Nucleic acid-polypeptide compositions and uses thereof

Abstract

Disclosed herein are compositions and pharmaceutical formulations that comprise a binding moiety conjugated to a polynucleic acid molecule and a polymer. Also described herein include methods for treating a cancer which utilize a composition or a pharmaceutical formulation comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer.

Description

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 11, 2018, is named 45532-707_303_SL.txt and is 615,705 bytes in size.

BACKGROUND OF THE DISCLOSURE

Gene suppression by RNA-induced gene silencing provides several levels of control: transcription inactivation, small interfering RNA (siRNA)-induced mRNA degradation, and siRNA-induced transcriptional attenuation. In some instances, RNA interference (RNAi) provides long lasting effect over multiple cell divisions. As such, RNAi represents a viable method useful for drug target validation, gene function analysis, pathway analysis, and disease therapeutics.

SUMMARY OF THE DISCLOSURE

Disclosed herein, in certain embodiments, are compositions and pharmaceutical formulations that comprise a binding moiety conjugated to a polynucleic acid molecule and a polymer. In some embodiments, also described herein include methods for treating a disease or condition (e.g., cancer) that utilize a composition or a pharmaceutical formulation comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer.

Disclosed herein, in certain embodiments, is a molecule of Formula (I):A-X—B—Y—C   Formula I

• • wherein,
    • A is a binding moiety;
    • B is a polynucleotide;
    • C is a polymer;
    • X is a bond or first linker; and
    • Y is a bond or second linker; and
  • wherein the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety.

In some embodiments, the at least one 2′ modified nucleotide comprises 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified nucleotide. In some embodiments, the at least one 2′ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA). In some embodiments, the at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the at least one inverted abasic moiety is at at least one terminus.

In some embodiments, the polynucleotide comprises a single strand. In some embodiments, the polynucleotide comprises two or more strands. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some embodiments, the second polynucleotide comprises at least one modification.

In some embodiments, the first polynucleotide and the second polynucleotide are RNA molecules. In some embodiments, the first polynucleotide and the second polynucleotide are siRNA molecules.

In some embodiments, the first polynucleotide comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117. In some embodiments, the first polynucleotide consists of a sequence selected from SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117.

In some embodiments, the second polynucleotide comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117. In some embodiments, the second polynucleotide consists of a sequence selected from SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117.

In some embodiments, X and Y are independently a bond or a non-polymeric linker group. In some embodiments, X is a bond. In some embodiments, X is a C₁-C₆ alkyl group. In some embodiments, Y is a C₁-C₆ alkyl group. In some embodiments, X is a homobifuctional linker or a heterobifunctional linker, optionally conjugated to a C₁-C₆ alkyl group. In some embodiments, Y is a homobifuctional linker or a heterobifunctional linker.

In some embodiments, the binding moiety is an antibody or binding fragment thereof. In some embodiments, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or binding fragment thereof. In some embodiments, the antibody or binding fragment thereof is an anti-EGFR antibody or binding fragment thereof.

In some embodiments, C is polyethylene glycol. In some embodiments, C has a molecular weight of about 5000 Da.

In some embodiments, A-X is conjugated to the 5′ end of B and Y—C is conjugated to the 3′ end of B. In some embodiments, Y—C is conjugated to the 5′ end of B and A-X is conjugated to the 3′ end of B. In some embodiments, A-X, Y—C or a combination thereof is conjugated to an internucleotide linkage group.

In some embodiments, the molecule further comprises D. In some embodiments, D is conjugated to C or to A.

In some embodiments, D is conjugated to the molecule of Formula (I) according to Formula (II):(A-X—B—Y—C_n)-L-D   Formula II

• • wherein,
    • A is a binding moiety;
    • B is a polynucleotide;
    • C is a polymer;
    • X is a bond or first linker;
    • Y is a bond or second linker;
    • L is a bond or third linker;
    • D is an endosomolytic moiety; and
    • n is an integer between 0 and 1; and
  • wherein the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety; and D is conjugated anywhere on A, B, or C.

In some embodiments, D is INF7 or melittin.

In some embodiments, D is an endosomolytic polymer.

In some embodiments, L is a C₁-C₆ alkyl group. In some embodiments, L is a homobifuctional linker or a heterobifunctional linker.

In some embodiments, the molecule further comprises at least a second binding moiety A. In some embodiments, the at least second binding moiety A is conjugated to A, to B, or to C. In some embodiments, the at least second binding moiety A is cholesterol.

In some embodiments, the molecule further comprises at least an additional polynucleotide B. In some embodiments, the at least an additional polynucleotide B is conjugated to A, to B, or to C.

In some embodiments, the molecule further comprises at least an additional polymer C. In some embodiments, the at least an additional polymer C is conjugated to A, to B, or to C.

