID NO. 159, or SEQ ID NO. 163. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising a nucleotide base sequence of SEQ ID NO. 88 and a sense strand comprising a nucleotide base sequence of SEQ ID NO. 179. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising a nucleotide base sequence of SEQ ID NO. 88 and a sense strand comprising a nucleotide base sequence of SEQ ID NO. 177. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising a nucleotide base sequence of SEQ ID NO. 157 and a sense strand comprising a nucleotide base sequence of SEQ ID NO. 175. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising a nucleotide base sequence of SEQ ID NO. 159 and a sense strand comprising a nucleotide base sequence of SEQ ID NO. 185. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising a nucleotide base sequence of SEQ ID NO. 163 and a sense strand comprising a nucleotide base sequence of SEQ ID NO. 185.


In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising SEQ ID NO. 88, SEQ ID NO. 157, SEQ ID NO. 159, or SEQ ID NO. 163. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising SEQ ID NO. 88 and a sense strand comprising SEQ ID NO. 179. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising SEQ ID NO. 88 and a sense strand comprising SEQ ID NO. 177. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising SEQ ID NO. 157 and a sense strand comprising SEQ ID NO. 175. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising SEQ ID NO. 159 and a sense strand comprising SEQ ID NO. 185. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising SEQ ID NO. 163 and a sense strand comprising SEQ ID NO. 185.


In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising a nucleotide base sequence of nucleotides 2-21 of SEQ ID NO. 38. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising a nucleotide base sequence of nucleotides 2-21 of SEQ ID NO. 38 and a sense strand comprising a nucleotide base sequence of nucleotides 1-19 of SEQ ID NO. 56.


In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising a nucleotide base sequence of SEQ ID NO. 86, SEQ ID NO. 155, SEQ ID NO. 156. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising a nucleotide base sequence of SEQ ID NO. 156 and a sense strand comprising a nucleotide base sequence of SEQ ID NO. 195. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising a nucleotide base sequence of SEQ ID NO. 86 and a sense strand comprising a nucleotide base sequence of SEQ ID NO. 197. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising a nucleotide base sequence of SEQ ID NO. 155 and a sense strand comprising a nucleotide base sequence of SEQ ID NO. 209.


In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising SEQ ID NO. 86, SEQ ID NO. 155, SEQ ID NO. 156. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising SEQ ID NO. 156 and a sense strand comprising SEQ ID NO. 195. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising SEQ ID NO. 86 and a sense strand comprising SEQ ID NO. 197. In some embodiments, a Hif2α RNAi trigger comprises an antisense strand comprising SEQ ID NO. 155 and a sense strand comprising SEQ ID NO. 209.


In some embodiments, an Hif2α RNAi trigger contains or is conjugated to a targeting group, linking group, delivery polymer, delivery vehicle, and/or other non-nucleotide group. The targeting group, linking group, delivery polymer, delivery vehicle, and/or other non-nucleotide group can be covalently linked to the 3′ and/or 5′ end of either the sense strand and/or the antisense strand. In some embodiments, an Hif2α RNAi trigger can contains a targeting group, linking group, delivery polymer, delivery vehicle, or other non-nucleotide group linked to the 3′ and/or 5′ end of the sense strand. In some embodiments a targeting group, linking group, delivery polymer, delivery vehicle, or other non-nucleotide group is linked to the 5′ end of an Hif2α RNAi trigger sense strand. In some embodiments, the targeting group, linking group, delivery polymer, delivery vehicle, and/or other non-nucleotide group is linked directly or indirectly to the trigger via a linker/linking group. In some embodiments, targeting group, linking group, delivery polymer, delivery vehicle, and/or other non-nucleotide group is linked to the trigger via a labile, cleavable, or reversible bond or linker.


A targeting group can enhance the pharmacokinetic or biodistribution properties of an RNAi trigger or conjugate to which it is attached to improve cell- or tissue-specific distribution and cell-specific uptake of the conjugate. In some instances, binding of a targeting group to a cell or cell receptor may initiate endocytosis. A targeting group can be monovalent, divalent, trivalent, tetravalent, or have higher valency. Representative targeting groups include, without limitation, compounds with affinity to cell surface molecule, cell receptor ligands, hapten, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics with affinity to cell surface molecules.


The RNAi trigger molecules described herein may be synthesized having a reactive group, such as an amine group, at the 5′-terminus. The reactive group may be used to subsequently attach a targeting moiety using methods typical in the art.


In some embodiments, an Hif2α RNAi trigger includes a linking group conjugated to the trigger. The linking group facilitates covalent linkage of the trigger to a targeting group or delivery polymer or delivery vehicle. The linking group can be linked to the 3′ or the 5′ end of the RNAi trigger sense strand or antisense strand. In some embodiments, the linking group is linked to the RNAi trigger sense strand. In some embodiments, the linking group is conjugated to the 5′ or 3′ end of an RNAi trigger sense strand. In some embodiments a linking group is conjugated to the 5′ end of an RNAi trigger sense strand. Examples of linking groups, include, but are not limited to: Alk-SMPT-C6, Alk-SS-C6, DBCO-TEG, Me-Alk-SS-C6, and C6-SS-Alk-Me, reactive groups such a primary amines and alkynes, alkyl groups, abasic ribose, ribitol, and/or PEG groups.


A linker or linking group is a connection between two atoms that links one chemical group (such as an RNAi trigger) or segment of interest to another chemical group (such as a targeting group or delivery polymer) or segment of interest via one or more covalent bonds. A labile linkage contains a labile bond. A linkage may optionally include a spacer that increases the distance between the two joined atoms. A spacer may further add flexibility and/or length to the linkage. Spacers may include, but are not be limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art and the preceding list is not meant to limit the scope of the description.


Targeting groups and linking groups include, but are not limited to, (Alk-C6), (Alk-C6-C6), (Alk-C6-SMPT-C6), (Alk-PEG5-C6), (Alk-PEG5-C6)(Alk-PEG5-Ser), (Alk-SMPT-C6), (Alk-SS-C6), (C6-SS-Alk-Me), (Chol-TEG), (DBCO-TEG), (Me-Alk-SS-C6), (NAG13), (NH2-C6). In some embodiments, any of the Hif2α RNAi trigger sense strands listed in Table 2B which contains a 3′ or 5′ targeting group or linking group, may alternatively contain no 3′ or 5′ targeting group or linking group, or may contain a different 3′ or 5′ targeting group or linking group including, but not limited to, those depicted in Table 4.


In some of the targeting group and linking group structures shown in Table 4, the RNAi trigger is shown and denoted by Trigger, RNA, R, or R1 or R2 (i.e. Trigger, RNA or R1 or R2 each comprises the RNAi trigger). For example, with respect to (Alk-C6-Ser), (Alk-PEG5-Ser), and (Alk-PEG13-Ser), one of R1 and R2 comprises the RNAi trigger and the other can be a hydrogen.









TABLE 4





Structures representing targeting groups and linking groups.









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In some embodiments, a delivery vehicle may be used to deliver an RNAi trigger to a cell or tissue. A delivery vehicle is a compound that improves delivery of the RNAi trigger to a cell or tissue. A delivery vehicle can include, or consist of, but is not limited to: a polymer, such as an amphipathic polymer, a membrane active polymer, a peptide, a melittin peptide, a melittin-like peptide, a lipid, a reversibly modified polymer or peptide, or a reversibly modified membrane active polyamine.


In some embodiments, the RNAi triggers can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs or other delivery systems available in the art. The RNAi triggers can also be chemically conjugated to targeting groups, lipids (including, but not limited to cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, for example WO 2000/053722, WO 2008/0022309, WO 2011/104169, and WO 2012/083185, WO 2013/032829, WO 2013/158141, each of which is incorporated herein by reference), or other delivery systems available in the art.


In some embodiments, pharmaceutical compositions for delivering an Hif2α RNAi trigger to a tumor cell in vivo are described. Such pharmaceutical compositions can include, but are not limited to, an Hif2α RNAi trigger conjugated to delivery polymer to form an RNAi trigger-delivery polymer conjugate. In some embodiments, the delivery polymer is a membrane active polyamine. In some embodiments, the delivery polymer is a reversibly modified membrane active polyamine.


Hif2α RNAi Trigger-Delivery Polymer Conjugates

In some embodiments, we describe compositions represented by the formula:




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wherein RNAi trigger is an Hif2α RNAi trigger as described herein, P is a membrane active polyamine, M1 comprises a targeting group linked to P via reversible physiologically labile linkage L1, and M2 comprises a steric stabilizer linked to P via reversible physiologically labile linkage L2, x is greater than 1, y is greater than or equal to 0. (M2-L2)y-P-(L1-M1)x, is not membrane active. As used herein, (M2-L2)y-P-(L1-M1)x refers to a delivery polymer. Cleavage of (L1-M1) and (M2-L2) restores P to a membrane active state. In some embodiments, the value of x+y is greater than 80%, greater than 90%, or greater than 95% of the number of primary amines of P. In some embodiments, the value of x+y is greater than 80%, greater than 90%, or greater than 95% of the number of primary amines on a population of P. The value of n can be from 0.25 to 5 (one (1) RNAi trigger per every 4 polymers to 5 RNAi triggers per polymer). In some embodiments, the value of n is 0.5 to 5. In some embodiments, n is 0.5-2. In some embodiments, n is 0.8-1.6. In some embodiments, x is 1-20, 2-20, 3-20, 4-20, 5-20, 6-20,7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, or 15-20.


In some embodiments, M1 comprises an integrin-binding compound. In some embodiments, the integrin-binding compound comprises an αvβ3-binding compound. In some embodiments, the integrin-binding compound comprises an RGD ligand. In some embodiments, the αvβ3-binding compound comprises an RGD ligand. In some embodiments the RGD ligand comprises an RGD mimic. In some embodiments, the steric stabilizer comprises a polyethylene glycol (PEG). In some embodiments, cleavage of L1 and/or L2 restores an unmodified amine on P. In some embodiments, (L1-M1) and (L2-M2) are independently tetrapeptide modifying agents and/or dipeptide modifying agents. In some embodiments, L1 and L2 are independently tetrapeptide linkages or dipeptide-PABC (p-amidobenzyl-carbamate) linkages. In some embodiments, L1 and L2 are tetrapeptide linkages. In other embodiments, L1 and L2 are dipeptide-PABC linkages. In some embodiments, L1 is a dipeptide-PABC linkage and L2 is a tetrapeptide linkage. In other embodiments, L1 is a tetrapeptide linkage and L2 is a dipeptide-PABC linkage. In some embodiments, a tetrapeptide linkage is an FCitFP (Phenylalanine-Citrulline-Phenylalanine-Proline) tetrapeptide linkage. In some embodiments, a dipeptide-PABC linkage is an ACit-PABC linkage. For x=2 or more, L can be all tetrapeptide linkages, all dipeptide-PABC linkages, or a combination tetrapeptide linkages and dipeptide-PABC linkages. For y=2 or more, L2 and be all tetrapeptide linkages, all dipeptide-PABC linkages, or a combination tetrapeptide linkages and dipeptide-PABC linkages.


In some embodiments, a described Hif2α RNAi trigger is conjugated to a reversibly modified membrane active polyamine to form an RNAi trigger-delivery polymer conjugate. In some embodiments, the RNAi trigger-delivery polymer conjugate comprises the formula represented by:





(RNAi trigger)n-poly(Aa-co-(Bb-graft-(Cc;Dd)))  (formula 2)

    • wherein
    • A is a hydrophobic group-containing monomeric unit,
    • B is a primary amine-containing monomeric unit,
    • C comprises an integrin-binding ligand linked (i.e., grafted) to a primary amine-containing monomeric unit via a reversible physiologically labile linkage,
    • D comprises a steric stabilizer linked (i.e., grafted) to a primary amine-containing monomeric unit via a reversible physiologically labile linkage,
    • a is an integer greater than zero,
    • b is an integer greater than or equal to two,
    • c is an integer greater than or equal to one,
    • d is an integer greater than or equal to one,
    • the value of c+d is greater than 80%, greater than 85%, greater than 90%, or greater than 95% of the value of b,
    • poly(Aa-co-Bb) is a membrane active polyamine copolymer having A and B monomeric units RNAi trigger comprises a Hif2α RNAi trigger described herein, and n has a value from 0.25 (i.e., conjugated to only one out of every four delivery polymers) to 5.0.


Poly(Aa-c(Bb-graft-(Cc; Dd))) is not membrane active. In some embodiments, the integrin-binding compound comprises an αvβ3-binding compound. In some embodiments, the integrin-binding compound comprises an RGD ligand, such as an RGD mimic. In some embodiments, the αvβ3-binding compound comprises an RGD ligand, such as an RGD mimic. In some embodiments, the steric stabilizer comprises a polyethylene glycol (PEG). In some embodiments, the PEG contains 2 to 25 ethylene glycol units. In some embodiments, c is any integer from 1-75, 1-50, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, 10-35, 10-30, 10-25, 10-20, or 15-20. In some embodiments, n has a value from 0.5 to 2. In some embodiments, the ratio A:B (i.e., a:b) is 30:70 to 60:40. In some embodiments, the ratio A:B is 60:40 to 40:60. In some embodiments, the ratio A:B is about 45+5:55+5. In some embodiments, the ratio A:B is about 44:56. In some embodiments, the ratio A:B is about 46:54. In some embodiments, the molecular weight (Mw) of the polymer is 30 kDa-70 kDa. In other embodiments, the Mw of the polymer is 40 kDa-60 kDa. In other embodiments, the Mw of the polymer is 40 kDa-50 kDa. In yet other embodiments, the Mw of the polymer about 43 kDa to about 48 kDa. In some embodiments, the polymer has a polydispersity index (PDI) less than 1.4, less than 1.3, 1.25, less than 1.2, less than 1.15, or less than 1.1. In some embodiments, the polymer contains a terminal azide group for attachment of an RNAi trigger. In some embodiments, n is 0.8-1.6. In some embodiments, n is 1+0.5. In some embodiments, c is 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, or 15-20. In some embodiments, the value of c+d is greater than 80%, greater than 90%, or greater than 95% of the value of b. In some embodiments, C is RGD-PEGx-FcitFPro and D is PEGy-ACit-PABC, wherein x is 1-50, y is 4-30. In some embodiments, x is greater than y.


In some embodiments, polyamine poly(Aa-co-Bb) is a poly(acrylate) random copolymer wherein A is a hydrophobic group-containing acrylate monomer and B is a primary amine-containing acrylate monomer. In some embodiments A is a propyl acrylate monomer and B is an ethoxy-ethylamine acrylate monomer.


Membrane active polyamines are membrane active and therefore capable of disrupting plasma membranes or lysosomal/endocytic membranes. As used herein, membrane active polyamines are surface active, amphipathic polymers that are able to induce one or more of the following effects upon a biological membrane: an alteration or disruption of the membrane that allows non-membrane permeable molecules to enter a cell or cross the membrane, pore formation in the membrane, fission of membranes, or disruption or dissolving of the membrane. As used herein, a membrane, or cell membrane, comprises a lipid bilayer. The alteration or disruption of the membrane can be functionally defined by the peptide's activity in at least one the following assays: red blood cell lysis (hemolysis), liposome leakage, liposome fusion, cell fusion, cell lysis, and endosomal release. Peptides, or modified peptides that preferentially cause disruption of endosomes or lysosomes over plasma membranes are considered endosomolytic. A reversibly modified membrane active polyamine is an example of an endosomolytic peptide. The effect of membrane active polymers on a cell membrane may be transient. Membrane active polymers possess affinity for the membrane and cause a denaturation or deformation of bilayer structures. Delivery of a RNAi trigger to a cell is mediated by the membrane active polyamine disrupting or destabilizing the plasma membrane or an internal vesicle membrane (such as an endosome or lysosome), including forming a pore in the membrane, or disrupting endosomal or lysosomal vesicles thereby permitting release of the contents of the vesicle into the cell cytoplasm. A preferred polymer is an amphipathic poly(acrylate) random copolymer.


Integrin-Binding Compound

An integrin-binding compound has affinity for one or more integrins expressed on a cell surface. A non-limiting example of an integrin includes an αvβ3 integrin. Examples of integrin-binding compounds include, but are not limited to: αvβ3-binding compounds, RGD ligand. RGD ligands include RGD peptide-containing compounds and RGD mimic-containing compounds. As used herein, an RGD peptide comprises an arginine-glycine-aspartate tripeptide. An RGD peptide may be conformationally constrained. An RGD peptide may have non-peptide components linked to the RGD amino acid sequence.


As used herein, an RGD ligand comprises an RGD peptide or RGD mimic<1500 kDa in size that binds to (has affinity for) an integrin, such as an alpha v/beta 3 (αvβ3 or αvβ3) integrin.


As used herein, an RGD mimic is a non-peptide synthetic molecule other than an RDG peptide that biologically mimics the active determinants of an RGD peptide, an integrin-binding RGD portion of an integrin-binding protein, or an αvβ3 integrin-binding RGD motif. An RGD mimic may contain one or two naturally occurring amino acids linked via amide bonds. An RGD mimetic may be a modified peptide, contain non-standard amino acids or non-standard amino acid side chains.


In one embodiment, an RGD ligand comprises a guanidinium group linked to a glycine-aspartate dipeptide via an amide bond. Guanidinium groups of the invention have the structure represented by:




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wherein R9 and R10 are independently hydrogen or alkyl and may by connected to form a ring, and R11 is a linker connecting the guanidinium group to the glycine-aspartate dipeptide. The guanidinium group includes both the structure represented above and its resonance structures. A preferred linker is: —(CRR′)—(CRR′HCRR′)— or —(CRR′)—(CRR′)—(CRR′)—(CRR′)—, wherein: a) each R is independently optional and if present is independently hydrogen, alkyl, or aryl, b) R′ is independently hydrogen, alkyl, aryl, or NH2, and c) each carbon (C) may be linked by single bonds, a single bond and a double bond, or aromatic bonds.


In some embodiments, an RGD mimic contains a phenoxy group attached to the aspartate amino acid. In some embodiments, an RGD mimic comprises a quanidinium-glycine-aspartate-4-aminophenoxy compound. In some embodiments, a quanidinium-glycine-aspartate-4-aminophenoxy compound comprises the structure represented by:




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wherein RD is:




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In some embodiments, a guanidinium is




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and their resonance structures.


In some embodiments, an RGD mimic comprises the structure represented by:




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    • wherein:
      • R14 is







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and


A comprises a linker. The linker connects the RGD mimic to another molecule such as a dipeptide amidobenzyl-carbonate or tetrapeptide, provides for increased solubility, or provides a means for covalent linkage to another molecule.


Steric Stabilizer

As used herein, a steric stabilizer is a non-ionic hydrophilic polymer (either natural, synthetic, or non-natural) that prevents or inhibits intramolecular or intermolecular interactions of a polymer to which it is attached relative to the polymer containing no steric stabilizer. A steric stabilizer hinders a polymer to which it is attached from engaging in electrostatic interactions. Electrostatic interaction is the non-covalent association of two or more substances due to attractive forces between positive and negative charges. Steric stabilizers can inhibit interaction with blood components and therefore opsonization, phagocytosis, and uptake by the reticuloendothelial system. Steric stabilizers can thus increase circulation time of molecules to which they are attached. Steric stabilizers can also inhibit aggregation of a polymer. In some embodiments, a steric stabilizer is a polyethylene glycol (PEG) or PEG derivative. In some embodiments, a PEG can have about 1-500 ethylene monomers or units. In some embodiments, the PEG contains 2-25 ethylene units. In some embodiments, the PEG contains 4-30 ethylene units. In some embodiments, PEG contains 5-24 ethylene units. In some embodiments, a PEG has a molecular weight average of about 85-20,000 Daltons (Da). In some embodiments a PEG has a molecular weight of about 85-1000 Da. As used herein, steric stabilizers prevent or inhibit intramolecular or intermolecular interactions of a polymer to which it is attached relative to the polymer containing no steric stabilizer in aqueous solution.


Reversible Physiologically Labile Linkages/Modifying Agents

A membrane active polyamine may be reversibly modified. Reversible modification can be accomplished through reversible attachment of modifying agents to primary amines of the membrane active polyamine.


In some embodiments, a reversible physiologically labile linkage comprises a tetrapeptide linkage. In some embodiments, P-(L1-M1)x and/or P-(L2-M2)y (of formula 1) comprises:





R5-A4-A3-A2-A1-R6


wherein

    • R5 comprises a targeting group (M) or a steric stabilizer (M2),
    • A4 is a natural, non-natural isomeric, or synthetic hydrophobic L amino acid wherein the hydrophobicity index (Monera et al, J. Protein Sci. 1995, 1, 319) at pH 7 is 41 or greater, normalized to glycine, as it relates to the composition of the amino acid side chain (R-group),
    • A3 is an uncharged hydrophilic L amino acid wherein the hydrophobicity index (Monera et al, J. Protein Sci. 1995, 1, 319) at pH 7 is −28 or less, normalized to glycine, as it relates to the composition of amino acid side chain (R-group),
    • A2 is a natural, non-natural isomeric, or synthetic hydrophobic L amino acid wherein the hydrophobicity index (Monera et al, J. Protein Sci. 1995, 1, 319) at pH 7 is 41 or greater, normalized to glycine, as it relates to the composition of the amino acid side chain (R-group),
    • A1 is L-proline, L-leucine, or L-N-methyl alanine, and
    • R6 is P, wherein P is a membrane active polyamine of formula 1.


