Patent Application
20210180066
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.
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.
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.
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:
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.
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.
In some embodiments, we describe compositions represented by the formula:
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)
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.
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:
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:
wherein RD is:
In some embodiments, a guanidinium is
and their resonance structures.
In some embodiments, an RGD mimic comprises the structure represented by:
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.
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.
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
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:
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:
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:
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:
wherein R1 comprises a targeting group or a steric stabilizer.
In some embodiments, the disubstituted alkylmaleic anhydride has the structure represented by:
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
In some embodiments, we describe compositions represented by the formula:
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.
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).
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.
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.
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.
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.
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.
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.
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.
A. RGD mimic #1-PEGn-HyNic, MW 1272.
B. RGD mimic #1a-HyNic, MW 802.8.
C. RGD mimic #1b-HyNic, MW 830.9 (RGD).
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).
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)).
(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)).
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).
PEGe-FCitFP modifying agents were made using similar chemistry.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 (
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).
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).
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.
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.
| Number | Date | Country | |
|---|---|---|---|
| 62168244 | May 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15954316 | Apr 2018 | US |
| Child | 17179605 | US | |
| Parent | 15166311 | May 2016 | US |
| Child | 15954316 | US |