Disclosed herein, in certain embodiments, is a molecule of Formula (I): A-X—B—Y—C (Formula I), wherein A is an antibody or its binding fragments thereof; B is a polynucleotide; C is a polymer; X is a bond or first non-polymeric linker; and Y is a bond or second linker; wherein the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety; and wherein A and C are not attached to B at the same terminus. In some embodiments, the at least one 2′ modified nucleotide comprises 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified nucleotide. In some embodiments, the at least one 2′ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA). In some embodiments, the at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the at least one inverted abasic moiety is at at least one terminus. In some embodiments, the polynucleotide comprises a single strand. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some embodiments, the second polynucleotide comprises at least one modification. In some embodiments, the first polynucleotide and the second polynucleotide are RNA molecules. In some embodiments, the first polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117. In some embodiments, the second polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117. In some embodiments, Y is a non-polymeric linker group. In some embodiments, X is a bond. In some embodiments, X is a C₁-C₆ alkyl group. In some embodiments, Y is a C₁-C₆ alkyl group. In some embodiments, X is a homobifuctional linker or a heterobifunctional linker, optionally conjugated to a C₁-C₆ alkyl group. In some embodiments, Y is a homobifuctional linker or a heterobifunctional linker. In some embodiments, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or binding fragment thereof. In some embodiments, C is polyethylene glycol. In some embodiments, C has a molecular weight of about 1000 Da, 2000 Da, or 5000 Da. In some embodiments, A-X is conjugated to the 5′ end of B and Y—C is conjugated to the 3′ end of B. In some embodiments, Y—C is conjugated to the 5′ end of B and A-X is conjugated to the 3′ end of B. In some embodiments, the molecule further comprises D. In some embodiments, D is conjugated to C or to A. In some embodiments, D is conjugated to the molecule of Formula (I) according to Formula (II): (A-X—B—Y—C_c)-L-D (Formula II), wherein A is an antibody or its binding fragments thereof; B is a polynucleotide; C is a polymer; X is a bond or first non-polymeric linker; Y is a bond or second linker; L is a bond or third linker; D is an endosomolytic moiety; and c is an integer between 0 and 1; wherein the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety; wherein A and C are not attached to B at the same terminus; and wherein D is conjugated anywhere on A or C or to a terminus of B. In some embodiments, D is INF7 or melittin. In some embodiments, D is an endosomolytic polymer. In some embodiments, L is a C₁-C₆ alkyl group. In some embodiments, L is a homobifuctional linker or a heterobifunctional linker. In some embodiments, the molecule further comprises at least a second binding moiety. In some embodiments, the at least second binding moiety is conjugated to A, to B, or to C. In some embodiments, the at least second binding moiety is cholesterol. In some embodiments, the molecule further comprises at least an additional polynucleotide B. In some embodiments, the at least an additional polynucleotide B is conjugated to A, to B, or to C. In some embodiments, the molecule further comprises at least an additional polymer C. In some embodiments, the at least an additional polymer C is conjugated to A, to B, or to C.

Disclosed herein, in certain embodiments, is a pharmaceutical composition comprising a molecule described above, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated as a nanoparticle formulation. In some embodiments, the pharmaceutical composition is formulated for parenteral, oral, intranasal, buccal, rectal, or transdermal administration.

Disclosed herein, in certain embodiments, is a method of treating a disease or disorder in a patient in need thereof, comprising administering to the patient a composition comprising a molecule described above. In some embodiments, the disease or disorder is a cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer comprises a KRAS-associated, an EGFR-associated, an AR-associated cancer, a β-catenin associated cancer, a PIK3C-associated cancer, or a MYC-associated cancer. In some embodiments, the cancer comprises bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, glioblastoma multiforme, head and neck cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, or thyroid cancer. In some embodiments, the cancer comprises acute myeloid leukemia, CLL, DLBCL, or multiple myeloma. In some embodiments, the method is an immuno-oncology therapy.

Disclosed herein, in certain embodiments, is a method of inhibiting the expression of a target gene in a primary cell of a patient, comprising administering a molecule described above to the primary cell. In some embodiments, the method is an in vivo method. In some embodiments, the patient is a human.

Disclosed herein, in certain embodiments, is an immuno-oncology therapy comprising a molecule described above for the treatment of a disease or disorder in a patient in need thereof.

Disclosed herein, in certain embodiments, is a kit comprising a molecule described above.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings below. The patent application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1A-FIG. 1V illustrate cartoon representations of molecules described herein.

FIG. 2 illustrates a structure of cholesterol conjugate passenger strand.

FIG. 3 shows an INF7 peptide sequence (SEQ ID NO: 2055) described herein.

FIG. 4 shows a melittin peptide sequence (SEQ ID NO: 2060) described herein.

FIG. 5 illustrates an analytical HPLC of EGFR antibody-PEG20kDa-EGFR.

FIG. 6 illustrates a SDS-PAGE analysis of EGFR antibody-PEG20kDa-EGFR conjugate.

FIG. 7 illustrates an analytical chromatogram of EGFR antibody-PEG10kDa-EGFR siRNA.

FIG. 8 shows an analytical chromatogram of EGFR antibody-PEG5kDa-EGFR siRNA.

FIG. 9 shows a SDS PAGE analysis of EGFR antibody-PEG10kDa-EGFR siRNA and EGFR antibody-PEG5kDa-EGFR siRNA conjugates.

FIG. 10 illustrates the overlay of EGFR antibody-PEG1kDa-EGFR siRNA conjugates with siRNA loading of 1, 2 and 3.

FIG. 11 shows a HPLC chromatogram of EGFR antibody-KRAS-PEG5kDa.

FIG. 12 shows a HPLC chromatogram of Panitumumab-KRAS-PEG5kDa.

FIG. 13 shows a HPLC chromatogram of EGFR antibody-S—S-siRNA-PEG5kDa (DAR=1).

FIG. 14 shows a HPLC chromatogram of EGFR antibody-PEG24-Melittin (loading=˜0.1).

FIG. 15 illustrates a HPLC chromatogram of EGFR antibody-Melittin (n=˜1).