In some embodiments, A1 is proline, A2 and A4 are independently alanine, valine, leucine, isoleucine or phenylalanine (side chains of —CH3, —CH(CH3)2, —CH2CH(CH3)2, —CH(CH3)CH2CH3, or —CH2C6H6, respectively), and A3 is citrulline or asparagine (side chains or —(CH2)3NHCONH2 or —CH2CONH2, respectively).


In some embodiments, A1 is proline, A2 and A4 are phenylalanine, and A3 is citrulline (FCitFPro). In some embodiments, A1 is proline, A2 is phenylalanine, and A3 is citrulline, and A4 is alanine (ACitFPro).


In some embodiments, a tetrapeptide modifying agent has the structure represented by:




embedded image




    • wherein,

    • R5 comprises a targeting group (M) or a steric stabilizer (M2),

    • R4 is a side chain of a natural, non-natural isomeric, or synthetic hydrophobic amino acid,

    • R3 is a side chain of an uncharged hydrophilic amino acid, preferably citrulline,

    • R2 is a side chain of a natural, non-natural isomeric, or synthetic hydrophobic amino acid, preferably phenylalanine,

    • X and Y are:
      • a) (CH2)2(CH3)2 and H, respectively (tetrapeptide A′ is Leucine),
      • b) CH3— and CH3—, respectively (tetrapeptide A′ is N-methyl alanine), or
      • c) CH2— and CH2—CH2—, respectively (tetrapeptide A′ is proline); and

    • R is







embedded image


Reaction of the tetrapeptide modifying agent with a polyamine yields P-(L-M).


In some embodiments, R4 is a side chain of phenylalanine or alanine. In some embodiments, R3 is a side chain of citrulline. In some embodiments, R2 is a side chain of phenylalanine.


In some embodiments, the membrane active polyamine is modified with dipeptide modifying agents (dipeptide-PABC-PNP modifying agent) having the general form:





R-A1A2-amidobenzyl-carbonate.


wherein R comprises a steric stabilizer or targeting group, A1 is a hydrophobic amino acid, and A2 is a hydrophilic uncharged amino acid. Reaction of the modifying agent carbonate with a polymer amine yields a carbamate linkage. In some embodiments, the amidobenzyl group is a p-amidobenzyl group. In some embodiments, the carbonate is an activated amine reactive carbonate. In some embodiments, dipeptide-PABC cleavable linkers have the general structure:




embedded image


wherein R4 comprises a targeting group or steric stabilizer, R3 comprises an amine reactive carbonate moiety, such as a para-nitrophenyl group, R1 is the side chain of a hydrophobic amino acid, such as Phenylalanine or Alanine and R2 is the side chain of a hydrophilic uncharged are amino acid, such as citrulline (Cit). In some embodiments, R1 is the side chain of Phenylalanine or Alanine. In some embodiments, R2 is the side chain of citrulline (Cit).


In some embodiments, an RGD modifying agent comprises the structure represented by:




embedded image


wherein R14 is a guanidinium-containing group as defined above, A1 comprises a PEG-containing linker, R1 is a side chain of a Phenylalanine or Alanine, R2 is a side chain of citrulline, and R3 is an amine-reactive carbonate.


A delivery polymer can include a polyamine reversibly modified by reaction of primary amines on the polymer with a disubstituted alkylmaleic anhydride:




embedded image


wherein R1 comprises a targeting group or a steric stabilizer.


In some embodiments, the disubstituted alkylmaleic anhydride has the structure represented by:




embedded image


wherein R1 comprises an targeting group or a steric stabilizer.


In some embodiments, a targeting group (e.g., RGD ligand) is linked to a modifying agent via a linker, such as a PEG linker. The PEG linker can have 1-50 ethylene units.


RGD and PEG modifying agents are shown in FIGS. 1-7.


In some embodiments, we describe compositions represented by the formula:




embedded image


wherein: RNAi trigger is an Hif2α RNAi trigger, n is 0.5-5, P is a membrane active polyamine, L1-M1 comprises RGD-PEGa-FCitFPro-, a is 1-50, x is 1-20, L2-M2 comprises PEGb-ACit-PABC-, b is 4-30, and y is greater than or equal to 0, and (M2-L2)y-P-(L-M). is not membrane active. In some embodiments, the value of x+y is greater than 80%, greater than 90%, or greater than 95% of the number of primary amines of P. In some embodiments, n is 0.5-2. In some embodiments, n is 0.8-1.6. In some embodiments, x is 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, or 15-20. In some embodiments, the value of a is greater than the value of b.


Pharmaceutical Compositions

In some embodiments, at least one of the described Hif2α RNAi triggers is used in the preparation of a pharmaceutical composition (i.e., medicament) for treatment of a subject that would benefit from reduction or inhibition in Hif2α expression. These pharmaceutical compositions are useful in the inhibition of the expression of the Hif2α gene in a cell, a tissue, or an organism. In some embodiments, the described pharmaceutical compositions are used to treat a subject having a disease or disorder that would benefit from reduction or inhibition in Hif2α expression.


As used herein, a pharmaceutical composition or medicament comprises a pharmacologically effective amount of at least one of the described Hif2α RNAi triggers or Hif2α RNAi trigger-containing conjugates and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the Active Pharmaceutical ingredient (API, therapeutic product, e.g., RNAi trigger) that have been appropriately evaluated for safety and are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients may act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.


Excipients include, but are not limited to: absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.


A pharmaceutical composition can contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to: anti-pruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine, etc.). It is also envisioned that cells, tissues or isolated organs that express or comprise the herein defined RNAi triggers may be used as “pharmaceutical compositions”. As used herein, “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to that amount of an RNAi trigger to produce the intended pharmacological, therapeutic or preventive result.


In some embodiments, a described Hif2α RNAi trigger is combined one or more additional therapeutics or treatments including, but not limited to: a second Hif2α RNAi trigger or other RNAi agent, a small molecule drug, an antibody, an antibody fragment, and/or a vaccine.


The described RNAi triggers and pharmaceutical compositions comprising Hif2α RNAi triggers disclosed herein may be packaged or included in a kit, container, pack, or dispenser. The Hif2α RNAi triggers and pharmaceutical compositions comprising said Hif2α RNAi triggers may be packaged in pre-filled syringes or vials.


Cells, tissues, and non-human organisms that include at least one of the Hif2α RNAi triggers described herein is contemplated. The cell, tissue, or non-human organism is made by delivering the RNAi trigger to the cell, tissue, or non-human organism by any means available in the art. In some embodiments, the cell is a mammalian cell, including, but no limited to, a human cell. The cell, tissue, or non-human organisms are useful for research or as research tools (e.g., drug testing or diagnoses).


Method of Treatment

In some embodiments, the Hif2α RNAi triggers described herein are used to treat a subject having a disease or disorder that would benefit from reduction or inhibition in Hif2α expression. In some embodiments, the described Hif2α RNAi triggers are used to treat or prevent at least one symptom in a subject having a disease or disorder that would benefit from reduction or inhibition in Hif2α expression. The subject is administered a therapeutically effective amount of any one or more of the described RNAi triggers thereby treating the symptom.


In some embodiments, the Hif2α RNAi triggers are used to treat or manage a clinical presentation wherein a subject in need of such treatment, prevention or management is administered a therapeutically or prophylactically effective amount of one or more of the Hif2α RNAi triggers or Hif2α RNAi trigger-containing compositions described herein. In some embodiments, the method comprises administering a composition comprising an Hif2α RNAi trigger molecule described herein to a mammal to be treated.


Representative diseases that would benefit from a reduction and/or inhibition of Hif2α gene expression include, but are not limited to, cancer, renal cancer, clear cell renal cell carcinoma, non-small cell lung cancer, astrocytoma (brain cancer), bladder cancer, breast cancer, chondrosarcoma, colorectal carcinoma, gastric carcinoma, glioblastoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, neuroblastoma, melanoma, multiple myeloma, ovarian cancer, rectal cancer, metastases, gingivitis, psoriasis, Kaposi's sarcoma-associated herpesvirus, preeclampsia, inflammation, chronic inflammation, neovascular diseases, and rheumatoid arthritis.


In some embodiments, an Hif2α RNAi trigger can be used to inhibit expression of Hif2α in a cell, group of cells, or a tissue, e.g., in a subject. In some embodiments, an Hif2α RNAi trigger can be used to formulate a composition for inhibiting expression of Hif2α in a cell, group of cells, or a tissue, e.g., in a subject. In some embodiments, a therapeutically effective amount of one type (or several different types) of Hif2α RNAi triggers as described herein is administered to a subject, thereby inhibiting expression of Hif2α in the subject (e.g., an amount effective to inhibit expression of Hif2α in the subject).


As used herein, the terms “silence,” “reduce,” “inhibit,” “down-regulate,” or “knockdown gene expression,” when referring to an Hif2α gene, mean that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein, or protein subunit translated from the mRNA in a cell, group of cells, or tissue, in which the Hif2α gene is transcribed, is reduced when the cell, group of cells, or tissue, is treated with the described Hif2α RNAi triggers as compared to a second cell, group of cells, or tissue that has or has not been so treated or compared to the same cell, group of cells, or tissue, prior to administration of the Hif2α RNAi trigger.


In some embodiments, the gene expression level and/or mRNA level of Hif2α in a subject to whom a described Hif2α RNAi trigger is administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% relative to the subject prior to being administered the Hif2α RNAi trigger or to a subject not receiving the Hif2α RNAi trigger. The gene expression level and/or mRNA level in the subject may be reduced in a cell, group of cells, and/or tissue of the subject. In some embodiments, the protein level of Hif2α in a subject to whom a described Hif2α RNAi trigger has been administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% relative to the subject prior to being administered the Hif2α RNAi trigger or to a subject not receiving the Hif2α RNAi trigger. The protein level in the subject may be reduced in a cell, group of cells, tissue, blood, and/or other fluid of the subject. A reduction in gene expression, mRNA, or protein levels can be assessed by any methods known in the art. Reduction or decrease in Hif2α mRNA level and/or protein level are collectively referred to herein as a reduction or decrease in Hif2α or inhibiting or reducing the expression of Hif2α.


“Introducing into a cell”, when referring to an RNAi trigger, means functionally delivering the RNAi trigger into a cell. By functional delivery, it is meant that the RNAi trigger is delivered to the cell and has the expected biological activity, (e.g., sequence-specific inhibition of gene expression).


The route of administration is the path by which an RNAi trigger is brought into contact with the body. In general, methods of administering drugs and nucleic acids for treatment of a subject are well known in the art and can be applied to administration of the compositions described herein. The compounds described herein can be administered via any suitable route in a preparation appropriately tailored to the particular route. Thus, the compounds described herein can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, or intraperitoneally.


In some embodiments, the Hif2α RNAi trigger molecules or compositions described herein can be delivered to a cell, group of cells, tissue, or subject using oligonucleotide delivery technologies known in the art. In general, any suitable method recognized in the art for delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an Hif2α RNAi trigger described herein. For example, delivery can be by local administration, (e.g., direct injection, implantation, or topical administering), systemic administration, or subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal, or topical (including buccal and sublingual) administration, In certain embodiments, the compositions are administered by subcutaneous or intravenous infusion or injection.


The above provided embodiments and items are now illustrated with the following, non-limiting examples.


EXAMPLES
Example 1. RNAi Trigger Synthesis

A) Synthesis. RNAi trigger molecules were synthesized according to phosphoramidite technology on solid phase used in oligonucleotide synthesis. Depending on the scale either a MerMade96E (Bioautomation) or a MerMade12 (Bioautomation) was used. Syntheses were performed on a solid support made of controlled pore glass (CPG, 500 Å or 600 Å, obtained from Prime Synthesis, Aston, Pa., USA). All DNA, 2′-modified RNA, and UNA phosphoramidites were purchased from Termo Fisher Scientific (Milwaukee, Wis., USA). Specifically, the following 2′-O-Methyl phosphoramidites were used: (5′-O-dimethoxytrityl-N6-(benzoyl)-2′-O-methyl-adenosine-3′-O-(2-cyanoethyl-N,N-diisopropy-lamino) phosphoramidite, 5′-O-dimethoxy-trityl-N4-(acetyl)-2′-O-methyl-cytidine-3′-O-(2-cyanoethyl-N,N-diisopropyl-amino) phosphoramidite, (5′-O-dimethoxytrityl-N2-(isobutyryl)-2′-O-methyl-guanosine-3′-O-(2-cyano-ethyl-N,N-diisopropylamino)phosphoramidite, and 5′-O-dimethoxy-trityl-2′-O-methyl-uridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite. The 2′-Deoxy-2′-fluoro-phosphor-amidites carried the same protecting groups as the 2′-O-methyl RNA amidites. The following UNA phosphoramidites were used: 5′-(4,4′-Dimethoxytrityl)-N-benzoyl-2′,3′-seco-adenosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphor-amidite, 5′-(4,4′-Dimethoxytrityl)-N-acetyl-2′,3′-seco-cytosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′-(4,4′-Dimethoxytrityl)-N-isobutyryl-2′,3′-seco-guanosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5′-(4,4′-Dimethoxy-trityl)-2′,3′-seco-uridine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. All amidites were dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3 Å) were added. In order to introduce the TEG-Cholesterol at the 5′-end of the oligomers, the 1-Dimethoxytrityloxy-3-O—(N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-2-O-(2-cyanoethyl)-(N,N,-diisopropyl)-phosphoramidite from Glen Research (Sterling, Va., USA) was employed. The 5′-modifications were introduced without any modification of the synthesis cycle. 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) was used as activator solution.


Coupling times were 10 min (RNA), 180 sec (Cholesterol), 90 sec (2′OMe and UNA), and 60 sec (2′F and DNA). In order to introduce phosphorothioate linkages, a 100 mM solution of 3-phenyl 1,2,4-dithiazoline-5-one (POS, obtained from PolyOrg, Inc., Leominster, Mass., USA) in anhydrous Acetonitrile was employed. See Tables 1-2 and 5 for specific sequences.


B. Cleavage and deprotection of support bound oligomer. After finalization of the solid phase synthesis, the dried solid support was treated with a 1:1 volume solution of 40 wt. % methylamine in water and 28% ammonium hydroxide solution (Aldrich) for two hours at 30° C. The solution was evaporated and the solid residue was reconstituted in water (see below).


C. Purification. Crude Cholesterol containing oligomers were purified by reverse phase HPLC using a Waters XBridge BEH300 C4 5u Prep column and a Shimadzu LC-8 system. Buffer A was 100 mM TEAA, pH 7.5 and contained 5% Acetonitrile and buffer B was 100 mM TEAA and contained 95% Acetonitrile. UV traces at 260 nm were recorded. Appropriate fractions were then run on size exclusion HPLC using a GE Healthcare XK 16/40 column packed with Sephadex G-25 medium with a running buffer of 100 mM ammonium bicarbonate, pH 6.7 and 20% Acetonitrile. Other crude oligomers were purified by anionic exchange HPLC using a TKSgel SuperQ-5PW 13u column and Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0 and contained 20% Acetonitrile and buffer B was the same as buffer A with the addition of 1.5 M sodium chloride. UV traces at 260 nm were recorded. Appropriate fractions were pooled then run on size exclusion HPLC as described for cholesterol containing oligomers.


D. Annealing. Complementary strands were mixed by combining equimolar solutions (sense and antisense) in 0.2×PBS (Phosphate-Buffered Saline, 1×, Corning, Cellgro) to form the RNAi triggers. This solution was placed into a thermomixer at 70° C., heated to 95° C., held at 95° C. for 5 min, and cooled to room temperature slowly. Some RNAi triggers were lyophilized and stored at −15 to −25° C. Duplex concentration was determined by measuring the solution absorbance on a UV-Vis spectrometer in 0.2×PBS. The solution absorbance at 260 nm was then multiplied by a conversion factor and the dilution factor to determine the duplex concentration. Unless otherwise stated, all conversion factor was 0.037 mg/(mL cm). For some experiments, a conversion factor was calculated from an experimentally determined extinction coefficient.


Example 2. Synthesis of APN 1170-100A (100A). APN 1203-006 (006). APN 1203-064 (064) Amphipathic Membrane Active Polyamines




















MW
Theoretical MW

% Amine
% Alkyl
% End Group
Azides/


Polymer
(protected)
(deprotected)
PDI
Incorp.
Incorp.
Removal
Polymer






















APN 1170-100A
64,430
45,765
1.22
56
44
0
1.25


APN 1203-006
60,330
43,578
1.05
56
44
99
1.14


APN 1203-062
65,170
46,736
1.05
54
46
99
0.96









A. Materials. 2,2′-Azobis(2,4-dimethyl valeronitrile) (V-65, radical initiator) was purchased from Wako Pure Chemical Industries. Propyl acrylate was purchased from Polysciences Inc. N-Boc-ethoxy-ethylamine acrylate was obtained from WuXi Inc. 2-(Dodecylthio-carbonothioylthio)-2-methylpropionic acid (DDMAT, RAFT Agent), 1,1′-Azobis-(cyclo-hexanecarbonitrile) (ACHN), 1-Ethylpiperidine hypophosphite (EPHP), Pentafluorophenol, N,N′-Dicyclohexylcarbodiimide and N,N-diisopropyl-ethylamine were purchased from Sigma Aldrich. O-(2-Aminoethyl)-O′-(2-azidoethyl)triethylene Glycol (azido-PEG4-amine) was purchased from Biomatrik Inc.


B. RAFT copolymer of N-Boc-ethoxyethylamine acrylate and propyl acrylate (EAP). Solutions of V-65 (2 mg/mL) and RAFT agent DDMAT (10 mg/mL) in butyl acetate were prepared. Monomer molar feed was 52% N-Boc-ethoxyethylamine acrylate, 48% propyl acrylate. Theoretical Mw was 75,000. RAFT agent (DDMAT) to initiator (V-65) molar ratio was 6.67:1.


N-Boc-ethoxyethylamine acrylate (1.778 g, 6.86 mmol), propyl acrylate (0.794 mL, 0.722 g, 6.33 mmol), DDMAT solution (1.215 mL, 0.0333 mmol), V-65 solution (0.621 mL, 0.005 mmol), and butyl acetate (10.2 mL) were added to a 20 mL glass vial with a stir bar. The vial was sealed with a septa cap and the solution bubbled with nitrogen using a long needle with a second needle as the outlet for 1 h. The needles were removed and the vial was heated to 50° C. for 24 h with stirring. The solution was allowed to cool to room temperature and transferred equally between two 50 mL centrifuge tube before hexane (35 mL) was added to both tubes. The solution was centrifuged for 2 min at 4400 rpm. The supernatant layer was carefully decanted and the bottom layer rinsed with hexane. The bottom layer of each tube was then re-dissolved in dichloromethane (7 mL), precipitated in hexane (40 mL) and centrifuged once more. The supernatant was decanted and the bottom layer rinsed with hexane before the layers were combined to one 50 mL centrifuge tube and the polymer was dried under reduced pressure for several hours. The yield of crude EAP copolymer was 2.1 g. Samples of the copolymer were taken for multi-angle light scattering (MALS), and 1H-NMR.

    • Polymer 006: The composition determined by 1H-NMR was 55% N-Boc-ethoxyethylamine acrylate and 45% propyl acrylate. The Mw for 006 determined by MALS was 58,600 g/mol with a polydispersity index (PDI) of 1.04.
    • Polymer 100A: Composition by 1H-NMR: 56% N-Boc-ethoxyethylamine acrylate and 44% propyl acrylate. MW by MALS: 65,150, PDI of 1.122.
    • Polymer 064: Composition by 1H-NMR: 54% N-Boc-ethoxyethylamine acrylate and 46% propyl acrylate. The Mw for 064 determined by MALS was 57,957 g/mol with a polydispersity index (PDI) of 1.07.


C. Radical induced w-end group removal (polymers 006 and 064). Solutions of 1,1′-Azobis-(cyclohexanecarbonitrile) (ACHN, 20 mg/mL) and 1-Ethylpiperidine hypophosphite (EPHP, 100 mg/mL) were prepared in toluene. EAP (2 g, 0.035 mmol), ACHN (0.213 mL, 0.5 eq, 0.0174 mmol), EPHP (1.25 mL, 20 eq, 0.697 mmol), and toluene (25.2 mL) were added to a 40 mL glass vial with a stir bar. The vial was sealed with a septa cap and the solution bubbled with nitrogen using a long needle with a second needle as the outlet for 1 h. The needles were removed and the vial was heated to 100° C. for 2 h. The solution was allowed to cool to room temperature and −20 mL toluene was removed by rotary evaporation. The remaining solution was transferred to a 50 mL centrifuge vial, and hexane (35 mL) was added. The solution was centrifuged for 2 min at 4400 rpm. The supernatant layer was carefully decanted and the bottom layer rinsed with hexane. The bottom layer was then re-dissolved in dichloromethane (7 mL), precipitated in hexane (40 mL) and centrifuged once more. The supernatant was decanted and the bottom layer rinsed with hexane before the polymer was dried under reduced pressure for ˜1 h. The polymer was dissolved in methyl tert-butyl ether (80 mL) and transferred to a separatory funnel. The solution was then washed with 3×30 mL volumes of H2O followed by 3×30 mL volumes of saturated NaCl. The polymer solution was then dried over sodium sulfate, and vacuum filtered through 0.45 μm GHP filters. MTBE was removed via rotary evaporation and high vacuum. A sample was taken for monitoring of end group removal using a UV spectrophotometer. End group removal was calculated to be 99%. Samples were taken for MALS, GC-FID, and 1H-NMR. The composition of 006 by H-NMR was 55% N-Boc-eth oxyethylamine acrylate and 45% propyl acrylate. The conversion of 006 determined by GC-FID was 81.4% for the N-Boc-ethoxyethylamine acrylate and 77.3% for the propyl acrylate. The conversion of 100A determined by GC-FID conversion was 87% for N-Boc-ethoxyethylamine acrylate and 83% for propyl acrylate. The Mw for polymer 006 determined by MALS was 57,700 g/mol with a polydispersity index (PDI) of 1.06.


D. Pentafluorophenol activation of α-end group. EAP polymer (2 g, 0.0347 mmol), pentafluorophenol (63.8 mg, 0.3466 mmol), N,N′-Dicyclohexylcarbodiimide (71.5 mg, 0.3466 mmol), and dichloromethane (40 mL) were added to a 100 mL round bottom flask with a stir bar. The flask was stoppered with a rubber septum and the system was purged with nitrogen for 15 min. The solution was stirred for 16 h at room temperature. Additional Pentafluorophenol (63.8 mg, 0.3466 mmol) and N,N′-Dicyclohexylcarbodiimide (71.5 mg, 0.3466 mmol) were added, the flask stoppered with a rubber septum, and the system was purged with nitrogen for 15 min. The solution was stirred for 3 h at room temperature. The polymer was precipitated with hexane (˜10× volume), centrifuged, and the solvent was decanted. The polymer was dissolved in minimal dichloromethane, precipitated with hexane (˜10× volume), centrifuged, and the solvent was decanted. The polymer was dissolved in minimal ethyl acetate, precipitated with hexane (˜10× volume), centrifuged, and the solvent was decanted. The polymer precipitate was dried under high vacuum until the solid reached a constant weight.


E. Azide functionalization of α-end group. In a 100 ml round bottom flask equipped with a rubber septum and stir bar, polymer from the previous step (1.9 g, 0.0329 mmol) was dissolved in dichloromethane (38 mL). Azido-PEG4-Amine (86.4 mg, 0.3293 mmol) and N,N-Diisopropylethylamine (46.8 mg, 63.1 μL, 0.3622 mmol) were added to the flask with stirring. The system was purged with nitrogen for 15 min, and the reaction was left to stir at room temperature overnight Additional Azido PEG4 Amine (86.4 mg, 0.3293 mmol) and N,N-Diisopropylethylamine (46.8 mg, 63.1 μL, 0.3622 mmol) were added to the flask, the system was purged with N2 gas, and the reaction was stirred for 3 h at room temperature. The polymer was precipitated with hexane (˜10× volume), centrifuged, and the solvent was decanted. The polymer was dissolved in minimal dichloromethane, precipitated with hexane (˜10× volume), centrifuged, and the solvent was decanted. The polymer precipitate was dried under high vacuum until the solid reached a constant weight. The yield of Azide functionalized EAP was 1.77 g. Samples of the copolymer were taken for multi-angle light scattering (MALS), and 1H-NMR.

    • Polymer 006: The composition determined by 1H-NMR was 56% N-Boc-ethoxyethylamine acrylate and 44% propyl acrylate. The Mw determined by MALS was 60,330 g/mol with a polydispersity index (PDI) of 1.05.
    • Polymer 100A: The composition by H-NMR was 56% N-Boc-ethoxyethylamine acrylate and 44% propyl acrylate. The Mw determined by MALS: 64,430 with PDI of 1.217.
    • Polymer 064: The composition by 1H-NMR was 54% N-Boc-ethoxyethylamine acrylate and 46% propyl acrylate. The Mw determined by MALS: 65,170 with PDI of 1.05


Mono-Azide: The term “mono-azide” or “mono-azide polymer” indicates that steps D and E of the procedures above were done and an azide group was coupled to the α-end group of the polymer.


F. Boc Deprotection and Tangential Flow Filtration. In a 100 mL round bottom flask, 2M HCl in acetic acid (28 mL) was added to Azide functionalized EAP copolymer (1.67 g, 0.0277 mmol). The reaction was stirred at room temperature for 1 h. De-ionized H2O (56 mL) was added, and stirred for 10 min. The solution was then immediately exchanged with 10 equivalent volumes of 5 mM Phosphate-Citrate buffer (pH 5) using a mPES 30 kD 115 cm2 filter module equipped with a tangential flow filtration system (KrosFlo Research). The solution was then concentrated using the apparatus to 55 mL final volume. A pH value of 5.1 was recorded. Samples were taken for concentration determination by headspace gas chromatography. An aliquot was lyophilized and then reconstituted in 33.3% Acetonitrile-d in Deuterium Oxide at a concentration of 10 mg/mL for 1H-NMR analysis. Theoretical MW was calculated to be 43,026 g/mol 45,765 g/mol for 006 and 100A respectively.


G. Using similar techniques, similar amphipathic membrane active polyamines can be readily formed. Particularly, amphipathic membrane active polyamines with molecular weight (Mw) 40-120 k protected (25 k to 85 k deprotected), PDI ranges of 1.03 to 1.2, and monomer ratios of 35% amine monomer/65% hydrophobic group monomer to 70% amine monomer/30% hydrophobic group monomer.


Example 3. Synthesis of APN 1095-126 (126)























% End



MW
Theoretical MW

% Amine
% Alkyl
Group
Azides Per


(protected)
(deprotected)
PDI
Incorporation
Incorporation
Removal
Polymer







66,670
47,606
1.11
56
44
0
4.1









Synthesis of APN 1095-126 used dithiobenzoate moiety RAFT agent and AIBN RAFT initiator, compared to the trithiocarbonate moiety RAFT agent and V-65 RAFT initiator used for synthesis of 100A and 006. The conditions for this polymerization required different heating temperatures and times. In addition, this polymer required fractional precipitation. The polymer was not end capped, but the method of azide addition was the same as 100A and 006.


A. Materials. Propyl acrylate was purchased from Polysciences Inc. N-Boc-ethoxyethylamine acrylate was obtained from WuXi Inc. 4-Cyano-4-(phenylcarbonothioylthio) pentanoic acid (CPCPA, RAFT Agent), 2,2′-Azobis(2-methylpropionitrile) (AIBN, radical initiator), Pentafluorophenol, N,N′-Dicyclohexylcarbodiimide and N,N-diisopropylethylamine were purchased from Sigma Aldrich. O-(2-Aminoethyl)-O′-(2-azidoethyl)triethylene Glycol (azido-PEG4-amine) was purchased from Biomatrik Inc.


B. RAFT copolymer of N-Boc-ethoxyethylamine acrylate and propyl acrylate (EAP). The following procedure was repeated 8 times to yield a total of 4.5513 g fractionated EAP copolymer. Solutions of AIBN (1.035 mg/mL) and RAFT agent CPCPA (50.54 mg/mL) in butyl acetate were prepared. Monomer molar feed was 52% N-Boc-ethoxyethylamine acrylate, 48% propyl acrylate. Theoretical Mw was 75,000. RAFT agent (CPCPA) to initiator (AIBN) molar ratio was 6.67:1.


N-Boc-ethoxyethylamine acrylate (1.7879 g, 6.9 mmol), propyl acrylate (0.774 mL, 0.7121 g, 6.24 mmol), CPCPA solution (0.184 mL, 0.0333 mmol), AIBN solution (0.793 mL, 0.005 mmol), and butyl acetate (11.02 mL) were added to a 20 mL glass vial with a stir bar.


The vial was sealed with a septa cap and the solution bubbled with nitrogen using a long needle with a second needle as the outlet for 1 h. The needles were removed and the vial was heated to 50° C. for 24 h with stirring. The solution was allowed to cool to room temperature and transferred to a 50 mL centrifuge tube before hexane (35 mL) was added. The solution was centrifuged for 2 min at 4400 rpm. The supernatant layer was carefully decanted and the bottom layer rinsed with hexane. The bottom layer of each tube was then re-dissolved in dichloromethane (7 mL), precipitated in hexane (40 mL) and centrifuged once more. The supernatant was decanted and the bottom layer rinsed with Hexane before the polymer was dried under reduced pressure for several hours. The yield of crude EAP copolymer was 1.734 g. Samples of the crude copolymer were taken for multi-angle light scattering (MALS), and 1H-NMR. The dried, crude copolymer was dissolved in DCM (100 mg/mL). Hexane was added until just after the cloud point was reached. The resulting milky solution was centrifuged. The bottom layer was extracted and fully precipitated into hexane. The fraction was centrifuged, after which the copolymer was isolated and dried under vacuum. The yield of isolated fraction of EAP copolymer was 0.602 g. Samples of the fractionated copolymer were taken for 1H-NMR and MALS. The composition determined by 1H-NMR was 56% N-Boc-ethoxyethylamine acrylate and 44% propyl acrylate. The Mw determined by MALS was 62,010 g/mol with a polydispersity index (PDI) of 1.14.


C. Pentafluorophenol activation of α-end group. EAP polymer (2 g, 0.0347 mmol), pentafluorophenol (63.8 mg, 0.3466 mmol), N,N′-Dicyclohexylcarbodiimide (71.5 mg, 0.3466 mmol), and dichloromethane (40 mL) were added to a 100 mL round bottom flask with a stir bar. The flask was stoppered with a rubber septum and the system was purged with nitrogen for 15 min. The solution was stirred for 16 h at room temperature. Additional Pentafluorophenol (63.8 mg, 0.3466 mmol) and N,N′-Dicyclohexylcarbodiimide (71.5 mg, 0.3466 mmol) were added, the flask stoppered with a rubber septum, and the system was purged with nitrogen for 15 min. The solution was stirred for 3 h at room temperature. The polymer was precipitated with hexane (˜10× volume), centrifuged, and the solvent was decanted. The polymer was dissolved in minimal dichloromethane, precipitated with hexane (˜10× volume), centrifuged, and the solvent was decanted. The polymer was dissolved in minimal ethyl acetate, precipitated with hexane (˜10× volume), centrifuged, and the solvent was decanted. The polymer precipitate was dried under high vacuum until the solid reached a constant weight.


D. Azide functionalization of α-end group. In a 100 ml round bottom flask equipped with a rubber septum and stir bar, polymer from the previous step (1.9 g, 0.0329 mmol) was dissolved in dichloromethane (38 mL). Azido-PEG4-Amine (86.4 mg, 0.3293 mmol) and N,N-Diisopropyl-ethylamine (46.8 mg, 63.1 μL, 0.3622 mmol) were added to the flask with stirring.


The system was purged with nitrogen for 15 min, and the reaction was left to stir at room temperature overnight. Additional Azido PEG4 Amine (86.4 mg, 0.3293 mmol) and N,N-Diisopropyl-ethylamine (46.8 mg, 63.1 μL, 0.3622 mmol) were added to the flask, the system was purged with N2 gas, and the reaction was stirred for 3 h at room temperature. The polymer was precipitated with hexane (˜10× volume), centrifuged, and the solvent was decanted. The polymer was dissolved in minimal dichloromethane, precipitated with hexane (˜10× volume), centrifuged, and the solvent was decanted. The polymer precipitate was dried under high vacuum until the solid reached a constant weight. The yield of Azide functionalized EAP was 1.77 g. Samples of the copolymer were taken for multi-angle light scattering (MALS), and 1H-NMR. The composition determined by 1H-NMR was 56% N-Boc-ethoxyethylamine acrylate and 44% propyl acrylate. The Mw determined by MALS was 66,670 g/mol with a polydispersity index (PDI) of 1.11.


E. Boc Deprotection and Tangential Flow Filtration. In a 100 mL round bottom flask, 2M HCl in acetic acid (28 mL) was added to Azide functionalized EAP copolymer (1.67 g, 0.0277 mmol). The reaction was stirred at room temperature for 1 hour. De-ionized H2O (56 mL) was added, and stirred for 10 min. The solution was then immediately exchanged with 10 equivalent volumes of 5 mM Phosphate-Citrate buffer (pH 5) using a mPES 30 kD 115 cm2 filter module equipped with a tangential flow filtration system (KrosFlo Research). The solution was then concentrated using the apparatus to 55 mL final volume. A pH value of 5.1 was recorded. Samples were taken for concentration determination by headspace gas chromatography. An aliquot was lyophilized and then reconstituted in 33.3% Acetonitrile-d in Deuterium Oxide at a concentration of 10 mg/mL for 1H-NMR analysis. Theoretical MW was calculated to be 43,026 g/mol.


Example 4. Polymer Analytics





    • (i) MALS analysis. Approximately 10 mg of the copolymer was dissolved in 0.5 mL 75% dichloromethane, 20% tetrahydrofiran, and 5% acetonitrile. The molecular weight and polydispersity (PDI) were measured using a Wyatt Heleos II multiangle light scattering detector attached to a Shimadzu Prominence HPLC using a Jordi 5 μm 7.8×300 Mixed Bed LS DVB column. Molecular weight (polymer 006) before de-protection: 60,330 (PDI 1.05).

    • (ii) Monomer conversion by Gas Chromatography. Approximately 40 μL of copolymer solution (section B) was taken after mixing (pre-N2 bubbling), after N2 bubbling, and after reaction completion. Samples were diluted 100 fold into ethyl acetate. The samples were analyzed with a Shimadzu GC-2010 plus equipped with a flame ionization detector using a Phenomenex Zebron capillary column (ZB-5, 30 m, 0.25 mm ID, 0.5 μm film thickness). Using the pre-N2 bubbled sample as a single point calibration, monomer conversion was measured by comparing post reaction monomer concentrations with pre reaction/post N2 bubbling monomer concentrations.

    • (iii) Polymer concentration by propanol content using headspace gas chromatography (HS-GC). Deprotected polymer solution (˜20 mg/mL) was diluted 50 fold into 3M NaOH using 1-Butanol as an internal standard. The reaction tube was sealed and shaken for 1 h. The reaction was then incubated for at least 6 h at room temperature. In a 10 mL headspace vial, hydrolyzed test article (250 μL) was added to saturated NaCl (500 μL) and HCl (4M, 250 μL) and the system was sealed. Test articles were analyzed using a Shimadzu GC-2010 plus with HS-20 headspace sampler using a Phenomenex ZB-WAX plus gc column (30.0 m, 0.25 mm ID, 0.25 μm film thickness). Propanol concentration was then quantitated using an external standard curve or propanol containing the same NaCl/HCl/NaOH matrix. Polymer concentration was then calculated by dividing propanol concentration by the amount of propanol per polymer as determined by monomer incorporation.

    • (iv) Azide quantitation using UV Spectroscopy. Deprotected polymer solution (˜20 mg/mL) was diluted to −5 mg/mL in 60 mM MES, pH 6. The polymer was then reacted with DBCO-amine (2.5 molar eq.) at room temperature for at least 6 h. The difference in absorbance at 310 nm was calculated and azide content per polymer was determined.





Example 5. Tangential Flow Filtration and Analysis of Conjugate

Following Conjugate formation, i.e., modification of polymer by addition of RGD and PEG modifying agents and attachment of RNAi trigger (see example 9 below) the conjugate solution (2 mg/mL, 10 mL) was exchanged with 10 equivalent volumes of 10 mM Phosphate-Citrate buffer (pH 5) using a mPES 30 kD 20 cm2 filter module equipped with a tangential flow filtration system (KrosFlo Research). A pH value of 5.1 was recorded.


A. Conjugate characterization and analysis.

    • (i) Polymer Concentration Throughout Conjugation. The Same Method as Section G(iii) was used throughout the assembly of the conjugate to monitor polymer concentration.
    • (ii) Impurity quantitation by HPLC-reverse-phase chromatography. Polymer conjugate (after TFF purification) was diluted to 1 mg/mL with H2O and injected onto a Shimadzu Prominence HPLC with a SPD-20A UV detector and a Waters Xbridge C18 5 μm 4.6×250 mm column. The method used a binary gradient consisting of H2O/Acetonitrile/0.1% formic acid with detection set to 247 nm. Concentrations of PEGn-ACit-PABOH, RGD-PEGn-FCFP-COOH, and PNP were calculated using external standard quantitation.
    • (iii) RGD-PEGn-FCitFP-TFP and PEGn-ACit-PABC-PNP modification through amino acid analysis. Polymer conjugate (after TFF purification) with NorValine as an internal standard was hydrolyzed for 16 h in HCl (6 M) at 110° C. in a sealed hydrolysis tube. The hydrolysis solution was then neutralized with NaOH, diluted with borate buffer (pH 10.1), and derivatized with phthaldialdehyde/3-mercaptopropionic acid. The sample was then injected onto a Shimadzu Nexera HPLC system with SIL-30A autosampler, SPD-20A photo diode array detector, and a Waters Xbridge C18 5 μm 4.6×250 mm column. Sample was eluted using a 10 mM Sodium tetraborate decahydrate/10 mM dibasic sodium phosphate/5 mM Sodium azide and 45% Methanol/45% Acetonitrile/10% H2O binary gradient. UV detection was set to 338 nm. Alanine and Phenylalanine concentrations were calculated using external standard curves. Alanine and Phenylalanine concentrations along with polymer concentration and monomer incorporations were used to calculate total amine group modification, as well as the ratio between both ligands.
    • (iv) RNA trigger quantitation and conjugate purity by size exclusion chromatography. A Shimadzu Prominence HPLC equipped with SPD-20A UV detector and Acclaim SEC-300 4.6 mm×300 mm, 5 μm, 300 Å size exclusion column (11st in series) connected to Acclaim SEC-1000 4.6 mm×300 mm, 7 μm, 1000 Å (2nd in series) size exclusion column was assembled. The method used was isocratic, with 200 mM NaBr, 10 mM Tris pH 8, 1 mM EDTA, and 20% Acetonitrile as mobile phase and detection at 260 nm. A sample of polymer conjugate (after RNA trigger addition) was diluted into mobile phase and injected onto the system. Another sample of conjugate followed the same dilution scheme but was treated with 200 mM dithiothreitol for 2 h before injection onto the system. RNA trigger concentration for both samples was calculated using an external standard curve. Amount of conjugated RNA trigger was calculated by subtracting DTT treated RNA levels from untreated RNA levels. Post-TFF purity of the conjugate was also determined using this method.


Example 6. RGD Ligands (RGD Mimic)



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A. RGD mimic #1-PEGn-HyNic, MW 1272.




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    • n=4-24 (preferably 8-12) PGP





B. RGD mimic #1a-HyNic, MW 802.8.




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C. RGD mimic #1b-HyNic, MW 830.9 (RGD).




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Example 7. RGD and PEG Modifying Agents

A. Dipeptide RGD-dipeptide and PEG-dipeptide modifying agents were made as described in US-2012-0172412-A1 (WO 2012/092373) and US 2015-0045573 A1 (WO 2015/021092) (both of which are incorporated herein by reference). FIG. 3-7.


B. RGD-PEGn-FCitFP-TFP and PEGn-FCitFP-TFP modifying agent synthesis. The modifying agent precursor (di-Boc)RGD(OtBu)-APBA-PEGn-FCitFPro-COOH was prepared using general Fmoc chemistry solid phase synthesis using 2-Cl-Trt resin preloaded with Fmoc-Proline-OH. To Resin-Pro-Fmoc was added sequentially (following Fmoc deprotection at each step): FMoc-Phe-OH, Fmoc-Cit-OH, Fmoc-Phe-OH, Fmoc-NH-PEGn-COOH, 4-(N-Fmoc-p-aminophenoxy)-butyric acid (APBA), Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, and diboc-m-guanidino benzoic acid.


(diboc)RGD(OtBu)-APBA-PEGn-FCitFPro-COOH (458 mg, 0.200 mmols) and TFP (66.5 mg, 0.400 mmols) were dissolved in anhydrous DCM (5.0 mL) and cooled to 0° C. in an ice/water bath while stirring under Argon. EDC (77 mg, 0.400 mmols) was added and the reaction mixture stirred in an ice/water bath at 0° C. for 30 min. Reaction progress was monitored by TLC (8.5:1.5 CHCl3:MeOH) and was complete after 90 min with no starting material observed by TLC. The reaction mixture was diluted to 100 mL total volume with DCM, washed 3×40 mL with DI H2O (pH=5), and washed 1×40 mL aqueous saturated NaCl solution. The organics were then dried over Na2SO4, and concentrated on a rotovap to yield 448 mg (92% yield) of a tan/orange foam. The structure was confirmed by 1H NMR, and ESI MS (Reaction shown above for PEG20 (n=20)).




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    • (diboc)RGD(OtBu)-APBA-PEGn-FCitFPro-TFP
      • (shown for n=20)





(diboc)RGD(OtBu)-PEGn-FCitFPro-TFP (497 mg, 0.204 mmols) was dissolved in [9.25:0.75:0.50] TFA:H2O:Thioanisole (5.0 mL) and stirred at room temperature in a closed flask for 45 min. Reaction completion was confirmed by MS (ESI, scan neg, 300-3000) with no masses related to starting material or partially deprotected intermediates observed. The reaction mixture was then precipitated into 45 mL diethyl ether, spun down, the supernatant poured off, and washed 2×10 mL diethyl ether and dried on high vacuum overnight. The final product was purified on prep HPLC using a Thermo Aquasil C18 5 μm semi prep column, with mobile phases 0.1% TFA in H2O and ACN. Each injection was 50 mg of crude material dissolved in 3.0 mL of 0.1% TFA in [61:39] H2O:ACN run on a gradient of (indicated in % B) 39-(5)-39-(35)-43-(5)-95-(10)-95-(2)-39-(5)-39. Each sample for injection was prepared (dissolved) within 15 minutes of being injected and positive fractions were pooled in one flask and kept cold in the freezer until the last injection of the day had finished. The positive fractions were then concentrated on the rotovap with a bath temperature of 32° C. to dryness, then chased 2× with ACN/Toluene, then 3× with ACN and then dried on high vacuum overnight. Out of 257 mg injected crude, 180 mg (70%) was isolated as pure material (Reaction shown above for PEG20 (n=20)).