FIG. 16 illustrates a mass spectrum of EGFR antibody-Melittin (n=1).

FIG. 17 shows a HIC chromatogram of EGFR antibody-PEG1kDa-INF7 (Peptide loading=˜1).

FIG. 18 shows a HPLC chromatogram of EGFR antibody-INF7 (Peptide Loading=˜1).

FIG. 19 shows INF7-PEG1kDa-(EGFR antibody-KRAS-PEG5kDa).

FIG. 20 illustrates Melittin-PEG1kDa-(EGFR antibody-KRAS-PEG5kDa).

FIG. 21 illustrates plasma concentration-time profiles out to 96 h post-dose with the siRNA concentration expressed as a percent of injected dose (% ID).

FIG. 22 shows plasma concentration-time profiles out to 96 h post-dose with the siRNA concentration expressed as a percent of injected dose (% ID).

FIG. 23 shows plasma concentration-time profiles out to 96 h post-dose with the siRNA concentration expressed as a percent of injected dose (% ID).

FIG. 24 illustrates plasma concentration-time profiles out to 96 h post-dose with the siRNA concentration expressed as a percent of injected dose (% ID).

FIG. 25 illustrates plasma concentration-time profiles out to 24 h post-dose with the siRNA concentration expressed as a percent of injected dose (% ID).

FIG. 26A and FIG. 26B illustrate tissue concentration-time profiles in tumor or normal livers of mice. FIG. 26A shows tissue concentration-time profiles out to 168 h post-dose measured in s.c. flank H358 tumors in a mice model. FIG. 26B shows tissue concentration-time profiles out to 168 h post-dose measured in normal livers of mice.

FIG. 27 shows tissue concentration-time profiles out to 168 h post-dose measured in s.c. flank H358 tumors and normal livers of mice.

FIG. 28 illustrates tissue concentration-time profiles out to 168 h post-dose measured in s.c. flank H358 tumors and normal livers of mice.

FIG. 29 illustrates tissue concentration-time profiles out to 168 h post-dose measured in s.c. flank H358 tumors and normal livers of mice.

FIG. 30 shows tissue concentration-time profiles out to 168 h post-dose measured in s.c. flank H358 tumors and normal livers of mice.

FIG. 31A and FIG. 31B illustrate siRNA-mediated mRNA knockdown of human KRAS in human s.c. flank H358 tumors (FIG. 31A) or mouse KRAS in normal mouse liver (FIG. 31B).

FIG. 32 illustrates siRNA-mediated mRNA knockdown of human EGFR in human s.c. flank H358 tumors.

FIG. 33 illustrates siRNA-mediated mRNA knockdown of human KRAS in human s.c. flank H358 tumors.

FIG. 34 illustrates siRNA-mediated mRNA knockdown of human EGFR in human s.c. flank H358 tumors.

FIG. 35 shows siRNA-mediated mRNA knockdown of mouse KRAS in mouse liver.

FIG. 36 illustrates plasma concentration-time profiles out to 96 h post-dose with the siRNA concentration expressed as a percent of injected dose (% ID).

FIG. 37 illustrates tissue concentration-time profiles out to 144 h post-dose measured in liver, kidneys, and lungs of wild-type CD-1 mice.

FIG. 38A and FIG. 38B illustrate tissue concentration-time profiles out to 144 h post-dose measured in human s.c. flank H358 tumors for chol-KRAS mixed with either chol-INF7 peptide (FIG. 38A) or chol-melittin peptide (FIG. 38B).

FIG. 39A and FIG. 39B illustrate tissue concentration-time profiles out to 144 h post-dose measured in mouse liver for chol-KRAS mixed with either chol-INF7 peptide (FIG. 39A) or chol-melittin peptide (FIG. 39B).

FIG. 40A and FIG. 40B illustrate tissue concentration-time profiles out to 144 h post-dose measured in mouse kidneys for chol-KRAS mixed with either chol-INF7 peptide (FIG. 40A) or chol-melittin peptide (FIG. 40B).

FIG. 41A and FIG. 41B illustrate tissue concentration-time profiles out to 144 h post-dose measured in mouse lungs for chol-KRAS mixed with either chol-INF7 peptide (FIG. 41A) or chol-melittin peptide (FIG. 41B).

FIG. 42 illustrates siRNA-mediated mRNA knockdown of mouse KRAS in mouse liver.

FIG. 43A and FIG. 43B illustrate tissue concentration-time profiles out to 96 h post-dose measured in human s.c. flank H358 tumors (FIG. 43A) or mouse liver (FIG. 43B).

FIG. 44A and FIG. 44B show tissue concentration-time profiles out to 96 h post-dose measured in mouse kidneys (FIG. 44A) or mouse lungs (FIG. 44B).

FIG. 45 shows siRNA-mediated mRNA knockdown of mouse KRAS in human s.c. flank H358 tumors.

FIG. 46 shows tissue concentrations of siRNA at 96 h post-dose measured in human s.c. flank H358 tumors and mouse liver, kidneys, and lungs.

FIG. 47A and FIG. 47B show siRNA-mediated mRNA knockdown in human s.c. flank H358 tumors of EGFR (FIG. 47A) or KRAS (FIG. 47B).

FIG. 48 shows siRNA-mediated mRNA knockdown of human CTNNB1 in Hep3B orthotopic liver tumors.

FIG. 49 shows human alpha-Fetoprotein in serum from mice with Hep3B orthotopic liver tumors.