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    • RGD-PEGn-FCitFPro-TFP
      • (shown for n=20)





4-(N-Fmoc-p-aminophenoxy)-butyric acid 1 synthesis. p-Nitro-phenol (2) (7.5 g, 53.9 mmole) was combined with ethyl 4-bromobutyrate (8.45 ml, 59 mmol) and K2CO3 (7.5 g, 54 mmole) in DMF (75 mL). The mixture was stirred for 2 h at 100° C. DMF was removed and the crude product was diluted in a mixture of 3:1 mixture of 2 N NaOH and methanol and stirred 4 h at RT. The reaction mixture was acidified with 6 M HCl. The white precipitate was collected to yield 4-(p-Nitrophenyloxy)-butyric acid 3: (10.9 g, 90% yield). 4-(p-Nitrophenyloxy)-butyric acid 3 (37.1 g, 165 mmole) was dissolved in MeOH (1 L) with ammonium formate (35 g, 555 mmole) and 10% Pd/C (Degussa Type) (3.5 g) was added. The mixture was refluxed at 65° C. overnight. The reaction was filtered with celite to yield a reddish brown solid of product 4-(p-Aminophenyloxy)-butyric acid 4 (30.5 g, 95% yield).


4-(p-Aminophenyloxy)-butyric acid 4 (5.1 g, 26 mmole) was dissolved in 6:4 a mixture of an aqueous saturated NaHCO3(3.36 g, 40 mmol) in H2O (450 mL) and THF (300 ml) to make a white slurry. Fmoc-OSu (8.82 g, 26.1 mmole) was added and the reaction was stirred for 4 h. The acetone was removed, the reaction was acidified (HCl), and the off-white precipitate was collected and triturated in 1N HCl to yield 9.6 g of product 4-(N-Fmoc-p-aminophenoxy)-butyric acid 1 (88% yield).




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    • diBoc-m-guanidino-benzoic acid 5 was synthesized according to Riches A G et al. Tetrahedron (2012) 68, p. 9448-9455.





PEGe-FCitFP modifying agents were made using similar chemistry. FIG. 1-2.


Example 8. Tetrapeptide Peptide Linkages

A. Tetrapeptide Syntheses. All tetrapeptides were synthesized in the same manner using standard solid phase Fmoc procedures. Some peptides were synthesized from commercially available 2-Cl-Trt resin (EMD Millipore, Billerica, Mass.) containing either proline, leucine, or alanine. For other peptides, 2-Cl-Trt resin was loaded with either FMOC-PEGn-CO2H or FMOC-N-methyl-Ala-CO2H by adding a solution of DMF containing the amino acid or PEG (1 eq) and DIEA (2 eq) to 2-Cl-Trt resin for 16 h. Upon completion, resins were capped with MeOH. Stepwise addition was performed using PYBOP (4 eq), amino acid (4 eq), and DIEA (8 eq) for coupling and 20% piperdine in DMF for Fmoc de-protection.


After peptide syntheses, the tetrapeptides were reacted with 2 eq of N-Hydroxysuccinimide (NHS) activated esters of either protected N-Acetyl-galactosamine, NAG(OAc)3 (Rozema D B et al. “Protease-triggered siRNA Delivery Vehicles.” J Control Release. 2015 Vol. 209:57-66 and U.S. Pat. No. 8,802,773) or PEGn in DMF containing 4 eq DIEA. Following attachment of NAG(OAc)3 or PEG, the peptides were removed from resin using HFIP (30%) in DCM for 0.5 h. After solvent removal the residue was triturated with Et2O.


Tetrapeptides were either purified and conjugated to activated esters to form modifying agents or conjugated to chromophore N-(p-Nitrophenyl)ethylenediamine (pNA) without purification to form substrates for physiological lability testing. Prior to purification, NAG(OAc)3-containing substrates were de-acetylated by treatment with TEA (35%) in water (45%) and MeOH (20%) and stirred at room temp. For purification, tetrapeptide substrates were separated by HPLC using a Thermo Scientific Aquasil C18 reverse-phase column (250×21.2, Waltham, Mass.), eluting a gradient of acetonitrile and water buffered with 0.1% formic acid. Following purification, the substrates were lyophilized.


Attachment of amine-reactive groups to tetrapeptides. 1 eq HPLC purified peptide with N-terminal NAG (RS=NAG(OH)3 or PEG (R=PEGe) in DMF or DCM was added to a flame dried flask at 0° C. to give a 0.2 M concentration of peptide. NHS (3 eq) and N,N′-Dicyclohexylcarbodiimide (DCC) (3 eq) were added and allowed to stir at room temp. under argon overnight to yield the modifying agents. The mixture was partially concentrated, chilled to −20° C., and filtered. All solvents were then removed in vacuo. The residue was dissolved in a minimum of DCM and MeOH, precipitated into cold Et2O and collected by decantation of the solvent after centrifugation. Precipitation into Et2O was repeated until no residual DCU (dicyclohexylurea) was detectable. All prepared compounds were subsequently used without further purification.


Example 9. Polymer Modification

Formation of siRNA delivery conjugate using RGD-PEG-HyNic, RGD-PEG-ACit-PNP, or RDG-PEG-FCitFP-TFP and PEG-dipeptide modifying agents.


1) Protocol 1. The indicated polymer was reacted with SMPT at a weight ratio of 1:0.015 (polymer:SMPT) in 5 mM HEPES, pH 8.0 buffer for 1 h at RT. The SMPT-modified polymer was then reacted with aldehyde-PEG-dipeptide modifying agent (aldehyde-PEG12-FCit or aldehyde-PEG24-ACit) at desired ratios for 1 h at RT. The modified polymer was then reacted with PEG12-dipeptide modifying agent (PEG12-FCit, PEG12-ACit or PEG24-ACit) at a weight ratio of 1:2 (polymer:PEG) in 100 mM HEPES, pH 9.0 buffer for 1 h at RT. The modified polymer was then reacted overnight with SATA-RNAi trigger at a weight ratio of 1:0.2 (polymer:SATA-RNAi trigger) in 100 mM HEPES, pH 9.0 buffer at RT to attach the RNAi trigger. Next, the modified polymer was reacted with protease cleavable PEG (PEG12-FCit or PEG12-ACit or PEG24-ACit) at a weight ratio of 1:6 (polymer:PEG) in 100 mM HEPES, pH 9.0 buffer for 1 h at RT. The resultant conjugate was purified using a sephadex G-50 spin column.

    • RGD-HyNic (Example 6B) was attached to the modified polymer to form the full delivery conjugate by reaction with the modified polymer at a weight ratio of 1:0.7 (polymer:RGD-HyNic mimic) in 50 mM MES, pH 5.0 buffer for a minimum of 4 h at RT. The conjugate was purified using a sephadex G-50 spin column. RGD ligand attachment efficiency was determined as described above.


2) Protocol 2. The indicated polymer was reacted with SMPT at a weight ratio of 1:0.015 (polymer:SMPT) in 5 mM HEPES, pH 8.0 buffer for 1 h at RT. The SMPT-modified polymer was then reacted with aldehyde-PEG-dipeptide modifying agent (aldehyde-PEG24-ACit) at a weight ratio of 1:0.5 (polymer:PEG) and with PEG-dipeptide modifying agent (PEG12-FCit, PEG12-ACit or PEG24-ACit) at a weight ratio of 1:2 (polymer:PEG) in 100 mM HEPES, pH 9.0 buffer for 1 h at RT. The modified polymer was then reacted overnight with SATA-RNAi trigger at a weight ratio of 1:0.2 (polymer:SATA-RNAi trigger) in 100 mM HEPES, pH 9.0 buffer at RT to attach the RNAi trigger. Next, the modified polymer was reacted with protease cleavable-PEG (PEG12-FCit or PEG12-ACit or PEG24-ACit) at a weight ratio of 1:6 (polymer:PEG) in 100 mM HEPES, pH 9.0 buffer for 1 h at RT. RGD-HyNic (Example 6) was attached to the modified polymer to form the full conjugate by reaction with the modified polymer at a weight ratio of 1:0.7 (polymer:RGD-HyNic) in 69 mM hydrogen chloride solution (HCl) overnight at RT. RGD ligand attachment efficiency was determined as described above. 3) Protocol 3. The indicated polymer was reacted with SMPT at a weight ratio of 1:0.015 (polymer:SMPT) in 5 mM HEPES, pH 8.0 buffer for 1 h at RT. The SMPT-modified polymer was then reacted with aldehyde-PEG-dipeptide modifying agent (aldehyde-PEG24-ACit) at a weight ratio of 1:0.5 (polymer:PEG) and with PEG-dipeptide modifying agent (PEG12-FCit, PEG12-ACit or PEG24-ACit) at a weight ratio of 1:2 (polymer:PEG) in 50 mM HEPES, pH 9.0 buffer for 1 h at RT. The modified polymer was then reacted overnight with SATA-RNAi trigger at a weight ratio of 1:0.2 (polymer:SATA-RNAi trigger) in 50 mM HEPES, pH 9.0 buffer at RT to attach the RNAi trigger. Next, the modified polymer was reacted with protease cleavable-PEG (PEG12-FCit or PEG12-ACit or PEG24-ACit) at a weight ratio of 1:6 (polymer:PEG) in 50 mM HEPES, pH 9.0 buffer for 1 h at RT. RGD-HyNic (Example 6) was attached to the modified polymer to form the full delivery conjugate by reaction with the modified polymer at a weight ratio of 1:0.7 (polymer:RGD-HyNic mimic) in 100 mM MES free acid solution overnight at RT. RGD targeting ligand conjugation efficiency was determined as described above.


4) Protocol 4. The indicated polymer was reacted with Azido-PEG4-NHS at a weight ratio of 1:0.015 (polymer:Azido) in 5 mM HEPES, pH 8.0 buffer for 1 h at RT. The Azido-modified polymer was then reacted with aldehyde-PEG-dipeptide modifying agent (aldehyde-PEG24-ACit) at a weight ratio of 1:0.5 (polymer:PEG) and with PEG-dipeptide modifying agent (PEG12-ACit) at a weight ratio of 1:2 (polymer:PEG) in 50 mM HEPES, pH 9.0 buffer for 1 h at RT. The modified polymer was then reacted overnight with Alkyne-RNAi trigger at a weight ratio of 1:0.2 (polymer:Alkyne-RNAi trigger) in 50 mM HEPES, pH 9.0 buffer at RT to attach the RNAi trigger. Next, the modified polymer was reacted with protease cleavable-PEG (PEG12-ACit) at a weight ratio of 1:6 (polymer:PEG) in 50 mM HEPES, pH 9.0 buffer for 1 h at RT. RGD-HyNic (Example 6) was attached to the modified polymer to form the full delivery conjugate by reaction with the modified polymer at a weight ratio of 1:0.7 (polymer:RGD-HyNic mimic) in 100 mM sodium acetate-acetic acid buffer solution, pH 5.0 overnight at RT. RGD targeting ligand conjugation efficiency was determined as described above.


5) Protocol S. The mono azide-polymer was reacted with aldehyde-PEG-dipeptide modifying agent (aldehyde-PEG24-ACit) at a weight ratio of 1:0.5 (polymer:PEG) and with PEG-dipeptide modifying agent (PEG12-ACit) at a weight ratio of 1:2 (polymer:PEG) in 50 mM HEPES, pH 9.0 buffer for 1 h at RT. The modified polymer was then reacted overnight with Alkyne-RNAi trigger at a weight ratio of 1:0.2 (polymer:Alkyne-RNAi trigger) in 50 mM HEPES, pH 9.0 buffer at RT to attach the RNAi trigger. Next, the modified polymer was reacted with protease cleavable-PEG (PEG12-ACit) at a weight ratio of 1:6 (polymer:PEG) in 50 mM HEPES, pH 9.0 buffer for 1 h at RT. RGD-HyNic (Example 6) was attached to the modified polymer to form the full delivery conjugate by reaction with the modified polymer at a weight ratio of 1:0.7 (polymer:RGD-HyNic mimic) in 100 mM sodium acetate-acetic acid buffer solution, pH 5.0 overnight at RT. RGD targeting ligand conjugation efficiency was determined as described above.


6) Protocol 6. The mono azide-polymer was reacted with protease cleavable-RGD agent (RGD-PEG8-ACit-PNP, RDG-PEG8-FCitFP-TFP, RGD-PEG15-FCitFP-TFP, RGD-PEG19-FCitFP-TFP, or RGD-PEG20-FCitFP-TFP) at weight ratios of 1:0.125, 1:0.25, 1:0.5, 1:1, 1:1.5, 1:2 (polymer:RGD) in 50 mM HEPES, pH 8.5 buffer for 4 h at RT. The modified polymer was then reacted with protease cleavable-PEG agent (PEG6-ACit-PABC-PNP, PEG12-ACit-PABC-PNP, PEG12-FCit-PABC-PNP, PEG12-FCitFP-TFP) at a weight ratio of 1:8 (polymer:PEG) in 50 mM HEPES, pH 8.5 buffer for 2 h at RT. Alkyne-RNAi trigger at a weight ratio of 1:0.3 (polymer:Alkyne-RNAi trigger) was added to the modified polymer in 100 mM sodium acetate-acetic acid buffer solution, pH 5.0 for 5 days at RT. The completed conjugate was TFF purified and conjugation efficiency determined.


7) Protocol 7. The mono azide-polymer was reacted with protease cleavable-RGD agent (RGD-PEG20-FCitFP-TFP) at weight ratio of 1:1 (polymer:RGD) in 50 mM HEPES, pH 8.5 buffer for 2 h at RT. The modified polymer was then reacted with protease cleavable-PEG agent (PEG12-ACit-PABC-PNP) at a weight ratio of 1:8 (polymer:PEG) in 50 mM HEPES, pH 8.5 buffer for 2 h at RT. The modified polymer was then TFF purified. Alkyne-RNAi trigger at a weight ratio of 1:0.4 (polymer:Alkyne-RNAi trigger) was added to the TFF purified polymer for 3 days at 37° C.


Example 10. In Vitro Analysis of Hf2α RNAi Triggers

Candidate sequences were identified by in silico analysis and screened as chemically modified canonical siRNAs in vitro. For screening purposes, the human EPAS1 (Hif2a) cDNA sequence (accession #NM_001430) was synthesized and cloned (DNA 2.0, Menlo Park, Calif.) into a commercially-available reporter-based screening plasmid, psiCHECK2 (Promega, Madison, Wis.) which generated a Renilla luciferase/EPAS1 fusion mRNA. For RNAi trigger efficacy evaluation, Hep3B cells, a human hepatocellular carcinoma line, were plated at ˜10,000 cells per well in 96-well format. Each of the 187 EPAS1 RNAi triggers, in two subsets, was co-transfected at two concentrations, 1 nM and 0.1 nM, with 25 ng EPAS1-psiCHECK2 plasmid DNA per well and 0.2 μL LipoFectamine 2000 (Life Technologies) per well. Gene knockdown was determined by measuring Renilla luciferase levels normalized to the levels of constitutively-expressed firefly luciferase, also present on the psiCHECK-2 plasmid, using the Dual Luciferase Reporter Assay (Promega, Madison, Wis.) Table 5.









TABLE 5A







Unmodified Hif2α RNAi trigger antisense 


strand and sense strand sequences.










SEQ

SEQ



ID
Sense Strand
ID
Antisense Strand


NO.
Sequence (5′ → 3′)
NO.
Sequence (5′ → 3′)