FIG. 50A shows siRNA-mediated mRNA knockdown of human EGFR in LNCaP tumor.

FIG. 50B shows siRNA concentration in tumor or liver tissues at 96 hour post-dose.

FIG. 51A illustrates siRNA-mediated mRNA knockdown of human EGFR in LNCaP tumor at 96 hour.

FIG. 51B shows siRNA concentration in tumor or liver tissues at 96 hour post-dose.

FIG. 52 shows plasma siRNA concentration of exemplary molecules described herein.

FIG. 53A illustrates siRNA concentration of exemplary molecules described herein in HCC827 tumor or liver tissue.

FIG. 53B shows EGFR EGFR mRNA expression level of exemplary molecules described herein.

FIG. 54 illustrates exemplary As and Bs to generate molecules encompassed by Formula (I).

FIG. 55 illustrates EGFR mRNA expression level of exemplary molecules described herein.

FIG. 56A illustrates siRNA concentration of exemplary molecules described herein in HCC827 tumor or liver tissue.

FIG. 56B shows EGFR mRNA expression level of exemplary molecules described herein.

FIG. 57A-FIG. 57B illustrate siRNA concentration of exemplary molecules described herein in liver (FIG. 57A) and tumor (FIG. 57B).

FIG. 57C shows KRAS mRNA expression level of exemplary molecules described herein.

FIG. 58A illustrates plasma siRNA concentration of exemplary molecules described herein.

FIG. 58B shows plasma antibody concentration of exemplary molecules described herein.

FIG. 59A illustrates siRNA concentration of exemplary molecules described herein in tumor or liver tissue.

FIG. 59B shows mRNA expression level of exemplary molecules described herein in Hep3B tumor.

FIG. 60 shows CTNNB1 mRNA expression level of an exemplary molecule described herein in liver.

FIG. 61 shows KRAS mRNA expression level of an exemplary molecule described herein in liver.

FIG. 62 illustrates plasma siRNA or monoclonal antibody (mAb) concentration of exemplary molecules described herein.

FIG. 63A illustrates siRNA concentration of exemplary molecules described herein in tumor or liver tissue.

FIG. 63B shows EGFR mRNA expression level of exemplary molecules described herein in LNCaP tumor.

FIG. 64A-FIG. 64E illustrate HPRT mRNA expression level in heart (FIG. 64A), HPRT mRNA expression level in gastrointestinal tissue (FIG. 64B), HPRT mRNA expression level in liver (FIG. 64C), HPRT mRNA expression level in lung (FIG. 64D), and siRNA concentration in tissue (FIG. 64E) of exemplary molecules described herein.

FIG. 65A-FIG. 65E illustrate mRNA expression level in heart (FIG. 65A), mRNA expression level in gastrointestinal tissue (FIG. 65B), mRNA expression level in liver (FIG. 65C), mRNA expression level in lung (FIG. 65D), and siRNA concentration in tissue (FIG. 65E) of exemplary molecules described herein.

FIG. 66A-FIG. 66D illustrate siRNA concentration in heart (FIG. 66A), mRNA expression level in heart (FIG. 66B), mRNA expression level in gastrointestinal tissue (FIG. 66C), and siRNA concentration in muscle (FIG. 66D).

FIG. 67A illustrate mRNA expression level of exemplary molecules described herein.

FIG. 67B shows siRNA concentration of exemplary molecules described herein in tumor or liver tissues.

FIG. 68A-FIG. 68B illustrate anti-B cell antibody-siRNA conjugates which activate primary mouse B cells. FIG. 68A illustrates an anti-B cell Fab-siRNA conjugate. FIG. 68B shows an anti-B cell monoclonal antibody-siRNA conjugate.

FIG. 69A illustrates plasma siRNA concentration of exemplary molecules described herein.

FIG. 69B shows antibody zalutumumab concentration of exemplary molecules described herein in the plasma at a 5 mg/kg dose.

FIG. 70A shows mRNA expression level of exemplary molecules described herein.

FIG. 70B shows siRNA concentration of exemplary molecules described herein in tumor or liver tissues.

FIG. 70C shows plasma siRNA concentration of exemplary molecules described herein.

FIG. 71A illustrates siRNA concentration of exemplary molecules described herein in LNCaP tomor.

FIG. 71B-FIG. 71C illustrate mRNA expression level of exemplary molecules described herein in LNCaP tomor.

FIG. 72A illustrates siRNA concentration of exemplary molecules described herein in tissue.

FIG. 72B shows mRNA expression level of exemplary molecules described herein in HCC827 tumors at 96 h post-treatment.

FIG. 73A illustrates siRNA concentration of exemplary molecules described herein in the plasma at a 0.5 mg/kg dose.

FIG. 73B shows antibody zalutumumab concentration of exemplary molecules described herein in the plasma at a 5 mg/kg dose.

FIG. 74 illustrates plasma clearance of exemplary molecules encompassed by Formula (I) which contains different linkers.

FIG. 75A illustrates the mRNA expression level of exemplary molecules described herein in HCC827 tumor at a 0.5 mg/kg dose.

FIG. 75B-FIG. 75D illustrate siRNA concentration in tumor (FIG. 75B), liver (FIG. 75C), and plasma (FIG. 75D).

FIG. 76A-FIG. 76D illustrate mRNA expression levels of exemplary molecules described herein targeting HPRT. FIG. 76A shows the mRNA expression level in heart. FIG. 76B shows the mRNA expression level in muscle. FIG. 76C shows the mRNA expression level in liver. FIG. 76D shows the mRNA expression level in lung.