285
GAGACUGUAUGGUCAGCUC
478
GAGCUGACCAUACAGUCUC





286
CUCCGACUCCUUCCGACUC
479
GAGUCGGAAGGAGUCGGAG





287
UCCGACUCCCAGCAUUCGA
480
UCGAAUGCUGGGAGUCGGA





288
CGACUCCCAGCAUUCGAGC
481
GCUCGAAUGCUGGGAGUCG





289
GACUCCCAGCAUUCGAGCC
482
GGCUCGAAUGCUGGGAGUC





290
CAGGUGCUCGGCGUCUGAA
483
UUCAGACGCCGAGCACCUG





291
GUGCUCGGCGUCUGAACGU
484
ACGUUCAGACGCCGAGCAC





292
UCGGCGUCUGAACGUCUCA
485
UGAGACGUUCAGACGCCGA





293
GGCGUCUGAACGUCUCAAA
486
UUUGAGACGUUCAGACGCC





294
CGUCUGAACGUCUCAAAGG
487
CCUUUGAGACGUUCAGACG





295
AAAAGGAGUAGCUCGGAGA
488
UCUCCGAGCUACUCCUUUU





296
GGGUUUCAUUGCCGUGGUG
489
CACCACGGCAAUGAAACCC





297
UUCAUGGGACUUACACAGG
490
CCUGUGUAAGUCCCAUGAA





298
GGGACUUACACAGGUGGAG
491
CUCCACCUGUGUAAGUCCC





299
ACACAGGUGGAGCUAACAG
492
CUGUUAGCUCCACCUGUGU





300
GAGCUAACAGGACAUAGUA
493
UACUAUGUCCUGUUAGCUC





301
GCUAACAGGACAUAGUAUC
494
GAUACUAUGUCCUGUUAGC





302
CUAACAGGACAUAGUAUCU
495
AGAUACUAUGUCCUGUUAG





303
GGACAUAGUAUCUUUGACU
496
AGUCAAAGAUACUAUGUCC





304
UCUUUGACUUCACUCAUCC
497
GGAUGAGUGAAGUCAAAGA





305
UCACUCAUCCCUGCGACCA
498
UGGUCGCAGGGAUGAGUGA





306
GAGAUUCGUGAGAACCUGA
499
UCAGGUUCUCACGAAUCUC





307
UUCGUGAGAACCUGAGUCU
500
AGACUCAGGUUCUCACGAA





308
UCGUGAGAACCUGAGUCUC
501
GAGACUCAGGUUCUCACGA





309
GACAUGUCCACAGAGCGGG
502
CCCGCUCUGUGGACAUGUC





310
GCGGGACUUCUUCAUGAGG
503
CCUCAUGAAGAAGUCCCGC





311
GGAUGAAGUGCACGGUCAC
504
GUGACCGUGCACUUCAUCC





312
CACGGUCACCAACAGAGGC
505
GCCUCUGUUGGUGACCGUG





313
UCACCAACAGAGGCCGUAC
506
GUACGGCCUCUGUUGGUGA





314
CACCAACAGAGGCCGUACU
507
AGUACGGCCUCUGUUGGUG





315
AGGCCGUACUGUCAACCUC
508
GAGGUUGACAGUACGGCCU





316
UCCUCACAAUAGUCUGUGU
509
ACACAGACUAUUGUGAGGA





317
AAUAGUCUGUGUGGCUACA
510
UGUAGCCACACAGACUAUU





318
CAGAACUGAUUGGUUACCA
511
UGGUAACCAAUCAGUUCUG





319
AGAACUGAUUGGUUACCAC
512
GUGGUAACCAAUCAGUUCU





320
CUGAUUGGUUACCACCCUG
513
CAGGGUGGUAACCAAUCAG





321
UUGGCCGCUCAGCCUAUGA
514
UCAUAGGCUGAGCGGCCAA





322
UAUGAAUUCUACCAUGCGC
515
GCGCAUGGUAGAAUUCAUA





323
AUGAAUUCUACCAUGCGCU
516
AGCGCAUGGUAGAAUUCAU





324
UGAAUUCUACCAUGCGCUA
517
UAGCGCAUGGUAGAAUUCA





325
GAAUUCUACCAUGCGCUAG
518
CUAGCGCAUGGUAGAAUUC





326
AAUUCUACCAUGCGCUAGA
519
UCUAGCGCAUGGUAGAAUU





327
UCUACCAUGCGCUAGACUC
520
GAGUCUAGCGCAUGGUAGA





328
AUGCGCUAGACUCCGAGAA
521
UUCUCGGAGUCUAGCGCAU





329
UGCGCUAGACUCCGAGAAC
522
GUUCUCGGAGUCUAGCGCA





330
GUAAGUGGCCAGUACCGGA
523
UCCGGUACUGGCCACUUAC





331
UAAGUGGCCAGUACCGGAU
524
AUCCGGUACUGGCCACUUA





332
CCAGUACCGGAUGCUCGCA
525
UGCGAGCAUCCGGUACUGG





333
AGUACCGGAUGCUCGCAAA
526
UUUGCGAGCAUCCGGUACU





334
UACCGGAUGCUCGCAAAGC
527
GCUUUGCGAGCAUCCGGUA





335
UGCUCGCAAAGCAUGGGGG
528
CCCCCAUGCUUUGCGAGCA





336
CGCAAAGCAUGGGGGCUAC
529
GUAGCCCCCAUGCUUUGCG





337
AGCAUGGGGGCUACGUGUG
530
CACACGUAGCCCCCAUGCU





338
GCAUGGGGGCUACGUGUGG
531
CCACACGUAGCCCCCAUGC





339
CAUCUACAACCCUCGCAAC
532
GUUGCGAGGGUUGUAGAUG





340
AUCUACAACCCUCGCAACC
533
GGUUGCGAGGGUUGUAGAU





341
CUACAACCCUCGCAACCUG
534
CAGGUUGCGAGGGUUGUAG





342
UACAACCCUCGCAACCUGC
535
GCAGGUUGCGAGGGUUGUA





343
UUUGAUAGCAGUGGCAAGG
536
CCUUGCCACUGCUAUCAAA





344
AGUAACUUCCUAUUCACCA
537
UGGUGAAUAGGAAGUUACU





345
UCGGGAAUCAGAACUUCGA
538
UCGAAGUUCUGAUUCCCGA





346
CUGCUCCACGCCCAAUAGC
539
GCUAUUGGGCGUGGAGCAG





347
UGCUCCACGCCCAAUAGCC
540
GGCUAUUGGGCGUGGAGCA





348
GCUCCACGCCCAAUAGCCC
541
GGGCUAUUGGGCGUGGAGC





349
ACGCCCAAUAGCCCUGAAG
542
CUUCAGGGCUAUUGGGCGU





350
CAUCUUUGGAUAACGACCU
543
AGGUCGUUAUCCAAAGAUG





351
CAAUGCAGUACCCAGACGG
544
CCGUCUGGGUACUGCAUUG





352
AUGCAGUACCCAGACGGAU
545
AUCCGUCUGGGUACUGCAU





353
AGUACCCAGACGGAUUUCA
546
UGAAAUCCGUCUGGGUACU





354
CUGUAGCCCCGCACAGUCC
547
GGACUGUGCGGGGCUACAG





355
AUCUUCUUUGAUGCCGGAA
548
UUCCGGCAUCAAAGAAGAU





356
CUUUGAUGCCGGAAGCAAA
549
UUUGCUUCCGGCAUCAAAG





357
GAUGCCGGAAGCAAAGCAU
550
AUGCUUUGCUUCCGGCAUC





358
AUGCCGGAAGCAAAGCAUC
551
GAUGCUUUGCUUCCGGCAU





359
GCCGGAAGCAAAGCAUCCC
552
GGGAUGCUUUGCUUCCGGC





360
CCCCCAGAUCCACCAUUAC
553
GUAAUGGUGGAUCUGGGGG





361
AGAUCCACCAUUACAUUUU
554
AAAAUGUAAUGGUGGAUCU





362
AUUUUGGGCCCACAAAGUG
555
CACUUUGUGGGCCCAAAAU





363
UUUUGGGCCCACAAAGUGG
556
CCACUUUGUGGGCCCAAAA





364
UUUGGGCCCACAAAGUGGG
557
CCCACUUUGUGGGCCCAAA





365
CCACAAAGUGGGCCGUCGG
558
CCGACGGCCCACUUUGUGG





366
CACAAAGUGGGCCGUCGGG
559
CCCGACGGCCCACUUUGUG





367
AGUGGGCCGUCGGGGAUCA
560
UGAUCCCCGACGGCCCACU





368
AAAGGGUUUUGGGGCUCGA
561
UCGAGCCCCAAAACCCUUU





369
GGCUCGAGGCCCAGACGUG
562
CACGUCUGGGCCUCGAGCC





370
GCUCGAGGCCCAGACGUGC
563
GCACGUCUGGGCCUCGAGC





371
CUCGAGGCCCAGACGUGCU
564
AGCACGUCUGGGCCUCGAG





372
GGUAGCCCUCUCCAACAAG
565
CUUGUUGGAGAGGGCUACC





373
CUUUGAUGCCGGACAAGCC
566
GGCUUGUCCGGCAUCAAAG





374
UUUGAUGCCGGACAAGCCA
567
UGGCUUGUCCGGCAUCAAA





375
UUGAUGCCGGACAAGCCAC
568
GUGGCUUGUCCGGCAUCAA





376
GGACAAGCCACUGAGCGCA
569
UGCGCUCAGUGGCUUGUCC





377
ACAAGCCACUGAGCGCAAA
570
UUUGCGCUCAGUGGCUUGU





378
GGACUACAGCCUGUCGUCA
571
UGACGACAGGCUGUAGUCC





379
GACUACAGCCUGUCGUCAG
572
CUGACGACAGGCUGUAGUC





380
CUACAGCCUGUCGUCAGCC
573
GGCUGACGACAGGCUGUAG





381
CCUGUCGUCAGCCCACAAG
574
CUUGUGGGCUGACGACAGG





382
GCAUGGCAAGCCGGCUGCU
575
AGCAGCCGGCUUGCCAUGC





383
CUGACCAGAUAUGACUGUG
576
CACAGUCAUAUCUGGUCAG





384
GAUAUGACUGUGAGGUGAA
577
UUCACCUCACAGUCAUAUC





385
GGUGAACGUGCCCGUGCUG
578
CAGCACGGGCACGUUCACC





386
UACAAGAUGGACUUACCUG
579
CAGGUAAGUCCAUCUUGUA





387
GGACUUACCUGGCAGACUU
580
AAGUCUGCCAGGUAAGUCC





388
UUUUUCUGAGAUGCUCACU
581
AGUGAGCAUCUCAGAAAAA





389
AGUACACAAUUGUUUUACC
582
GGUAAAACAAUUGUGUACU





390
ACAAGUUUGGUGCAUGUCU
583
AGACAUGCACCAAACUUGU





391
ACUAAAAAGAUUCCUCGUU
584
AACGAGGAAUCUUUUUAGU





392
AGGGUCAACUCCAACGUAU
585
AUACGUUGGAGUUGACCCU





393
GGGUCAACUCCAACGUAUG
586
CAUACGUUGGAGUUGACCC





394
GUCAACUCCAACGUAUGUG
587
CACAUACGUUGGAGUUGAC





395
UCAACUCCAACGUAUGUGG
588
CCACAUACGUUGGAGUUGA





396
CAACUCCAACGUAUGUGGU
589
ACCACAUACGUUGGAGUUG





397
CUCCAACGUAUGUGGUUAU
590
AUAACCACAUACGUUGGAG





398
UCCAACGUAUGUGGUUAUC
591
GAUAACCACAUACGUUGGA





399
CCAACGUAUGUGGUUAUCU
592
AGAUAACCACAUACGUUGG





400
AACGUAUGUGGUUAUCUGU
593
ACAGAUAACCACAUACGUU





401
UUAUAUCUGGGUUAAGUGU
594
ACACUUAACCCAGAUAUAA





402
CCACGGCCUGUACGGACAC
595
GUGUCCGUACAGGCCGUGG





403
ACGGCCUGUACGGACACUG
596
CAGUGUCCGUACAGGCCGU





404
UGUCGGCUUUUUGCCAUCU
597
AGAUGGCAAAAAGCCGACA





405
GUCGGCUUUUUGCCAUCUG
598
CAGAUGGCAAAAAGCCGAC





406
AUCUGUGAUAUGCCAUAGG
599
CCUAUGGCAUAUCACAGAU





407
UGCCAUAGGUGUGACAAUC
600
GAUUGUCACACCUAUGGCA





408
CCAUAGGUGUGACAAUCCG
601
CGGAUUGUCACACCUAUGG





409
CAUAGGUGUGACAAUCCGA
602
UCGGAUUGUCACACCUAUG





410
AUAGGUGUGACAAUCCGAG
603
CUCGGAUUGUCACACCUAU





411
GGUGUGACAAUCCGAGCAG
604
CUGCUCGGAUUGUCACACC





412
ACAAUCCGAGCAGUGGAGU
605
ACUCCACUGCUCGGAUUGU





413
CCGAGCAGUGGAGUCAUUC
606
GAAUGACUCCACUGCUCGG





414
GGGAGCACUGCGCGCUAUC
607
GAUAGCGCGCAGUGCUCCC





415
GGAGCACUGCGCGCUAUCC
608
GGAUAGCGCGCAGUGCUCC





416
AGCACUGCGCGCUAUCCCC
609
GGGGAUAGCGCGCAGUGCU





417
UAUUGCUGCCAAGAGGGUC
610
GACCCUCUUGGCAGCAAUA





418
GGUCUGAUGGCACGUUGUG
611
CACAACGUGCCAUCAGACC





419
CUGAUGGCACGUUGUGGGG
612
CCCCACAACGUGCCAUCAG





420
GGCACGUUGUGGGGUCGGG
613
CCCGACCCCACAACGUGCC





421
GCACGUUGUGGGGUCGGGG
614
CCCCGACCCCACAACGUGC





422
CACGUUGUGGGGUCGGGGG
615
CCCCCGACCCCACAACGUG





423
GCGGGGAAGUGCUCUAACU
616
AGUUAGAGCACUUCCCCGC





424
CGGGGAAGUGCUCUAACUU
617
AAGUUAGAGCACUUCCCCG





425
UUAAGGUUUUGUUGCUAGC
618
GCUAGCAACAAAACCUUAA





426
GUUGCUAGCCCUUCAAGUG
619
CACUUGAAGGGCUAGCAAC





427
GAGCUAUGUGACUCGGAUG
620
CAUCCGAGUCACAUAGCUC





428
GCUAUGUGACUCGGAUGGU
621
ACCAUCCGAGUCACAUAGC





429
CGGAUGGUCUUUCACACGG
622
CCGUGUGAAAGACCAUCCG





430
GAUGGUCUUUCACACGGCA
623
UGCCGUGUGAAAGACCAUC





431
UGGUCUUUCACACGGCACA
624
UGUGCCGUGUGAAAGACCA





432
AACUACCAUGAGAUGGUUU
625
AAACCAUCUCAUGGUAGUU





433
UACCAUGAGAUGGUUUAGA
626
UCUAAACCAUCUCAUGGUA





434
CCAAGCUCACGACCUUGGA
627
UCCAAGGUCGUGAGCUUGG





435
ACGACCUUGGAGCCCCGUG
628
CACGGGGCUCCAAGGUCGU





436
GGGUAAGAGGGACGACACC
629
GGUGUCGUCCCUCUUACCC





437
GGUAAGAGGGACGACACCU
630
AGGUGUCGUCCCUCUUACC





438
GUAAGAGGGACGACACCUC
631
GAGGUGUCGUCCCUCUUAC





439
UGGUUUUUCAAUACCAAUU
632
AAUUGGUAUUGAAAAACCA





440
UUCAAUACCAAUUACAUGG
633
CCAUGUAAUUGGUAUUGAA





441
AUACCAAUUACAUGGAACU
634
AGUUCCAUGUAAUUGGUAU





442
CCAACUAUUUAGUAAGCCC
635
GGGCUUACUAAAUAGUUGG





443
AACUAUUUAGUAAGCCCGG
636
CCGGGCUUACUAAAUAGUU





444
ACUAUUUAGUAAGCCCGGA
637
UCCGGGCUUACUAAAUAGU





445
AGAAAUUCCUUAGUCAUGG
638
CCAUGACUAAGGAAUUUCU





446
CAUUAAGGGCAUUUUACCC
639
GGGUAAAAUGCCCUUAAUG





447
UAAGGGCAUUUUACCCUUG
640
CAAGGGUAAAAUGCCCUUA





448
AGCUUCAUAUUAACCCUAC
641
GUAGGGUUAAUAUGAAGCU





449
UAUUAACCCUACCUGUCAA
642
UUGACAGGUAGGGUUAAUA





450
UUAACCCUACCUGUCAACG
643
CGUUGACAGGUAGGGUUAA





451
ACCCUACCUGUCAACGUAA
644
UUACGUUGACAGGUAGGGU





452
CCCUACCUGUCAACGUAAC
645
GUUACGUUGACAGGUAGGG





453
CCUACCUGUCAACGUAACG
646
CGUUACGUUGACAGGUAGG





454
CUACCUGUCAACGUAACGA
647
UCGUUACGUUGACAGGUAG





455
UACCUGUCAACGUAACGAU
648
AUCGUUACGUUGACAGGUA





456
ACCUGUCAACGUAACGAUU
649
AAUCGUUACGUUGACAGGU





457
CCUGUCAACGUAACGAUUU
650
AAAUCGUUACGUUGACAGG





458
CUGUCAACGUAACGAUUUC
651
GAAAUCGUUACGUUGACAG





459
UGUCAACGUAACGAUUUCA
652
UGAAAUCGUUACGUUGACA





460
UCAACGUAACGAUUUCAUG
653
CAUGAAAUCGUUACGUUGA





461
ACGUAACGAUUUCAUGAAC
654
GUUCAUGAAAUCGUUACGU





462
UAUUAUAUUGUCGAAUUCC
655
GGAAUUCGACAAUAUAAUA





463
UUAUAUUGUCGAAUUCCUA
656
UAGGAAUUCGACAAUAUAA





464
UAUUGUCGAAUUCCUACUG
657
CAGUAGGAAUUCGACAAUA





465
GAAUUCCUACUGACAACAU
658
AUGUUGUCAGUAGGAAUUC





466
UCCUACUGACAACAUUAUA
659
UAUAAUGUUGUCAGUAGGA





467
UAUAACUGUAUGGGAGCUU
660
AAGCUCCCAUACAGUUAUA





468
UAACUGUAUGGGAGCUUAA
661
UUAAGCUCCCAUACAGUUA





469
UGUAUGGGAGCUUAACUUU
662
AAAGUUAAGCUCCCAUACA





470
UUGACACUGGUAUCUUAUU
663
AAUAAGAUACCAGUGUCAA





471
AAGUAUUCUGAUCCUACCA
664
UGGUAGGAUCAGAAUACUU





472
CAACGUAACGAUUUCAUGA
665
UUCAUGAAAUCGUUACGUU



AA

GGC





473
UAUAUCAACGUAACGAUUU
666
UUCAUGAAAUCGUUACGUU



CAUGAAA

GGCU





474
UAUAUCAACGUAACGAUUU
667
UUCAUGAAAUCGUUACGUU



CAUGAAA

GGCUAU





475
UAUAUCAACGUAACGAUUU
668
UUCAUGAAAUCGUUACGUU



CAUGAAA

GGCUGU





476
UAUAUCGACGUAACGAUUU
669
UUCAUGAAAUCGUUACGUC



CAUGAAA

GGCUAU





477
UAUACGUAACGAUUUCAUG
670
UUCAUGAAAUCGUUACGU



AAA
















TABLE 5B







Hif2α RNAi trigger sequences having modified


nucleotides.











Duplex
SEQ

SEQ



ID
ID
Sense Strand Sequence
ID
Antisense Strand Sequence


No.
No.
(5′ → 3′)
No.
(5′ → 3′)