FIG. 77A-FIG. 77D illustrate siRNA concentrations of exemplary molecules encompassed by Formula (I) in muscle (FIG. 77A), heart (FIG. 77B), liver (FIG. 77C), and lung (FIG. 77D).

FIG. 78A-FIG. 78D illustrate mRNA expression levels of exemplary molecules encompassed by Formula (I) in heart (FIG. 78A), gastrointestinal tissue (FIG. 78B), liver (FIG. 78C), and lung (FIG. 78D) at 96 h post-treatment.

FIG. 79 illustrates plasma siRNA concentration of exemplary molecules encompassed by Formula (I).

FIG. 80A shows mRNA expression level of exemplary molecules encompassed by Formula (I) in LNCaP tumor at 96 h post-treatment.

FIG. 80B shows siRNA concentration of exemplary molecules encompassed by Formula (I) in LNCaP tumor, liver, kidney, lung, and spleen tissue samples.

FIG. 81A shows mRNA expression level of exemplary molecules encompassed by Formula (I) in HCC827 tumor at 96 h post-treatment.

FIG. 81B illustrates siRNA concentrations of exemplary molecules encompassed by Formula (I) in tumor, liver, kidney, lung, and spleen tissue samples.

FIG. 82 illustrates plasma siRNA concentration of exemplary molecules encompassed by Formula (I).

FIG. 83 illustrates plasma siRNA concentration of exemplary molecules encompassed by Formula (I).

FIG. 84 illustrates mRNA expression levels of exemplary molecules encompassed by Formula (I) in HCC827 tumor at 96 h post treatment.

FIG. 85 illustrates siRNA concentration in HCC827 tumor or liver tissues at 96 hour post-dose.

FIG. 86 illustrates the relative mRNA expression levels of exemplary molecules encompassed by Formula (I) in mouse splenic B cells 48 h post treatment. Each exemplary molecule is further denoted with a number.

FIG. 87 illustrates stability of exemplary molecules encompassed by Formula (I) (or ASCs) in mouse plasma.

FIG. 88 illustrates Conjugation scheme 1.

FIG. 89 illustrates Conjugation scheme 2.

FIG. 90 illustrates Conjugation scheme 3.

FIG. 91 illustrates Conjugation scheme 4.

FIG. 92 illustrates Conjugation scheme 5.

FIG. 93 illustrates Conjugation scheme 6.

FIG. 94 illustrates Conjugation scheme 7.

FIG. 95 illustrates Conjugation scheme 8.

FIG. 96 illustrates Conjugation scheme 9.

FIG. 97 illustrates Conjugation scheme 10.

FIG. 98 illustrates Conjugation scheme 11.

FIG. 99 illustrates Conjugation scheme 12.

FIG. 100 illustrates Conjugation scheme 13.

FIG. 101 illustrates Conjugation scheme 14.

FIG. 102 illustrates Conjugation scheme 15.

FIG. 103 illustrates Conjugation scheme 16.

FIG. 104 illustrates a representative structure of siRNA with C6-NH₂ conjugation handle at the 5′ end and C6-SH at 3′end of the passenger strand.

FIG. 105 illustrates Antibody-Lys-SMCC-S-3′-Passenger strand.

FIG. 106 illustrates Antibody-Cys-SMCC-3′-Passenger strand.

FIG. 107 illustrates Antibody-Lys-SMCC-S-5′-passenger strand.

FIG. 108 illustrates Antibody-Cys-SMCC-5′-passenger strand.

FIG. 109 illustrates Antibody-Lys-PEG-5′-passenger strand.

FIG. 110 illustrates Antibody-Lys-PEG-5′-passenger strand.

FIG. 111 illustrates Antibody-Cys-PEG-5′-passenger strand without inverted abasic at 5′ end.

DETAILED DESCRIPTION OF THE DISCLOSURE

Nucleic acid (e.g., RNAi) therapy is a targeted therapy with high selectivity and specificity. However, in some instances, nucleic acid therapy is also hindered by poor intracellular uptake, limited blood stability and non-specific immune stimulation. To address these issues, various modifications of the nucleic acid composition are explored, such as for example, novel linkers for better stabilizing and/or lower toxicity, optimization of binding moiety for increased target specificity and/or target delivery, and nucleic acid polymer modifications for increased stability and/or reduced off-target effect.

In some embodiments, the arrangement or order of the different components that make-up the nucleic acid composition further effects intracellular uptake, stability, toxicity, efficacy, and/or non-specific immune stimulation. For example, if the nucleic acid component includes a binding moiety, a polymer, and a polynucleic acid molecule (or polynucleotide), the order or arrangement of the binding moiety, the polymer, and/or the polynucleic acid molecule (or polynucleotide) (e.g., binding moiety-polynucleic acid molecule-polymer, binding moiety-polymer-polynucleic acid molecule, or polymer-binding moiety-polynucleic acid molecule) further effects intracellular uptake, stability, toxicity, efficacy, and/or non-specific immune stimulation.

In some embodiments, described herein include a molecule those arrangement of the nucleic acid components effects intracellular uptake, stability, toxicity, efficacy, and/or non-specific immune stimulation. In some instances, the molecule comprises a binding moiety conjugated to a polynucleic acid molecule and a polymer. In some embodiments, the molecule comprises a molecule according to Formula (I): A-X—B—Y—C; in which A is a binding moiety, B is a polynucleotide, C is a polymer, X is a bond or first linker, and Y is a bond or second linker. In some instances, the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety. In some instances, the molecule of Formula (I) further comprises D, an endosomolytic moiety.

In some embodiments, a molecule comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer arranged as described herein enhances intracellular uptake, stability, and/or efficacy. In some instances, a molecule comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer arranged as described herein reduces toxicity and/or non-specific immune stimulation. In some cases, the molecule comprises a molecule according to Formula (I): A-X—B—Y—C; in which A is a binding moiety, B is a polynucleotide, C is a polymer, X is a bond or first linker, and Y is a bond or second linker. In some instances, the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety. In some instances, the molecule of Formula (I) further comprises D, an endosomolytic moiety.

In some embodiments, a molecule described herein is further used to treat a disease or disorder. In some instances, a molecule for the treatment of a disease or disorder is a molecule according to Formula (I): A-X—B—Y—C; in which A is a binding moiety, B is a polynucleotide, C is a polymer, X is a bond or first linker, and Y is a bond or second linker. In some instances, the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety. In some instances, the molecule of Formula (I) further comprises D, an endosomolytic moiety.

In some embodiments, a molecule described herein is also used for inhibiting the expression of a target gene in a primary cell of a patient in need thereof. In such instances, a molecule for such use is a molecule according to Formula (I): A-X—B—Y—C; in which A is a binding moiety, B is a polynucleotide, C is a polymer, X is a bond or first linker, and Y is a bond or second linker. In some instances, the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety. In some instances, the molecule of Formula (I) further comprises D, an endosomolytic moiety.

In some embodiments, a molecule described herein is additionally used as an immuno-oncology therapy for the treatment of a disease or disorder. In some instance, the molecule is a molecule according to Formula (I): A-X—B—Y—C; in which A is a binding moiety, B is a polynucleotide, C is a polymer, X is a bond or first linker, and Y is a bond or second linker. In some instances, the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety. In some instances, the molecule of Formula (I) further comprises D, an endosomolytic moiety.

In additional embodiments, described herein include a kit, which comprises one or more of the molecules described herein.

Therapeutic Molecule Platform

In some embodiments, a molecule (e.g., a therapeutic molecule) described herein comprises a binding moiety conjugated to a polynucleic acid molecule and a polymer. In some embodiments, a molecule (e.g., a therapeutic molecule) comprises a molecule according to Formula (I):A-X—B—Y—C   Formula Iwherein,

• • A is a binding moiety;
  • B is a polynucleotide;
  • C is a polymer;
  • X is a bond or first linker; and
  • Y is a bond or second linker; andwherein the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety.

In some instances, the molecule of Formula (I) further comprises D, an endosomolytic moiety.

In some embodiments, at least one A and/or at least one C are conjugated to the 5′ terminus of B, the 3′ terminus of B, an internal site on B, or in any combinations thereof. In some instances, at least one A is conjugated at one terminus of B while at least one C is conjugated at the opposite terminus of B. In some instances, at least one of A is conjugated at one terminus of B while at least one of C is conjugated at an internal site on B.

In some cases, A and C are not conjugated or attached to B at the same terminus. In some cases, A is attached or conjugated to B at a first terminus of B. In some cases, C is attached or conjugated to B at a second terminus of B, and the second terminus of B is different than the first terminus. In some cases, A is attached or conjugated to B at the 5′ terminus of B, and C is attached or conjugated to B at the 3′ terminus of B. In other cases, A is attached or conjugated to B at the 3′ terminus of B, and C is attached or conjugated to B at the 5′ terminus of B.

In some embodiments, A is an antibody or binding fragment thereof. In some cases, C is a polymer. In some cases, A and C are not conjugated or attached to B at the same terminus. In some cases, A is attached or conjugated to B at a first terminus of B. In some cases, C is attached or conjugated to B at a second terminus of B, and the second terminus of B is different than the first terminus. In some cases, A is attached or conjugated to B at the 5′ terminus of B, and C is attached or conjugated to B at the 3′ terminus of B. In other cases, A is attached or conjugated to B at the 3′ terminus of B, and C is attached or conjugated to B at the 5′ terminus of B. In some cases, X which connects A to B is a bond or a non-polymeric linker. In some cases, X is a non-peptide linker (or a linker that does not comprise an amino acid residue). In some cases, Y which connects B to C is a bond or a second linker. In some instances, X connects A to the 5′ terminus of B, and Y connects C to the 3′ terminus of B. In other instances, X connects A to the 3′ terminus of B, and Y connects C to the 5′ terminus of B.

In some embodiments, X—B is conjugated or attached to the N-terminus, C-terminus, a constant region, a hinge region, or a Fc region of A. In some instances, X—B is conjugated or attached to the N-terminus of A. In some instances, X—B is conjugated or attached to the C-terminus of A. In some instances, X—B is conjugated or attached to a hinge region of A. In some instances, X—B is conjugated or attached to a constant region of A. In some instances, X—B is conjugated or attached to the Fc region of A.