2231
671
GfaGfaCfuGfuAfuGfgUfcAfgCfuAfdT
 858
dTAfgCfuGfaCfcAfuAfcAfgUfcUfcdTsdT





2232
672
CfuCfcGfaCfuCfcUfuCfcGfaCfuAfdT
 859
dTAfgUfcGfgAfaGfgAfgUfcGfgAfgdTsdT





2233
673
UfcCfgAfcUfcCfcAfgCfaUfuCfgAfdT
 860
dTCfgAfaUfgCfuGfgGfaGfuCfgGfadTsdT





2234
674
CfgAfcUfcCfcAfgCfaUfuCfgAfgAfdT
 861
dTCfuCfgAfaUfgCfuGfgGfaGfuCfgdTsdT





2235
675
GfaCfuCfcCfaGfcAfuUfcGfaGfcAfdT
 862
dTGfcUfcGfaAfuGfcUfgGfgAfgUfcdTsdT





2236
676
CfaGfgUfgCfuCfgGfcGfuCfuGfaAfdT
 863
dTUfcAfgAfcGfcCfgAfgCfaCfcUfgdTsdT





2237
677
GfuGfcUfcGfgCfgUfcUfgAfaCfgAfdT
 864
dTCfgUfuCfaGfaCfgCfcGfaGfcAfcdTsdT





2238
678
UfcGfgCfgUfcUfgAfaCfgUfcUfcAfdT
 865
dTGfaGfaCfgUfuCfaGfaCfgCfcGfadTsdT





2239
679
GfgCfgUfcUfgAfaCfgUfcUfcAfaAfdT
 866
dTUfuGfaGfaCfgUfuCfaGfaCfgCfcdTsdT





2240
680
CfgUfcUfgAfaCfgUfcUfcAfaAfgAfdT
 867
dTCfuUfuGfaGfaCfgUfuCfaGfaCfgdTsdT





2241
681
AfaAfaGfgAfgUfaGfcUfcGfgAfgAfdT
 868
dTCfuCfcGfaGfcUfaCfuCfcUfuUfudTsdT





2242
682
GfgGfuUfuCfaUfuGfcCfgUfgGfuAfdT
 869
dTAfcCfaCfgGfcAfaUfgAfaAfcCfcdTsdT





2243
683
UfuCfaUfgGfgAfcUfuAfcAfcAfgAfdT
 870
dTCfuGfuGfuAfaGfuCfcCfaUfgAfadTsdT





2244
684
GfgGfaCfuUfaCfaCfaGfgUfgGfaAfdT
 871
dTUfcCfaCfcUfgUfgUfaAfgUfcCfcdTsdT





2245
685
AfcAfcAfgGfuGfgAfgCfuAfaCfaAfdT
 872
dTUfgUfuAfgCfuCfcAfcCfuGfuGfudTsdT





2246
686
GfaGfcUfaAfcAfgGfaCfaUfaGfuAfdT
 873
dTAfcUfaUfgUfcCfuGfuUfaGfcUfcdTsdT





2247
687
GfcUfaAfcAfgGfaCfaUfaGfuAfuAfdT
 874
dTAfuAfcUfaUfgUfcCfuGfuUfaGfcdTsdT





2248
688
CfuAfaCfaGfgAfcAfuAfgUfaUfcAfdT
 875
dTGfaUfaCfuAfuGfuCfcUfgUfuAfgdTsdT





2249
689
GfgAfcAfuAfgUfaUfcUfuUfgAfcAfdT
 876
dTGfuCfaAfaGfaUfaCfuAfuGfuCfcdTsdT





2250
690
UfcUfuUfgAfcUfuCfaCfuCfaUfcAfdT
 877
dTGfaUfgAfgUfgAfaGfuCfaAfaGfadTsdT





2251
691
UfcAfcUfcAfuCfcCfuGfcGfaCfcAfdT
 878
dTGfgUfcGfcAfgGfgAfuGfaGfuGfadTsdT





2252
692
GfaGfaUfuCfgUfgAfgAfaCfcUfgAfdT
 879
dTCfaGfgUfuCfuCfaCfgAfaUfcUfcdTsdT





2253
693
UfuCfgUfgAfgAfaCfcUfgAfgUfcAfdT
 880
dTGfaCfuCfaGfgUfuCfuCfaCfgAfadTsdT





2254
694
UfcGfuGfaGfaAfcCfuGfaGfuCfuAfdT
 881
dTAfgAfcUfcAfgGfuUfcUfcAfcGfadTsdT





2255
695
GfaCfaUfgUfcCfaCfaGfaGfcGfgAfdT
 882
dTCfcGfcUfcUfgUfgGfaCfaUfgUfcdTsdT





2256
696
GfcGfgGfaCfuUfcUfuCfaUfgAfgAfdT
 883
dTCfuCfaUfgAfaGfaAfgUfcCfcGfcdTsdT





2257
697
GfgAfuGfaAfgUfgCfaCfgGfuCfaAfdT
 884
dTUfgAfcCfgUfgCfaCfuUfcAfuCfcdTsdT





2258
698
CfaCfgGfuCfaCfcAfaCfaGfaGfgAfdT
 885
dTCfcUfcUfgUfuGfgUfgAfcCfgUfgdTsdT





2259
699
UfcAfcCfaAfcAfgAfgGfcCfgUfaAfdT
 886
dTUfaCfgGfcCfuCfuGfuUfgGfuGfadTsdT





2260
700
CfaCfcAfaCfaGfaGfgCfcGfuAfcAfdT
 887
dTGfuAfcGfgCfcUfcUfgUfuGfgUfgdTsdT





2261
701
AfgGfcCfgUfaCfuGfuCfaAfcCfuAfdT
 888
dTAfgGfuUfgAfcAfgUfaCfgGfcCfudTsdT





2262
702
UfcCfuCfaCfaAfuAfgUfcUfgUfgAfdT
 889
dTCfaCfaGfaCfuAfuUfgUfgAfgGfadTsdT





2263
703
AfaUfaGfuCfuGfuGfuGfgCfuAfcAfdT
 890
dTGfuAfgCfcAfcAfcAfgAfcUfaUfudTsdT





2264
704
CfaGfaAfcUfgAfuUfgGfuUfaCfcAfdT
 891
dTGfgUfaAfcCfaAfuCfaGfuUfcUfgdTsdT





2265
705
AfgAfaCfuGfaUfuGfgUfuAfcCfaAfdT
 892
dTUfgGfuAfaCfcAfaUfcAfgUfuCfudTsdT





2266
706
CfuGfaUfuGfgUfuAfcCfaCfcCfuAfdT
 893
dTAfgGfgUfgGfuAfaCfcAfaUfcAfgdTsdT





2267
707
UfuGfgCfcGfcUfcAfgCfcUfaUfgAfdT
 894
dTCfaUfaGfgCfuGfaGfcGfgCfcAfadTsdT





2268
708
UfaUfgAfaUfuCfuAfcCfaUfgCfgAfdT
 895
dTCfgCfaUfgGfuAfgAfaUfuCfaUfadTsdT





2269
709
AfuGfaAfuUfcUfaCfcAfuGfcGfcAfdT
 896
dTGfcGfcAfuGfgUfaGfaAfuUfcAfudTsdT





2270
710
UfgAfaUfuCfuAfcCfaUfgCfgCfuAfdT
 897
dTAfgCfgCfaUfgGfuAfgAfaUfuCfadTsdT





2271
711
GfaAfuUfcUfaCfcAfuGfcGfcUfaAfdT
 898
dTUfaGfcGfcAfuGfgUfaGfaAfuUfcdTsdT





2272
712
AfaUfuCfuAfcCfaUfgCfgCfuAfgAfdT
 899
dTCfuAfgCfgCfaUfgGfuAfgAfaUfudTsdT





2273
713
UfcUfaCfcAfuGfcGfcUfaGfaCfuAfdT
 900
dTAfgUfcUfaGfcGfcAfuGfgUfaGfadTsdT





2274
714
AfuGfcGfcUfaGfaCfuCfcGfaGfaAfdT
 901
dTUfcUfcGfgAfgUfcUfaGfcGfcAfudTsdT





2275
715
UfgCfgCfuAfgAfcUfcCfgAfgAfaAfdT
 902
dTUfuCfuCfgGfaGfuCfuAfgCfgCfadTsdT





2276
716
GfuAfaGfuGfgCfcAfgUfaCfcGfgAfdT
 903
dTCfcGfgUfaCfuGfgCfcAfcUfuAfcdTsdT





2277
717
UfaAfgUfgGfcCfaGfuAfcCfgGfaAfdT
 904
dTUfcCfgGfuAfcUfgGfcCfaCfuUfadTsdT





2278
718
CfcAfgUfaCfcGfgAfuGfcUfcGfcAfdT
 905
dTGfcGfaGfcAfuCfcGfgUfaCfuGfgdTsdT





2279
719
AfgUfaCfcGfgAfuGfcUfcGfcAfaAfdT
 906
dTUfuGfcGfaGfcAfuCfcGfgUfaCfudTsdT





2280
720
UfaCfcGfgAfuGfcUfcGfcAfaAfgAfdT
 907
dTCfuUfuGfcGfaGfcAfuCfcGfgUfadTsdT





2281
721
UfgCfuCfgCfaAfaGfcAfuGfgGfgAfdT
 908
dTCfcCfcAfuGfcUfuUfgCfgAfgCfadTsdT





2282
722
CfgCfaAfaGfcAfuGfgGfgGfcUfaAfdT
 909
dTUfaGfcCfcCfcAfuGfcUfuUfgCfgdTsdT





2283
723
AfgCfaUfgGfgGfgCfuAfcGfuGfuAfdT
 910
dTAfcAfcGfuAfgCfcCfcCfaUfgCfudTsdT





2284
724
GfcAfuGfgGfgGfcUfaCfgUfgUfgAfdT
 911
dTCfaCfaCfgUfaGfcCfcCfcAfuGfcdTsdT





2285
725
CfaUfcUfaCfaAfcCfcUfcGfcAfaAfdT
 912
dTUfuGfcGfaGfgGfuUfgUfaGfaUfgdTsdT





2286
726
AfuCfuAfcAfaCfcCfuCfgCfaAfcAfdT
 913
dTGfuUfgCfgAfgGfgUfuGfuAfgAfudTsdT





2287
727
CfuAfcAfaCfcCfuCfgCfaAfcCfuAfdT
 914
dTAfgGfuUfgCfgAfgGfgUfuGfuAfgdTsdT





2288
728
UfaCfaAfcCfcUfcGfcAfaCfcUfgAfdT
 915
dTCfaGfgUfuGfcGfaGfgGfuUfgUfadTsdT





2289
729
UfuUfgAfuAfgCfaGfuGfgCfaAfgAfdT
 916
dTCfuUfgCfcAfcUfgCfuAfuCfaAfadTsdT





2290
730
AfgUfaAfcUfuCfcUfaUfuCfaCfcAfdT
 917
dTGfgUfgAfaUfaGfgAfaGfuUfaCfudTsdT





2291
731
UfcGfgGfaAfuCfaGfaAfcUfuCfgAfdT
 918
dTCfgAfaGfuUfcUfgAfuUfcCfcGfadTsdT





2292
732
CfuGfcUfcCfaCfgCfcCfaAfuAfgAfdT
 919
dTCfuAfuUfgGfgCfgUfgGfaGfcAfgdTsdT





2293
733
UfgCfuCfcAfcGfcCfcAfaUfaGfcAfdT
 920
dTGfcUfaUfuGfgGfcGfuGfgAfgCfadTsdT





2294
734
GfcUfcCfaCfgCfcCfaAfuAfgCfcAfdT
 921
dTGfgCfuAfuUfgGfgCfgUfgGfaGfcdTsdT





2295
735
AfcGfcCfcAfaUfaGfcCfcUfgAfaAfdT
 922
dTUfuCfaGfgGfcUfaUfuGfgGfcGfudTsdT





2296
736
CfaUfcUfuUfgGfaUfaAfcGfaCfcAfdT
 923
dTGfgUfcGfuUfaUfcCfaAfaGfaUfgdTsdT





2297
737
CfaAfuGfcAfgUfaCfcCfaGfaCfgAfdT
 924
dTCfgUfcUfgGfgUfaCfuGfcAfuUfgdTsdT





2298
738
AfuGfcAfgUfaCfcCfaGfaCfgGfaAfdT
 925
dTUfcCfgUfcUfgGfgUfaCfuGfcAfudTsdT





2299
739
AfgUfaCfcCfaGfaCfgGfaUfuUfcAfdT
 926
dTGfaAfaUfcCfgUfcUfgGfgUfaCfudTsdT





2300
740
CfuGfuAfgCfcCfcGfcAfcAfgUfcAfdT
 927
dTGfaCfuGfuGfcGfgGfgCfuAfcAfgdTsdT





2301
741
AfuCfuUfcUfuUfgAfuGfcCfgGfaAfdT
 928
dTUfcCfgGfcAfuCfaAfaGfaAfgAfudTsdT





2302
742
CfuUfuGfaUfgCfcGfgAfaGfcAfaAfdT
 929
dTUfuGfcUfuCfcGfgCfaUfcAfaAfgdTsdT





2303
743
GfaUfgCfcGfgAfaGfcAfaAfgCfaAfdT
 930
dTUfgCfuUfuGfcUfuCfcGfgCfaUfcdTsdT





2304
744
AfuGfcCfgGfaAfgCfaAfaGfcAfuAfdT
 931
dTAfuGfcUfuUfgCfuUfcCfgGfcAfudTsdT





2305
745
GfcCfgGfaAfgCfaAfaGfcAfuCfcAfdT
 932
dTGfgAfuGfcUfuUfgCfuUfcCfgGfcdTsdT





2306
746
CfcCfcCfaGfaUfcCfaCfcAfuUfaAfdT
 933
dTUfaAfuGfgUfgGfaUfcUfgGfgGfgdTsdT





2307
747
AfgAfuCfcAfcCfaUfuAfcAfuUfuAfdT
 934
dTAfaAfuGfuAfaUfgGfuGfgAfuCfudTsdT





2308
748
AfuUfuUfgGfgCfcCfaCfaAfaGfuAfdT
 935
dTAfcUfuUfgUfgGfgCfcCfaAfaAfudTsdT





2309
749
UfuUfuGfgGfcCfcAfcAfaAfgUfgAfdT
 936
dTCfaCfuUfuGfuGfgGfcCfcAfaAfadTsdT





2310
750
UfuUfgGfgCfcCfaCfaAfaGfuGfgAfdT
 937
dTCfcAfcUfuUfgUfgGfgCfcCfaAfadTsdT





2311
751
CfcAfcAfaAfgUfgGfgCfcGfuCfgAfdT
 938
dTCfgAfcGfgCfcCfaCfuUfuGfuGfgdTsdT





2312
752
CfaCfaAfaGfuGfgGfcCfgUfcGfgAfdT
 939
dTCfcGfaCfgGfcCfcAfcUfuUfgUfgdTsdT





2313
753
AfgUfgGfgCfcGfuCfgGfgGfaUfcAfdT
 940
dTGfaUfcCfcCfgAfcGfgCfcCfaCfudTsdT





2314
754
AfaAfgGfgUfuUfuGfgGfgCfuCfgAfdT
 941
dTCfgAfgCfcCfcAfaAfaCfcCfuUfudTsdT





2315
755
GfgCfuCfgAfgGfcCfcAfgAfcGfuAfdT
 942
dTAfcGfuCfuGfgGfcCfuCfgAfgCfcdTsdT





2316
756
GfcUfcGfaGfgCfcCfaGfaCfgUfgAfdT
 943
dTCfaCfgUfcUfgGfgCfcUfcGfaGfcdTsdT





2317
757
CfuCfgAfgGfcCfcAfgAfcGfuGfcAfdT
 944
dTGfcAfcGfuCfuGfgGfcCfuCfgAfgdTsdT





2318
758
GfgUfaGfcCfcUfcUfcCfaAfcAfaAfdT
 945
dTUfuGfuUfgGfaGfaGfgGfcUfaCfcdTsdT





2319
759
CfuUfuGfaUfgCfcGfgAfcAfaGfcAfdT
 946
dTGfcUfuGfuCfcGfgCfaUfcAfaAfgdTsdT





2320
760
UfuUfgAfuGfcCfgGfaCfaAfgCfcAfdT
 947
dTGfgCfuUfgUfcCfgGfcAfuCfaAfadTsdT





2321
761
UfuGfaUfgCfcGfgAfcAfaGfcCfaAfdT
 948
dTUfgGfcUfuGfuCfcGfgCfaUfcAfadTsdT





2322
762
GfgAfcAfaGfcCfaCfuGfaGfcGfcAfdT
 949
dTGfcGfcUfcAfgUfgGfcUfuGfuCfcdTsdT





2323
763
AfcAfaGfcCfaCfuGfaGfcGfcAfaAfdT
 950
dTUfuGfcGfcUfcAfgUfgGfcUfuGfudTsdT





2324
764
GfgAfcUfaCfaGfcCfuGfuCfgUfcAfdT
 951
dTGfaCfgAfcAfgGfcUfgUfaGfuCfcdTsdT





2325
765
GfaCfuAfcAfgCfcUfgUfcGfuCfaAfdT
 952
dTUfgAfcGfaCfaGfgCfuGfuAfgUfcdTsdT





2326
766
CfuAfcAfgCfcUfgUfcGfuCfaGfcAfdT
 953
dTGfcUfgAfcGfaCfaGfgCfuGfuAfgdTsdT





2327
767
CfcUfgUfcGfuCfaGfcCfcAfcAfaAfdT
 954
dTUfuGfuGfgGfcUfgAfcGfaCfaGfgdTsdT





2328
768
GfcAfuGfgCfaAfgCfcGfgCfuGfcAfdT
 955
dTGfcAfgCfcGfgCfuUfgCfcAfuGfcdTsdT





2329
769
CfuGfaCfcAfgAfuAfuGfaCfuGfuAfdT
 956
dTAfcAfgUfcAfuAfuCfuGfgUfcAfgdTsdT





2330
770
GfaUfaUfgAfcUfgUfgAfgGfuGfaAfdT
 957
dTUfcAfcCfuCfaCfaGfuCfaUfaUfcdTsdT





2331
771
GfgUfgAfaCfgUfgCfcCfgUfgCfuAfdT
 958
dTAfgCfaCfgGfgCfaCfgUfuCfaCfcdTsdT





2332
772
UfaCfaAfgAfuGfgAfcUfuAfcCfuAfdT
 959
dTAfgGfuAfaGfuCfcAfuCfuUfgUfadTsdT





2333
773
GfgAfcUfuAfcCfuGfgCfaGfaCfuAfdT
 960
dTAfgUfcUfgCfcAfgGfuAfaGfuCfcdTsdT





2334
774
UfuUfuUfcUfgAfgAfuGfcUfcAfcAfdT
 961
dTGfuGfaGfcAfuCfuCfaGfaAfaAfadTsdT





2335
775
AfgUfaCfaCfaAfuUfgUfuUfuAfcAfdT
 962
dTGfuAfaAfaCfaAfuUfgUfgUfaCfudTsdT





2336
776
AfcAfaGfuUfuGfgUfgCfaUfgUfcAfdT
 963
dTGfaCfaUfgCfaCfcAfaAfcUfuGfudTsdT





2337
777
AfcUfaAfaAfaGfaUfuCfcUfcGfuAfdT
 964
dTAfcGfaGfgAfaUfcUfuUfuUfaGfudTsdT





2338
778
AfgGfgUfcAfaCfuCfcAfaCfgUfaAfdT
 965
dTUfaCfgUfuGfgAfgUfuGfaCfcCfudTsdT





2339
779
GfgGfuCfaAfcUfcCfaAfcGfuAfuAfdT
 966
dTAfuAfcGfuUfgGfaGfuUfgAfcCfcdTsdT





2340
780
GfuCfaAfcUfcCfaAfcGfuAfuGfuAfdT
 967
dTAfcAfuAfcGfuUfgGfaGfuUfgAfcdTsdT





2341
781
UfcAfaCfuCfcAfaCfgUfaUfgUfgAfdT
 968
dTCfaCfaUfaCfgUfuGfgAfgUfuGfadTsdT





2342
782
CfaAfcUfcCfaAfcGfuAfuGfuGfgAfdT
 969
dTCfcAfcAfuAfcGfuUfgGfaGfuUfgdTsdT





2343
783
CfuCfcAfaCfgUfaUfgUfgGfuUfaAfdT
 970
dTUfaAfcCfaCfaUfaCfgUfuGfgAfgdTsdT





2344
784
UfcCfaAfcGfuAfuGfuGfgUfuAfuAfdT
 971
dTAfuAfaCfcAfcAfuAfcGfuUfgGfadTsdT





2345
785
CfcAfaCfgUfaUfgUfgGfuUfaUfcAfdT
 972
dTGfaUfaAfcCfaCfaUfaCfgUfuGfgdTsdT





2346
786
AfaCfgUfaUfgUfgGfuUfaUfcUfgAfdT
 973
dTCfaGfaUfaAfcCfaCfaUfaCfgUfudTsdT





2347
787
UfuAfuAfuCfuGfgGfuUfaAfgUfgAfdT
 974
dTCfaCfuUfaAfcCfcAfgAfuAfuAfadTsdT





2348
788
CfcAfcGfgCfcUfgUfaCfgGfaCfaAfdT
 975
dTUfgUfcCfgUfaCfaGfgCfcGfuGfgdTsdT





2349
789
AfcGfgCfcUfgUfaCfgGfaCfaCfuAfdT
 976
dTAfgUfgUfcCfgUfaCfaGfgCfcGfudTsdT





2350
790
UfgUfcGfgCfuUfuUfuGfcCfaUfcAfdT
 977
dTGfaUfgGfcAfaAfaAfgCfcGfaCfadTsdT





2351
791
GfuCfgGfcUfuUfuUfgCfcAfuCfuAfdT
 978
dTAfgAfuGfgCfaAfaAfaGfcCfgAfcdTsdT





2352
792
AfuCfuGfuGfaUfaUfgCfcAfuAfgAfdT
 979
dTCfuAfuGfgCfaUfaUfcAfcAfgAfudTsdT





2353
793
UfgCfcAfuAfgGfuGfuGfaCfaAfuAfdT
 980
dTAfuUfgUfcAfcAfcCfuAfuGfgCfadTsdT





2354
794
CfcAfuAfgGfuGfuGfaCfaAfuCfcAfdT
 981
dTGfgAfuUfgUfcAfcAfcCfuAfuGfgdTsdT





2355
795
CfaUfaGfgUfgUfgAfcAfaUfcCfgAfdT
 982
dTCfgGfaUfuGfuCfaCfaCfcUfaUfgdTsdT





2356
796
AfuAfgGfuGfuGfaCfaAfuCfcGfaAfdT
 983
dTUfcGfgAfuUfgUfcAfcAfcCfuAfudTsdT





2357
797
GfgUfgUfgAfcAfaUfcCfgAfgCfaAfdT
 984
dTUfgCfuCfgGfaUfuGfuCfaCfaCfcdTsdT





2358
798
AfcAfaUfcCfgAfgCfaGfuGfgAfgAfdT
 985
dTCfuCfcAfcUfgCfuCfgGfaUfuGfudTsdT





2359
799
CfcGfaGfcAfgUfgGfaGfuCfaUfuAfdT
 986
dTAfaUfgAfcUfcCfaCfuGfcUfcGfgdTsdT





2360
800
GfgGfaGfcAfcUfgCfgCfgCfuAfuAfdT
 987
dTAfuAfgCfgCfgCfaGfuGfcUfcCfcdTsdT





2361
801
GfgAfgCfaCfuGfcGfcGfcUfaUfcAfdT
 988
dTGfaUfaGfcGfcGfcAfgUfgCfuCfcdTsdT





2362
802
AfgCfaCfuGfcGfcGfcUfaUfcCfcAfdT
 989
dTGfgGfaUfaGfcGfcGfcAfgUfgCfudTsdT





2363
803
UfaUfuGfcUfgCfcAfaGfaGfgGfuAfdT
 990
dTAfcCfcUfcUfuGfgCfaGfcAfaUfadTsdT





2364
804
GfgUfcUfgAfuGfgCfaCfgUfuGfuAfdT
 991
dTAfcAfaCfgUfgCfcAfuCfaGfaCfcdTsdT





2365
805
CfuGfaUfgGfcAfcGfuUfgUfgGfgAfdT
 992
dTCfcCfaCfaAfcGfuGfcCfaUfcAfgdTsdT





2366
806
GfgCfaCfgUfuGfuGfgGfgUfcGfgAfdT
 993
dTCfcGfaCfcCfcAfcAfaCfgUfgCfcdTsdT





2367
807
GfcAfcGfuUfgUfgGfgGfuCfgGfgAfdT
 994
dTCfcCfgAfcCfcCfaCfaAfcGfuGfcdTsdT





2368
808
CfaCfgUfuGfuGfgGfgUfcGfgGfgAfdT
 995
dTCfcCfcGfaCfcCfcAfcAfaCfgUfgdTsdT





2369
809
GfcGfgGfgAfaGfuGfcUfcUfaAfcAfdT
 996
dTGfuUfaGfaGfcAfcUfuCfcCfcGfcdTsdT





2370
810
CfgGfgGfaAfgUfgCfuCfuAfaCfuAfdT
 997
dTAfgUfuAfgAfgCfaCfuUfcCfcCfgdTsdT





2371
811
UfuAfaGfgUfuUfuGfuUfgCfuAfgAfdT
 998
dTCfuAfgCfaAfcAfaAfaCfcUfuAfadTsdT





2372
812
GfuUfgCfuAfgCfcCfuUfcAfaGfuAfdT
 999
dTAfcUfuGfaAfgGfgCfuAfgCfaAfcdTsdT





2373
813
GfaGfcUfaUfgUfgAfcUfcGfgAfuAfdT
1000
dTAfuCfcGfaGfuCfaCfaUfaGfcUfcdTsdT





2374
814
GfcUfaUfgUfgAfcUfcGfgAfuGfgAfdT
1001
dTCfcAfuCfcGfaGfuCfaCfaUfaGfcdTsdT





2375
815
CfgGfaUfgGfuCfuUfuCfaCfaCfgAfdT
1002
dTCfgUfgUfgAfaAfgAfcCfaUfcCfgdTsdT





2376
816
GfaUfgGfuCfuUfuCfaCfaCfgGfcAfdT
1003
dTGfcCfgUfgUfgAfaAfgAfcCfaUfcdTsdT





2377
817
UfgGfuCfuUfuCfaCfaCfgGfcAfcAfdT
1004
dTGfuGfcCfgUfgUfgAfaAfgAfcCfadTsdT





2378
818
AfaCfuAfcCfaUfgAfgAfuGfgUfuAfdT
1005
dTAfaCfcAfuCfuCfaUfgGfuAfgUfudTsdT





2379
819
UfaCfcAfuGfaGfaUfgGfuUfuAfgAfdT
1006
dTCfuAfaAfcCfaUfcUfcAfuGfgUfadTsdT





2380
820
CfcAfaGfcUfcAfcGfaCfcUfuGfgAfdT
1007
dTCfcAfaGfgUfcGfuGfaGfcUfuGfgdTsdT





2381
821
AfcGfaCfcUfuGfgAfgCfcCfcGfuAfdT
1008
dTAfcGfgGfgCfuCfcAfaGfgUfcGfudTsdT





2382
822
GfgGfuAfaGfaGfgGfaCfgAfcAfcAfdT
1009
dTGfuGfuCfgUfcCfcUfcUfuAfcCfcdTsdT





2383
823
GfgUfaAfgAfgGfgAfcGfaCfaCfcAfdT
1010
dTGfgUfgUfcGfuCfcCfuCfuUfaCfcdTsdT





2384
824
GfuAfaGfaGfgGfaCfgAfcAfcCfuAfdT
1011
dTAfgGfuGfuCfgUfcCfcUfcUfuAfcdTsdT





2385
825
UfgGfuUfuUfuCfaAfuAfcCfaAfuAfdT
1012
dTAfuUfgGfuAfuUfgAfaAfaAfcCfadTsdT





2386
826
UfuCfaAfuAfcCfaAfuUfaCfaUfgAfdT
1013
dTCfaUfgUfaAfuUfgGfuAfuUfgAfadTsdT





2387
827
AfuAfcCfaAfuUfaCfaUfgGfaAfcAfdT
1014
dTGfuUfcCfaUfgUfaAfuUfgGfuAfudTsdT





2388
828
CfcAfaCfuAfuUfuAfgUfaAfgCfcAfdT
1015
dTGfgCfuUfaCfuAfaAfuAfgUfuGfgdTsdT





2389
829
AfaCfuAfuUfuAfgUfaAfgCfcCfgAfdT
1016
dTCfgGfgCfuUfaCfuAfaAfuAfgUfudTsdT





2390
830
AfcUfaUfuUfaGfuAfaGfcCfcGfgAfdT
1017
dTCfcGfgGfcUfuAfcUfaAfaUfaGfudTsdT





2391
831
AfgAfaAfuUfcCfuUfaGfuCfaUfgAfdT
1018
dTCfaUfgAfcUfaAfgGfaAfuUfuCfudTsdT





2392
832
CfaUfuAfaGfgGfcAfuUfuUfaCfcAfdT
1019
dTGfgUfaAfaAfuGfcCfcUfuAfaUfgdTsdT





2393
833
UfaAfgGfgCfaUfuUfuAfcCfcUfuAfdT
1020
dTAfaGfgGfuAfaAfaUfgCfcCfuUfadTsdT





2394
834
AfgCfuUfcAfuAfuUfaAfcCfcUfaAfdT
1021
dTUfaGfgGfuUfaAfuAfuGfaAfgCfudTsdT





2395
835
UfaUfuAfaCfcCfuAfcCfuGfuCfaAfdT
1022
dTUfgAfcAfgGfuAfgGfgUfuAfaUfadTsdT





2396
836
UfuAfaCfcCfuAfcCfuGfuCfaAfcAfdT
1023
dTGfuUfgAfcAfgGfuAfgGfgUfuAfadTsdT





2397
837
AfcCfcUfaCfcUfgUfcAfaCfgUfaAfdT
1024
dTUfaCfgUfuGfaCfaGfgUfaGfgGfudTsdT





2398
838
CfcCfuAfcCfuGfuCfaAfcGfuAfaAfdT
1025
dTUfuAfcGfuUfgAfcAfgGfuAfgGfgdTsdT





2399
839
CfcUfaCfcUfgUfcAfaCfgUfaAfcAfdT
1026
dTGfuUfaCfgUfuGfaCfaGfgUfaGfgdTsdT





2400
840
CfuAfcCfuGfuCfaAfcGfuAfaCfgAfdT
1027
dTCfgUfuAfcGfuUfgAfcAfgGfuAfgdTsdT





2401
841
UfaCfcUfgUfcAfaCfgUfaAfcGfaAfdT
1028
dTUfcGfuUfaCfgUfuGfaCfaGfgUfadTsdT





2402
842
AfcCfuGfuCfaAfcGfuAfaCfgAfuAfdT
1029
dTAfuCfgUfuAfcGfuUfgAfcAfgGfudTsdT





2403
843
CfcUfgUfcAfaCfgUfaAfcGfaUfuAfdT
1030
dTAfaUfcGfuUfaCfgUfuGfaCfaGfgdTsdT





2404
844
CfuGfuCfaAfcGfuAfaCfgAfuUfuAfdT
1031
dTAfaAfuCfgUfuAfcGfuUfgAfcAfgdTsdT





2405
845
UfgUfcAfaCfgUfaAfcGfaUfuUfcAfdT
1032
dTGfaAfaUfcGfuUfaCfgUfuGfaCfadTsdT





2406
846
UfcAfaCfgUfaAfcGfaUfuUfcAfuAfdT
1033
dTAfuGfaAfaUfcGfuUfaCfgUfuGfadTsdT





2407
847
AfcGfuAfaCfgAfuUfuCfaUfgAfaAfdT
1034
dTUfuCfaUfgAfaAfuCfgUfuAfcGfudTsdT





2408
848
UfaUfuAfuAfuUfgUfcGfaAfuUfcAfdT
1035
dTGfaAfuUfcGfaCfaAfuAfuAfaUfadTsdT





2409
849
UfuAfuAfuUfgUfcGfaAfuUfcCfuAfdT
1036
dTAfgGfaAfuUfcGfaCfaAfuAfuAfadTsdT





2410
850
UfaUfuGfuCfgAfaUfuCfcUfaCfuAfdT
1037
dTAfgUfaGfgAfaUfuCfgAfcAfaUfadTsdT





2411
851
GfaAfuUfcCfuAfcUfgAfcAfaCfaAfdT
1038
dTUfgUfuGfuCfaGfuAfgGfaAfuUfcdTsdT





2412
852
UfcCfuAfcUfgAfcAfaCfaUfuAfuAfdT
1039
dTAfuAfaUfgUfuGfuCfaGfuAfgGfadTsdT





2413
853
UfaUfaAfcUfgUfaUfgGfgAfgCfuAfdT
1040
dTAfgCfuCfcCfaUfaCfaGfuUfaUfadTsdT





2414
854
UfaAfcUfgUfaUfgGfgAfgCfuUfaAfdT
1041
dTUfaAfgCfuCfcCfaUfaCfaGfuUfadTsdT





2415
855
UfgUfaUfgGfgAfgCfuUfaAfcUfuAfdT
1042
dTAfaGfuUfaAfgCfuCfcCfaUfaCfadTsdT





2416
856
UfuGfaCfaCfuGfgUfaUfcUfuAfuAfdT
1043
dTAfuAfaGfaUfaCfcAfgUfgUfcAfadTsdT





2417
857
AfaGfuAfuUfcUfgAfuCfcUfaCfcAfdT
1044
dTGfgUfaGfgAfuCfaGfaAfuAfcUfudTsdT
















TABLE 5C







Efficacy screen results of Hif2α RNAi triggers in viro, as determined by dual-luciferase reporter assay.










duplex
Relative Rluc-Hif2α
AD
Relative Rluc-Hif2α












number
1 nM
0.1 nM
number
1 nM
0.1 nM





2231
0.491 ± 0.198
0.544 ± 0.368
2325
1.379 ± 0.275
1.304 ± 0.134


2232
0.468 ± 0.032
0.684 ± 0.061
2326
0.546 ± 0.083
0.660 ± 0.114


2233
0.862 ± 0.125
0.913 ± 0.019
2327
1.073 ± 0.089
1.339 ± 0.179


2234
0.388 ± 0.046
0.508 ± 0.148
2328
1.192 ± 0.144
1.291 ± 0.109


2235
0.857 ± 0.090
0.743 ± 0.129
2329
0.456 ± 0.064
0.807 ± 0.117


2236
0.761 ± 0.042
0.693 ± 0.071
2330
0.464 ± 0.035
1.097 ± 0.416


2237
0.978 ± 0.083
0.746 ± 0.029
2331
0.783 ± 0.080
1.002 ± 0.321


2238
0.148 ± 0.024
0.208 ± 0.023
2332
0.871 ± 0.235
1.174 ± 0.027


2239
0.157 ± 0.017
0.225 ± 0.022
2333
0.291 ± 0.019
0.684 ± 0.147


2240
0.845 ± 0.052
0.841 ± 0.048
2334
0.389 ± 0.044
0.672 ± 0.082


2241
0.270 ± 0.068
0.385 ± 0.028
2335
0.104 ± 0.013
0.311 ± 0.032


2242
0.222 ± 0.064
0.411 ± 0.019
2336
0.299 ± 0.034
0.783 ± 0.037


2243
0.694 ± 0.107
0.688 ± 0.059
2337
0.131 ± 0.033
0.334 ± 0.066


2244
0.915 ± 0.115
0.760 ± 0.050
2338
0.334 ± 0.091
0.743 ± 0.070


2245
0.727 ± 0.066
0.761 ± 0.039
2339
0.189 ± 0.028
0.523 ± 0.134


2246
0.327 ± 0.042
0.509 ± 0.044
2340
0.444 ± 0.039
0.920 ± 0.114


2247
0.231 ± 0.048
0.439 ± 0.082
2341
0.202 ± 0.018
0.378 ± 0.116


2248
0.148 ± 0.036
0.215 ± 0.007
2342
0.549 ± 0.041
1.058 ± 0.064


2249
0.190 ± 0.028
0.303 ± 0.042
2343
0.254 ± 0.009
0.620 ± 0.208


2250
0.139 ± 0.046
0.255 ± 0.020
2344
0.276 ± 0.033
0.570 ± 0.071


2251
0.872 ± 0.121
0.857 ± 0.087
2345
0.129 ± 0.026
0.296 ± 0.096


2252
0.592 ± 0.061
0.696 ± 0.096
2346
0.273 ± 0.012
0.523 ± 0.133


2253
0.564 ± 0.043
0.646 ± 0.144
2347
0.530 ± 0.040
0.753 ± 0.092


2254
0.641 ± 0.054
0.756 ± 0.080
2348
0.454 ± 0.096
0.840 ± 0.270


2255
0.721 ± 0.055
0.653 ± 0.130
2349
0.504 ± 0.062
0.674 ± 0.172


2256
0.765 ± 0.124
0.796 ± 0.028
2350
0.678 ± 0.105
0.755 ± 0.085


2257
0.802 ± 0.044
0.647 ± 0.127
2351
1.306 ± 0.216
1.216 ± 0.415


2258
0.972 ± 0.168
0.882 ± 0.098
2352
0.749 ± 0.049
1.012 ± 0.048


2259
0.836 ± 0.110
0.743 ± 0.086
2353
0.324 ± 0.075
0.635 ± 0.156


2260
1.030 ± 0.187
0.861 ± 0.031
2354
0.741 ± 0.189
1.043 ± 0.239


2261
0.657 ± 0.067
0.568 ± 0.065
2355
0.248 ± 0.063
0.457 ± 0.066


2262
0.277 ± 0.071
0.755 ± 0.031
2356
0.210 ± 0.023
0.574 ± 0.006


2263
0.875 ± 0.059
1.126 ± 0.083
2357
0.435 ± 0.062
0.737 ± 0.082


2264
0.183 ± 0.019
0.381 ± 0.023
2358
0.731 ± 0.123
0.647 ± 0.113


2265
0.186 ± 0.045
0.448 ± 0.042
2359
0.354 ± 0.022
0.576 ± 0.208


2266
0.368 ± 0.046
0.808 ± 0.062
2360
0.962 ± 0.167
1.102 ± 0.119


2267
0.651 ± 0.049
1.029 ± 0.104
2361
0.603 ± 0.133
0.800 ± 0.198


2268
0.210 ± 0.037
0.409 ± 0.023
2362
0.454 ± 0.069
0.673 ± 0.089


2269
0.302 ± 0.080
0.530 ± 0.097
2363
0.452 ± 0.091
0.653 ± 0.199


2270
0.613 ± 0.147
0.658 ± 0.149
2364
0.855 ± 0.282
1.070 ± 0.042


2271
0.930 ± 0.094
1.056 ± 0.291
2365
1.259 ± 0.247
1.132 ± 0.107


2272
0.864 ± 0.197
1.028 ± 0.281
2366
0.695 ± 0.115
0.879 ± 0.223


2273
0.588 ± 0.057
0.574 ± 0.033
2367
0.376 ± 0.039
0.665 ± 0.064


2274
0.506 ± 0.074
0.870 ± 0.108
2368
1.140 ± 0.221
1.036 ± 0.063


2275
0.464 ± 0.048
0.515 ± 0.264
2369
0.338 ± 0.055
0.464 ± 0.032


2276
0.330 ± 0.080
0.543 ± 0.029
2370
0.294 ± 0.026
0.498 ± 0.128


2277
0.702 ± 0.091
0.625 ± 0.384
2371
0.166 ± 0.037
0.193 ± 0.030


2278
0.564 ± 0.070
0.947 ± 0.044
2372
0.514 ± 0.044
0.831 ± 0.103


2279
0.280 ± 0.042
0.506 ± 0.086
2373
0.162 ± 0.032
0.369 ± 0.082


2280
0.911 ± 0.086
0.871 ± 0.147
2374
0.192 ± 0.014
0.355 ± 0.020


2281
0.651 ± 0.041
0.914 ± 0.177
2375
0.305 ± 0.040
0.481 ± 0.062


2282
0.824 ± 0.155
1.209 ± 0.132
2376
0.228 ± 0.029
0.546 ± 0.115


2283
0.882 ± 0.026
1.175 ± 0.194
2377
0.958 ± 0.189
0.900 ± 0.189


2284
0.787 ± 0.062
1.227 ± 0.266
2378
0.734 ± 0.170
0.994 ± 0.246


2285
0.723 ± 0.088
1.109 ± 0.247
2379
0.939 ± 0.204
0.858 ± 0.098


2286
0.878 ± 0.078
1.291 ± 0.367
2380
1.722 ± 0.101
1.128 ± 0.285


2287
0.751 ± 0.011
0.871 ± 0.102
2381
1.063 ± 0.191
0.756 ± 0.109


2288
0.932 ± 0.118
0.943 ± 0.029
2382
1.151 ± 0.365
1.070 ± 0.064


2289
1.039 ± 0.146
0.992 ± 0.153
2383
1.060 ± 0.292
0.833 ± 0.082


2290
0.353 ± 0.036
0.698 ± 0.155
2384
0.439 ± 0.101
0.533 ± 0.067


2291
0.434 ± 0.046
0.911 ± 0.092
2385
0.515 ± 0.089
0.701 ± 0.178


2292
0.832 ± 0.000
1.135 ± 0.048
2386
0.274 ± 0.087
0.603 ± 0.032


2293
0.925 ± 0.126
1.010 ± 0.045
2387
0.201 ± 0.063
0.436 ± 0.028


2294
0.546 ± 0.033
0.743 ± 0.100
2388
0.436 ± 0.021
0.901 ± 0.318


2295
0.863 ± 0.122
1.047 ± 0.184
2389
0.820 ± 0.184
1.085 ± 0.307


2296
0.213 ± 0.028
0.411 ± 0.079
2390
0.568 ± 0.108
0.772 ± 0.175


2297
0.643 ± 0.028
0.704 ± 0.083
2391
0.129 ± 0.015
0.273 ± 0.006


2298
0.695 ± 0.037
0.729 ± 0.092
2392
0.283 ± 0.052
0.636 ± 0.039


2299
0.827 ± 0.067
0.907 ± 0.171
2393
1.049 ± 0.089
0.862 ± 0.388


2300
0.786 ± 0.181
1.090 ± 0.106
2394
0.254 ± 0.026
0.533 ± 0.076


2301
0.423 ± 0.102
0.558 ± 0.034
2395
0.218 ± 0.029
0.494 ± 0.104


2302
0.147 ± 0.028
0.414 ± 0.019
2396
0.939 ± 0.299
1.321 ± 0.070


2303
0.294 ± 0.034
0.414 ± 0.002
2397
0.119 ± 0.024
0.254 ± 0.048


2304
0.302 ± 0.025
0.528 ± 0.116
2398
0.534 ± 0.043
1.047 ± 0.047


2305
0.992 ± 0.217
0.961 ± 0.120
2399
0.418 ± 0.081
0.784 ± 0.071


2306
0.613 ± 0.031
0.596 ± 0.038
2400
0.165 ± 0.020
0.478 ± 0.028


2307
0.702 ± 0.142
0.800 ± 0.248
2401
0.174 ± 0.003
0.375 ± 0.062


2308
0.998 ± 0.059
0.799 ± 0.084
2402
0.128 ± 0.015
0.389 ± 0.060


2309
1.081 ± 0.135
0.823 ± 0.096
2403
0.568 ± 0.106
0.930 ± 0.132


2310
0.923 ± 0.101
0.785 ± 0.122
2404
0.104 ± 0.019
0.234 ± 0.045


2311
0.841 ± 0.130
0.888 ± 0.051
2405
0.138 ± 0.035
0.261 ± 0.044


2312
0.725 ± 0.173
0.899 ± 0.124
2406
0.168 ± 0.044
0.241 ± 0.022


2313
0.886 ± 0.243
0.853 ± 0.133
2407
0.124 ± 0.021
0.222 ± 0.027


2314
0.920 ± 0.150
0.692 ± 0.141
2408
0.282 ± 0.004
0.730 ± 0.045


2315
0.876 ± 0.099
0.781 ± 0.206
2409
0.104 ± 0.020
0.301 ± 0.085


2316
0.579 ± 0.055
0.797 ± 0.211
2410
0.154 ± 0.022
0.228 ± 0.033


2317
0.843 ± 0.157
0.842 ± 0.239
2411
0.410 ± 0.083
0.796 ± 0.110


2318
0.780 ± 0.103
0.885 ± 0.370
2412
0.291 ± 0.014
0.515 ± 0.037


2319
0.234 ± 0.055
0.454 ± 0.101
2413
0.317 ± 0.055
0.675 ± 0.104


2320
0.486 ± 0.059
0.770 ± 0.312
2414
0.167 ± 0.038
0.422 ± 0.070


2321
0.665 ± 0.033
0.561 ± 0.043
2415
0.119 ± 0.015
0.180 ± 0.097


2322
0.306 ± 0.059
0.468 ± 0.012
2416
0.166 ± 0.031
0.247 ± 0.031


2323
0.664 ± 0.166
0.631 ± 0.085
2417
0.253 ± 0.037
0.295 ± 0.057


2324
1.046 ± 0.208
1.081 ± 0.293









Example 11. Hif2α RNAi Trigger EC50 Determination

The eight best canonical sequences were further evaluated by determining the EC50 concentration. Each trigger was assessed for knockdown under the same conditions and assays as above, but at 10 different concentrations ranging from 0.00051 nM to 10 nM. EC50 were determined using GraphPad Prism software. Each of the top five canonical sequences were modified to contain UNA at sites 6 and 7. These triggers, along with their parent canonical sequences, were evaluated side-by-side for EC50 concentration determination using the same conditions and assays as above, Table 6.









TABLE 6







EC50 values (nM) determined in vitro for the indicated RNAi triggers.








EC50
EC50 UNAs











ID number
EC50 (nM)
ID number
Mod.
EC50














XD-02335
0.0593
AD00988
UNA6
0.8406




AD00993
UNA7
0.5504


XD-02337
0.1010





XD-02371
0.0592
AD00989
UNA6
0.3754




AD00994
UNA7
0.08068


XD-02391
0.1554





XD-02397
0.0858





XD-02404
0.0570
AD00990
UNA6
0.1534




AD00995
UNA7
0.1689


XD-02407
0.0287
AD00991
UNA6
0.3503




AD00996
UNA7
0.1176


XD-02415
0.0892
AD00992
UNA6
0.1419




AD00997
UNA7
0.1827









Example 12. Creation of SEAP-Expressing Clear Cell Renal Cell Carcinoma (ccRCC) A498 Cells

A pCR3.1 expression vector expressing the reporter gene secreted alkaline phosphatase (SEAP) under the CMV promoter was prepared by directional cloning of the SEAP coding sequence PCR amplified from Clontech's pSEAP2-basic vector. Convenient restriction sites were added onto primers used to amplify the SEAP coding sequence for cloning into the pCR3.1 vector (Invitrogen). The resultant construct pCR3-SEAP was used to create a SEAP-expressing A498 ccRCC cell line. Briefly, pCR3-SEAP plasmid was transfected into A498 ccRCC cells by electroporation following manufacturer's recommendation. Stable transfectants were selected by G418 resistance. Selected A498-SEAP clones were evaluated for SEAP expression and integration stability.


Example 13. Orthotopic RCC Tumor Bearing Mice with A498 Cell Stably Expressing SEAP

Female athymic nude mice were anesthetized with ˜3% isoflourane and placed in the right lateral decubitus position. A small, 0.5-1 cm, longitudinally abdominal incision in the left flank was made. Using a moist cotton swab, the left kidney was lifted out of the peritoneum and gently stabilized. Just before injection, a 1.0 ml syringe was filled with the cell/Matrigel mixture and a 27 gauge needle catheter was attached to the syringe tip. The filled syringe was then attached to a syringe pump (Harvard Apparatus, model PHD2000) and primed to remove air. The tip of a 27-gauge needle catheter attached to a syringe was inserted just below the renal capsule near the caudal pole and the tip of the needle was then carefully advanced cranially along the capsule 3-4 mm. A 10 μl aliquot of 2:1 (vol:vol) cell/matrigel mixture containing about 300,000 cells was slowly injected into the kidney parenchyma using a syringe pump. The needle was left in the kidney for 15-20 seconds to ensure the injection was complete. The needle was then removed from the kidney and a cotton swab was placed over the injection site for 30 seconds to prevent leakage of the cells or bleeding. The kidney was then gently placed back into the abdomen and the abdominal wall was closed. Serum was collected every 7-14 days after implantation to monitor tumor growth using a commercial SEAP assay kit. For most studies, tumor mice were used 5-6 weeks after implantation, when tumor measurements were typically around 4-8 mm.


Example 14. Evaluation of HiF2α-RNAi Triggers in Orthotopic RCC Tumor Bearing Mice

RGD targeted HiF2α-RNAi trigger delivery conjugates. Delivery polymers were modified using RGD-PEG-HyNic, RGD-PEG-ACit-PNP, or RDG-PEG-FCitFP-TFP and PEG-dipeptide modifying agents. The indicated amount of polymer 126 or 100A polymer was modified with 8×PEG12-ACit-PABC-PNP/0.5× aldehyde-PEG24-FCit-PABC-PNP (with RGD mimic #1-PEG-HyNic using protocol #1) and the indicated amount of the indicated Hif2α RNAi trigger.


Polymer 064 was modified according to protocol 7. Kidney RCC tumor-bearing mice were generated as described and treated with a single tail vein injection of isotonic glucose (G1) or the indicated Hif2α RNAi trigger-delivery polymer conjugate. Mice were euthanized at the indicated time after injection and total RNA was prepared from kidney tumor using Trizol reagent following manufacturer's recommendation. Relative HiF2α mRNA levels were determined by RT-qPCR as described below and compared to mice treated with delivery buffer (isotonic glucose) only.









TABLE 7







Hif2α knockdown in mice following Hif2α RNAi trigger delivery.


RNAi triggers were conjugated to the


indicated reversibly modified delivery polymer.