In some instances, at least one B and/or at least one C, and optionally at least one D are conjugated to a first A. In some instances, the at least one B is conjugated at a terminus (e.g., a 5′ terminus or a 3′ terminus) to the first A or are conjugated via an internal site to the first A. In some cases, the at least one C is conjugated either directly to the first A or indirectly via the two or more Bs. If indirectly via the two or more Bs, the two or more Cs are conjugated either at the same terminus as the first A on B, at opposing terminus from the first A, or independently at an internal site. In some instances, at least one additional A is further conjugated to the first A, to B, or to C. In additional instances, the at least one D is optionally conjugated either directly or indirectly to the first A, to the at least one B, or to the at least one C. If directly to the first A, the at least one D is also optionally conjugated to the at least one B to form a A-D-B conjugate or is optionally conjugated to the at least one B and the at least one C to form a A-D-B—C conjugate. In some cases, the at least one additional A is different than the first A.

In some cases, two or more Bs and/or two or more Cs are conjugated to a first A. In some instances, the two or more Bs are conjugated at a terminus (e.g., a 5′ terminus or a 3′ terminus) to the first A or are conjugated via an internal site to the first A. In some instances, the two or more Cs are conjugated either directly to the first A or indirectly via the two or more Bs. If indirectly via the two or more Bs, the two or more Cs are conjugated either at the same terminus as the first A on B, at opposing terminus from the first A, or independently at an internal site. In some instances, at least one additional A is further conjugated to the first A, to two or more Bs, or to two or more Cs. In additional instances, at least one D is optionally conjugated either directly or indirectly to the first A, to the two or more Bs, or to the two or more Cs. If indirectly to the first A, the at least one D is conjugated to the first A through the two or more Bs, through the two or more Cs, through a B—C orientation to form a A-B—C-D type conjugate, or through a C—B orientation to form a A-C—B-D type conjugate. In some cases, the at least one additional A is different than the first A. In some cases, the two or more Bs are different. In other cases, the two or more Bs are the same. In some instances, the two or more Cs are different. In other instances, the two or more Cs are the same. In additional instances, the two or more Ds are different. In additional instances, the two or more Ds are the same.

In other cases, two or more Bs and/or two or more Ds, optionally two or more Cs are conjugated to a first A. In some instances, the two or more Bs are conjugated at a terminus (e.g., a 5′ terminus or a 3′ terminus) to the first A or are conjugated via an internal site to the first A. In some instances, the two or more Ds are conjugated either directly to the first A or indirectly via the two or more Bs. If indirectly via the two or more Bs, the two or more Ds are conjugated either at the same terminus as the first A on B, at opposing terminus from the first A, or independently at an internal site. In some instances, at least one additional A is further conjugated to the first A, to the two or more Bs, or to the two or more Ds. In additional instances, the two or more Cs are optionally conjugated either directly or indirectly to the first A, to the two or more Bs, or to the two or more Ds. In some cases, the at least one additional A is different than the first A. In some cases, the two or more Bs are different. In other cases, the two or more Bs are the same. In some instances, the two or more Cs are different. In other instances, the two or more Cs are the same. In additional instances, the two or more Ds are different. In additional instances, the two or more Ds are the same.

In some embodiments, a molecule (e.g., a therapeutic molecule) described herein comprises a molecule according to Formula (II):(A-X—B—Y—C_c)-L-D   Formula IIwherein,

• • A is a binding moiety;
  • B is a polynucleotide;
  • C is a polymer;
  • X is a bond or first linker;
  • Y is a bond or second linker;
  • L is a bond or third linker;
  • D is an endosomolytic moiety; and
  • c is an integer between 0 and 1; andwherein the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety; and D is conjugated anywhere on A, B, or C.

In some embodiments, a molecule (e.g., a therapeutic molecule) described herein comprises a molecule according to Formula (III):A_a-X—B_b—Y—C_c-L-D_n   Formula IIIwherein,

• • A is a binding moiety;
  • B is a polynucleotide;
  • C is a polymer;
  • D is an endosomolytic moiety;
  • X is a bond or first linker;
  • Y is a bond or second linker;
  • L is a bond or third linker;
  • a and b are independently an integer between 1-3;
  • c is an integer between 0 and 3; and
  • n is an integer between 0 and 10; andwherein the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety; A is conjugated anywhere on B, C, or D; B is conjugated anywhere on A, C, or D; C is conjugated anywhere on A, B, or D; and D is conjugated anywhere on A, B, or C.

In some embodiments, a molecule (e.g., a therapeutic molecule) described herein comprises a molecule according to Formula (IIIa): A-X—B-L-D-Y—C.

In some embodiments, a molecule (e.g., a therapeutic molecule) described herein comprises a molecule according to Formula (Mb): A_a-X—B_b-L-D_n.

In some embodiments, a molecule (e.g., a therapeutic molecule) described herein comprises a molecule according to Formula (IV):A-X—(B_b—Y—C_c-D_n)_m wherein,

• • A is a binding moiety;
  • B is a polynucleotide;
  • C is a polymer;
  • D is an endosomolytic moiety;
  • X is a bond or first linker;
  • Y is a bond or second linker;
  • L is a bond or third linker;
  • a and b are independently an integer between 1-3;
  • c is an integer between 0 and 3;
  • n is an integer between 0 and 10; and
  • m is an integer between 1-3; andwherein the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety; C is conjugated anywhere on B or D; and D is conjugated anywhere on B or C.

In some embodiments, a molecule (e.g., a therapeutic molecule) described herein comprises a molecule according to Formula (IVa): A-X—(B_b-L-D_n-Y—C_c)_m.