RNAi trigger


Relative Expression











duplex

Polymer
day
low error/












number
μg
number
μg
4
high error















isotonic glucose
0

0
1.00
0.06/0.06


AD01031
80
126
400
0.20
0.04/0.05


AD01214
80
126
400
0.29
0.08/0.12


AD01214
112.5
006
300
0.36
0.07/0.09


AD01255
80
126
400
0.28
0.05/0.05


AD01476
115
100A
375
0.32
0.04/0.04


AD01291
80
126
400
0.19
0.03/0.03


AD01292
80
126
400
0.27
0.06/0.08


AD01293
80
126
400
0.20
0.01/0.01


AD01294
80
126
400
0.17
0.01/0.02


AD01295
80
126
400
0.22
0.02/0.02


AD01296
80
126
400
0.21
0.04/0.06


AD01029
80
126
400
0.94
0.08/0.09


AD01030
80
126
400
0.47
0.08/0.10


AD01256
80
126
400
0.22
0.05/0.07


AD01257
80
126
400
0.24
0.04/0.05


AD01258
80
126
400
0.38
0.03/0.03


AD01424
150
100A
300
0.54
0.06/0.07


AD01404
150
100A
300
0.58
0.11/0.13


AD01405
150
100A
300
0.51
0.10/0.12


AD01406
150
100A
300
0.45
0.06/0.07


AD01407
150
100A
300
0.47
0.06/0.07


AD01408
150
100A
300
0.50
0.07/0.09


AD01409
150
100A
300
0.55
0.03/0.03


AD01410
150
100A
300
0.41
0.10/0.12


AD01411
150
100A
300
0.36
0.01/0.01


AD01288
115
100A
375
0.32
0.02/0.02


AD01522
115
100A
375
0.44
0.07/0.09


AD01289
115
100A
375
0.28
0.06/0.08


AD01290
115
100A
375
0.39
0.02/0.02


AD01523
115
100A
375
0.49
0.04/0.05


AD01524
115
100A
375
0.26
0.05/0.07


AD01554
115
100A
375
0.29
0.04/0.04


AD01555
115
100A
375
0.20
0.03/0.03


AD01025
80
126
400
0.34
0.02/0.02


AD01023
80
126
400
0.93
0.10/0.12


AD01024
80
126
400
0.51
0.04/0.05


AD01028
80
126
400
0.39
0.04/0.04


AD01026
80
126
400
0.97
0.12/0.13


AD01027
80
126
400
0.92
0.08/0.09


AD01034
80
126
400
0.35
0.07/0.09


AD01032
80
126
400
1.00
0.09/0.10


AD01033
80
126
400
1.08
0.08/0.09


AD01022
80
126
400
0.53
0.07/0.09


AD01020
80
126
400
0.94
0.06/0.06


AD01021
80
126
400
0.90
0.05/0.05


AD01654
100
100A
250
0.185
0.01/0.01


AD01655
100
100A
250
0.234
0.02/0.02


AD01656
100
100A
250
0.184
0.01/0.01


AD01657
100
100A
250
0.256
0.03/0.03


AD01658
100
100A
250
0.138
0.01/0.01


AD01659
100
100A
250
0.249
0.04/0.05


AD01884
100
064
250
0.091
0.01/0.01


AD01885
100
064
250
0.146
0.02/0.02


AD01886
100
064
250
0.292
0.04/0.05


AD01887
100
064
250
0.329
0.05/0.06


AD01888
100
064
250
0.209
0.04/0.08


AD01889
100
064
250
0.282
0.04/0.05


AD01890
100
064
250
0.256
0.02/0.03


AD01891
100
064
250
0.189
0.02/0.03


AD01892
100
064
250
0.146
0.03/0.03


AD01893
100
064
250
0.115
0.02/0.03


AD02691
75
064
187.5
0.124
0.04/0.07


AD02692
75
064
187.5
0.089
0.03/0.04


AD02693
75
064
187.5
0.122
0.01/0.01


AD02694
75
064
187.5
0.099
0.01/0.01


AD02695
75
064
187.5
0.101
0.02/0.02


AD02733
75
064
187.5
0.283
0.02/0.03


AD02734
75
064
187.5
0.262
0.03/0.03


AD02735
75
064
187.5
0.080
0.01/0.02


AD01884
75
064
187.5
0.227
0.02/0.02


AD01884
50
064
125
0.203
0.03/0.03


AD02692
75
064
187.5
0.121
0.02/0.02


AD02692
50
064
125
0.092
0.01/0.01


AD02695
75
064
187.5
0.155
0.02/0.02


AD02695
50
064
125
0.123
0.03/0.03


AD02735
75
064
187.5
0.222
0.07/0.10


AD02735
50
064
125
0.144
0.03/0.03


AD02857
50
064
125
0.192
0.03/0.03


AD02858
50
064
125
0.192
0.04/0.05


AD02859
50
064
125
0.214
0.03/0.03


AD02860
50
064
125
0.165
0.02/0.02


AD02949
50
064
125
0.176
0.05/0.07


AD02074
50
064
125
0.566
0.01/0.13


AD02861
50
064
125
0.103
0.03/0.05


AD02862
50
064
125
0.093
0.05/0.11


AD02873
50
064
125
0.079
0.04/0.06


AD02875
50
064
125
0.101
0.01/0.02


AD03011
50
064
125
0.183
0.01/0.02


AD02874
50
064
125
0.138
0.05/0.07


AD03187
50
064
125
1.000
0.06/0.07


AD03188
50
064
125
0.308
0.08/0.07


AD03189
50
064
125
0.245
0.02/0.02


AD03190
50
064
125
0.269
0.08/0.12


AD03191
50
064
125
0.307
0.13/0.24


AD03192
50
064
125
0.286
0.01/0.01


AD03193
50
064
125
0.275
0.07/0.09


AD03125
50
064
125
0.205
0.04/0.05


AD03126
50
064
125
0.172
0.04/0.04


AD03253
50
064
125
0.188
0.04/0.05


AD03264
50
064
125
0.264
0.09/0.13


AD03265
50
064
125
0.294
0.07/0.08


AD03266
50
064
125
0.408
0.07/0.08


AD03254
50
064
125
0.295
0.06/0.07


AD03255
50
064
125
0.264
0.07/0.10


AD03256
50
064
125
0.333
0.06/0.08


AD03257
50
064
125
0.203
0.04/0.05


AD03258
50
064
125
0.137
0.02/0.02


AD03259
50
064
125
0.179
0.03/0.04


AD03260
50
064
125
0.175
0.03/0.04


AD03261
50
064
125
0.177
0.02/0.02


AD03262
50
064
125
0.197
0.01/0.01


AD03263
50
064
125
0.134
0.04/0.06









Quantitative Real-Time PCR assay. In preparation for quantitative PCR, total RNA was isolated from tissue samples homogenized in TrReagent (Molecular Research Center, Cincinnati, Ohio) following the manufacturers protocol. Approximately 500 ng RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit (Life Technologies). For human (tumor) Hif2α (EPAS 1) expression, pre-manufactured TaqMan gene expression assays for human Hif2α (Catalog #4331182) and CycA (PPIA) Catalog #: 4326316E) were used in biplex reactions in triplicate using TaMan Gene Expression Master Mix (Life Technologies) or VeriQuest Probe Master Mix (Affymetrix). For human (tumor) VegFa (VEGFA) expression, pre-manufactured TaqMan gene expression assays for human VegFa (Catalog #4331182, Assay ID: Hs00900055) and CycA (Part #: 4326316E) were used in biplex reactions in triplicate using TaqMan Gene Expression Master Mix (Life Technologies) or VeriQuest Probe Master Mix (Affymetrix). Quantitative PCR was performed by using a 7500 Fast or StepOnePlus Real-Time PCR system (Life Technologies). The AACT method was used to calculate relative gene expression.


Example 15. Multi-Dose Hif2α RNAi Trigger-Delivery Polymer Conjugate Inhibits Tumor Growth in Orthotopic RCC Tumor Bearing Mice

Hif2α RNAi trigger-delivery polymer conjugate was prepared using protocol #1 with RNAi trigger duplex ID AD01031 and polymer Ant 126. The conjugate was then TFF purified and polymer concentration, RNAi trigger, RGD and modifying conjugation efficiency was determined as described above. Weekly doses of Hif2α RNAi trigger-delivery polymer conjugate containing either 400 μg (polymer weight) or 280 μg (polymer weight) were administered intravenously to 2 different groups of tumor bearing mice. Tumor bearing mice receiving isotonic glucose (IG) were used as treatment control. A total of 3 weekly doses were administered during the course of study. Tumor growth rates were evaluated by serum SEAP collected at 5-7 days interval during treatment. Tumor weight and volume was determined at necropsy. Gross tumor morphology and H&E histopathology were evaluated.









TABLE 8







Hif2α RNAi trigger-delivery polymer conjugate


knockdown HiF2α and VEGFa










Hif2α expression
VEGFa expression











Treat-
Relative
low/high
Relative
low/high


ment
Expression
error
Expression
error





G1-IG
1.00
0.07/0.07
1.00
0.07/0.08


G2-400 μg
0.18
0.03/0.04
0.45
0.07/0.09


G3-280 μg
0.19
0.04/0.05
0.39
0.10/0.13









Expression of Hif2α in the 400 μg or 280 μg Hif2α RNAi trigger-delivery polymer conjugate group was 82% and 81% decreased, respectively, compared to control treatment (Table 8). Expression of VEGFa, a well characterized down-stream Hif2α regulated gene, was also decreased by 55% and 61%, respectively (Table 8).


Collectively, after 3 weekly Hif2α RNAi trigger-delivery polymer conjugate injections, tumor growth was dramatically inhibited in both dosages evaluated. This is supported by the overall tumor sizes and serum SEAP levels (FIGS. 8-9 and 12, Table 9). A downward trend of the SEAP levels after the third injections suggest beginning of tumor regression. In addition, tumor histopathology examination from H&E stained formalin fixed paraffin sections showed destruction of typical RCC tubular structure. The number of apoptotic cells was increased in treatment groups. Some tumor sample contained large areas of tumor necrosis (FIG. 10).









TABLE 9







Serum SEAP levels during treatment, G1 and G2, n = 4; G3, n = 3









SEAP (fold-changed relative to day −1)











Treatment
Day −1
Day 7
Day 14
Day 21





G1-IG
1.00 ± 0.19
1.65 ± 0.22
3.44 ± 0.23
7.48 ± 0.27


G2-400 μg
1.00 ± 0.45
0.79 ± 0.43
1.36 ± 0.61
1.25 ± 0.68


G3-280 μg
1.00 ± 0.14
1.46 ± 0.09
2.55 ± 0.29
1.82 ± 0.35









Example 16. Evaluation of HiF2α-RNAi triggers in orthotopic RCC tumor bearing mice. RGD targeted HiF2α-RNAi trigger delivery polymer conjugates were formed using polymer 126, 100A, or 006. The RNAi trigger, μg indicates the quantity of trigger reacted with polymer. The polymer was modified with the indicated RGD mimic and PEG modifying agents as described above. Kidney RCC tumor-bearing mice were generated as described and treated with a single tail vein injection of isotonic glucose (G1) or the indicated Hif2α RNAi trigger-delivery polymer conjugate. Mice were euthanized 72 h (day 4) after injection and total RNA was prepared from kidney tumor using Trizol reagent following manufacturer's recommendation. Relative HiF2α mRNA levels were determined by RT-qPCR as described and compared to mice treated with delivery buffer (isotonic glucose) only (Table 11).


Example 17. HiF2α RNAi Trigger/Second Therapeutic Combination Study

HiF2α RNAi trigger-delivery polymer conjugate (125 μg polymer) was prepared using protocol 7 Duplex ID No. AD1884 and polymer 064. HiF2α RNAi trigger-delivery polymer conjugate was dosed every 4 weeks by iv injection, 4 doses total. Sunitinib (Malate salt) obtained from LC laboratories was suspended in Ora-plus/Ora sweet (50:50, vol:vol). Sunitinib treatment started 2 weeks after the first HiF2α RNAi trigger dose was administered. Mice were dosed by oral gavage 5 days/week for 2 weeks, then off 2 weeks, 3 cycles total.


Tumor growth rates were evaluated by serum SEAP collected at 5-7 days interval during treatment. Tumor weight and volume was determined at necropsy. Gross tumor morphology and H&E histopathology were evaluated. Relative HiF2a expression levels were of were 11.4%, 73.8%, and 77.6% decreased in the sunitinib alone, DPC+sunitinib and DPC alone treated groups, respectively (Table 10A). Combined HiF2a RNAi trigger and sunitinib treatments resulted in increased tumor growth inhibition response. Overall smaller tumor sizes were smaller and lower overall growth (as measured by overall-fold increase in SEAP) was observed (Table 10B).









TABLE 10A







HiF2α expression in RCC tumors in animal models treated with


Sunitinib, HiF2α RNAi trigger, or HiF2α RNAi trigger + Sunitinib.









HiF2α expression










Treatment
Relative expression
Low error
High error





isotonic glucose
1.000
0.153
0.180


Sunitinib
0.886
0.070
0.076


HiF2α RNAi trigger
0.262
0.071
0.097


HiF2α RNAi trigger +
0.224
0.081
0.126


Sunitinib
















TABLE 10B







Tumor size and SEAP expression in RCC tumors in animal models treated with


Sunitinib, HiF2α RNAi trigger, or HiF2α RNAi trigger + Sunitinib.










Treatment
Tumor weight (mg)
Tumor volume (mm3)
Fold increase in SEAP













isotonic glucose
4158.5 ± 865 
3576.3 ± 279
 21.8 ± 11


Sunitinib
2385.7 ± 845 
2113.5 ± 368
  18.1 ± 9.8


HiF2α RNAi trigger
2130.9 ± 1066
1537.4 ± 999
20.54 ± 20


HiF2α RNAi trigger +
1075.8 ± 600 
1008.8 ± 650
  5.7 ± 3.3


Sunitinib












Other Embodiments: It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.









TABLE 11







Hif2α expression in RCC cells in RCC tumor


bearing mice following Hif2α RNAi trigger delivery.













HiF2α
polymer




Relative Expression














RNAi trigger


purifi-
RGD
PEG

error


















duplex ID
μg
ID
μg
cation
modifying agent
amt.
no.
modifying agent
day 4
low
high




















isotonic glucose







1.000
0.060
0.064


















AD01031
80
126
280

RGD-PEG8-HyNic
0.5×

PEG12-ACit-PABC-PNP
0.300
0.074
0.098


AD01031
80
126
280

RGD-PEG8-ACit-
0.4×

PEG6-ACit-PABC-PNP
0.311
0.055
0.067







PABC-PNP








AD01214
80
126
280
TFF
RGD-PEG8-HyNic
0.5×

PEG12-FCit-PABC-PNP
0.489
0.022
0.023


AD01214
80
126
280

RGD-PEG8-HyNic
0.5×

PEG12-FCitFP-TFP
0.210
0.032
0.038


AD01214
80
126
280

RGD-PEG8-ACit-
0.4×

PEG6-ACit-PABC-PNP
0.360
0.019
0.021







PABC-PNP








AD01214
80
126
280

RGD-PEG8-ACitFP-NHS
  1×

PEG6-ACit-PABC-PNP
0.288
0.005
0.005


AD01214
115
100A
375

RGD-PEG8-HyNic
0.5×

PEG12-FCitFP-TFP
0.258
0.033
0.038


AD01214
112.5
100A
375
TFF
RGD-PEG15-FCitFP-TFP
0.5×
10.8
PEG12-ACit-PABC-PNP
0.193
0.046
0.061


AD01214
112.5
100A
375
TFF
RGD-PEG15-FCitFP-TFP
  1×
16.1
PEG12-ACit-PABC-PNP
0.182
0.007
0.008


AD01214
112.5
100A
375
TFF
RGD-PEG15-FCitFP-TFP
  2×
29.0
PEG12-ACit-PABC-PNP
0.182
0.031
0.038


AD01214
112.5
100A
375
TFF
RGD-PEG19-FCitFP-TFP
0.5×
10.7
PEG12-ACit-PABC-PNP
0.163
0.023
0.027


AD01214
112.5
100A
375
TFF
RGD-PEG19-FCitFP-TFP
  1×
18.5
PEG12-ACit-PABC-PNP
0.114
0.011
0.012


AD01214
112.5
100A
375
TFF
RGD-PEG19-FCitFP-TFP
  2×
31.1
PEG12-ACit-PABC-PNP
0.182
0.047
0.063


AD01214
112.5
100A
375

RGD-PEG19-FCitFP-TFP
  1×

PEG12-FCitFP-TFP
0.148
0.079
0.169


AD01214
112.5
100A
375

RGD-PEG19-FCitFP-TFP
  1×

PEG12-FCitFP-TFP
0.188
0.026
0.03


AD01214
112.5
100A
375

RGD-PEG19-FCitFP-TFP
  1×

PEG12-ACit-PABC-PNP
0.195
0.043
0.055


AD01214
112.5
006
300

RGD-PEG8-HyNic


PEG12-ACit-PABC-PNP
0.357
0.069
0.086


AD01214
112.5
100A
375
TFF
RGD-PEG20-FCitFP-TFP
0.125× 
1.9
PEG12-ACit-PABC-PNP
0.169
0.052
0.075


AD01214
112.5
100A
375
TFF
RGD-PEG20-FCitFP-TFP
0.25× 
3.4
PEG12-ACit-PABC-PNP
0.168
0.029
0.035


AD01214
112.5
100A
375
TFF
RGD-PEG20-FCitFP-TFP
0.5×
6.6
PEG12-ACit-PABC-PNP
0.130
0.004
0.005


AD01214
112.5
100A
375
TFF
RGD-PEG20-FCitFP-TFP
  1×
12.7
PEG12-ACit-PABC-PNP
0.121
0.016
0.018


AD01214
112.5
100A
375
TFF
RGD-PEG20-FCitFP-TFP
1.5×
20.3
PEG12-ACit-PABC-PNP
0.135
0.018
0.020








Claims
  • 1. A composition comprising an RNA interference (RNAi) trigger for inhibiting the expression of an Hif2α gene, wherein the RNAi trigger comprises a sense strand and an antisense strand, wherein said antisense strand comprises the base sequence of nucleotides 2-19 of any of the antisense strands of Table 1, Table 2 or Table 5.
  • 2. The composition of claim 1, wherein the antisense strand comprises a nucleotide base sequence of nucleotides 2-21 of SEQ ID NO. 4 (UUCAUGAAAUCGUUACGUUG) or SEQ ID NO. 38 (GUAAAACAAUUGUGUACUUU).
  • 3. The composition of claim 2, wherein a) the antisense strand comprises a nucleotide base sequence of nucleotides 2-21 of SEQ ID NO. 4 (UUCAUGAAAUCGUUACGUUG) and the sense strand comprises a nucleotide base sequence of SEQ ID NO. 53 (ACGUAACGAUUUCAUGAAU), orb) the antisense strand comprises a nucleotide base sequence of nucleotides SEQ ID NO. 38 (GUAAAACAAUUGUGUACUUU) and the sense strand comprises a nucleotide base sequence of nucleotides 1-19 of SEQ ID NO. 56 (AGUACACAAUUGUUUUACA).
  • 4. The composition of claim 1, wherein the sense strand and/or the antisense strand further comprises a 3′ and/or 5′ extension of 1-6 nucleotides in length.
  • 5. The composition of claim 1, wherein a targeting group is conjugated to the RNAi trigger.
  • 6. The composition of claim 5, wherein the targeting group comprises a compound selected from the group consisting of: integrin-binding compound, αvβ3 integrin-binding ligand, RGD peptide ligand, and RGD mimic.
  • 7. The composition of claim 1, wherein a delivery polymer is conjugated to the RNAi trigger.
  • 8. The composition of claim 1, wherein a linking group is conjugated to the RNAi trigger.
  • 9. The composition of claim 1, wherein the sense strand and/or antisense strand independently comprises one or more modified nucleotides or nucleotide mimics.
  • 10. The composition of claim 9, wherein the sense strand contains one, two, or three 2′-deoxy-2′-fluoro modified nucleotides at positions 11, 12, and/or 13 from the 3′ end.
  • 11. The composition of claim 9, wherein the antisense strand contains a 2′-deoxy-2′-fluoro modified nucleotide at position 2 from the 5′ end.
  • 12. The composition of claim 9, wherein the antisense strand contains a 2′-deoxy-2′-fluoro modified nucleotide at position 14 from the 5′ end.
  • 13. The composition of claim 9, wherein the antisense strand contains one, two, three, or four 2′-F nucleotides at positions 4, 6, 8, 10, and 12 from the 5′ end.
  • 14. The composition of claim 1, wherein the RNAi trigger comprises one or more phosphorothioate internucleotide linkages.
  • 15. The composition of claim 14, wherein the antisense strand contains one, two, three, or four phosphorothioate internucleotide linkages.
  • 16. The composition of claim 1, further comprising one or more additional therapeutics or treatments.
  • 17. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.
  • 18. The composition of claim 1, wherein said composition is packaged in a kit, container, pack, dispenser, pre-filled syringes, or vials.
  • 19. A method for inhibiting Hif2α expression in a cell, tissue, or subject, the method comprising: administering to the subject a therapeutically effective amount of a composition of claim 1.
  • 20. The method of claim 19, wherein the composition is administered via subcutaneous injection.
  • 21. The method of claim 19, wherein the cell or tissue is a renal cell carcinoma cell.
  • 22. An (RNAi) trigger capable of inhibiting the expression of an Hif2α gene, wherein the RNAi trigger comprises a sense strand and an antisense strand, wherein said antisense strand comprises any of the antisense sequences in Table 1, Table 2, or Table 5.
CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser. No. 15/954,316, filed Apr. 16, 2018 which is a divisional of U.S. patent application Ser. No. 15/166,311, filed May 27, 2016 (now U.S. Pat. No. 9,976,141), which claims benefit to U.S. Provisional Patent Application Ser. No. 62/168,244 filed on May 29, 2015, the disclosure of each of which is specifically incorporated by reference herein.

Provisional Applications (1)
Number Date Country
62168244 May 2015 US
Divisions (2)
Number Date Country
Parent 15954316 Apr 2018 US
Child 17179605 US
Parent 15166311 May 2016 US
Child 15954316 US