In some embodiment, a molecule (e.g., a therapeutic molecule) described herein is a molecule as illustrated in FIGS. 1A-1T. In some instances, a molecule (e.g., a therapeutic molecule) described herein is a molecule as illustrated in FIGS. 1A-1G. In some cases, a molecule (e.g., a therapeutic molecule) described herein is a molecule as illustrated in FIGS. 1H-1N. In additional cases, a molecule (e.g., a therapeutic molecule) described herein is a molecule as illustrated in FIGS. 1O-1T.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1A.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1B.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1C.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1D.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1E.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1F.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1G.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1H.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1I.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1J.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1K.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1L.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1M.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1N.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1O.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1P.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1Q.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated in FIG. 1R.

The antibody as illustrated in FIGS. 1A-1R is for representation purposes only and encompasses a humanized antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or binding fragment thereof.

Polynucleic Acid Molecule Targets

In some embodiments, the polynucleic acid molecule B is a polynucleic acid molecule (or polynucleotide) that hybridizes to a target region on an oncogene. In some instances, oncogenes are further classified into several categories: growth factors or mitogens, receptor tyrosine kinases, cytoplasmic tyrosine kinases, cytoplasmic serine/threonine kinases, regulatory GTPases, and transcription factors. Exemplary growth factors include c-Sis. Exemplary receptor tyrosine kinases include epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), and HER2/neu. Exemplary cytoplasmic tyrosine kinases include Src-family tyrosine kinases, Syk-ZAP-70 family of tyrosine kinases, BTK family of tyrosine kinases, and Abl gene in CML. Exemplary cytoplasmic serine/threonine kinases include Raf kinase and cyclin-dependent kinases. Exemplary regulatory GTPases include Ras family of proteins such as KRAS. Exemplary transcription factors include MYC gene. In some instances, an oncogene described herein comprises an oncogene selected from growth factors or mitogens, receptor tyrosine kinases, cytoplasmic tyrosine kinases, cytoplasmic serine/threonine kinases, regulatory GTPases, or transcription factors. In some embodiments, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of an oncogene selected from growth factors or mitogens, receptor tyrosine kinases, cytoplasmic tyrosine kinases, cytoplasmic serine/threonine kinases, regulatory GTPases, or transcription factors.

In some embodiments, an oncogene described herein comprises Abl, AKT-2, ALK, AML1 (or RUNX1), AR, AXL, BCL-2, 3, 6, BRAF, c-MYC, EGFR, ErbB-2 (Her2, Neu), Fms, FOS, GLI1, HPRT1, IL-3, INTS2, JUN, KIT, KS3, K-sam, LBC (AKAP13), LCK, LMO1, LMO2, LYL1, MAS1, MDM2, MET, MLL (KMT2A), MOS, MYB, MYH11/CBFB, NOTCH1 (TAN1), NTRK1 (TRK), OST (SLC51B), PAX5, PIM1, PRAD-1, RAF, RAR/PML, HRAS, KRAS, NRAS, REL/NRG, RET, ROS, SKI, SRC, TIAM1, or TSC2. In some embodiments, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of Abl, AKT-2, ALK, AML1 (or RUNX1), AR, AXL, BCL-2, 3, 6, BRAF, c-MYC, EGFR, ErbB-2 (Her2, Neu), Fms, FOS, GLI1, HPRT1, IL-3, INTS2, JUN, KIT, KS3, K-sam, LBC (AKAP13), LCK, LMO1, LMO2, LYL1, MAS1, MDM2, MET, MLL (KMT2A), MOS, MYB, MYH11/CBFB, NOTCH1 (TAN1), NTRK1 (TRK), OST (SLC51B), PAX5, PIM1, PRAD-1, RAF, RAR/PML, HRAS, KRAS, NRAS, REL/NRG, RET, ROS, SKI, SRC, TIAM1, or TSC2.

In some embodiments, an oncogene described herein comprises KRAS, EGFR, AR, HPRT1, CNNTB1 (β-catenin), or β-catenin associated genes. In some embodiments, the polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to a target region of KRAS, EGFR, AR, HPRT1, CNNTB1 (β-catenin), or β-catenin associated genes. In some embodiments, the polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to a target region of KRAS. In some embodiments, the polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to a target region of EGFR. In some embodiments, the polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to a target region of AR. In some embodiments, the polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to a target region of CNNTB1 (β-catenin). In some embodiments, the polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to a target region of CNNTB1 (β-catenin) associated genes. In some instances, the β-catenin associated genes comprise PIK3CA, PIK3CB, and Myc. In some instances, the polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to a target region of HPRT1.

Polynucleic Acid Molecules that Target Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS)

Kirsten Rat Sarcoma Viral Oncogene Homolog (also known as GTPase KRas, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog, or KRAS) is involved in regulating cell division. The K-Ras protein is a GTPase belonging to the Ras superfamily. In some instances, K-Ras modulates cell cycle progression, as well as induces growth arrest, apoptosis, and replicative senescence under different environmental triggers (e.g., cellular stress, ultraviolet, heat shock, or ionizing irradiation). In some cases, wild type KRAS gene has been shown to be frequently lost during tumor progression in different types of cancer, while mutations of KRAS gene have been linked to cancer development. In some instances, KRAS amplification has also been implicated in cancer development (see, for example, Valtorta et al. “KRAS gene amplification in colorectal cancer and impact on response to EGFR-targeted therapy,” Int. J. Cancer 133: 1259-1266 (2013)). In such cases, the cancer pertains to a refractory cancer in which the patient has acquired resistance to a particular inhibitor or class of inhibitors.

In some embodiments, the KRAS gene is wild type or comprises a mutation. In some instances, KRAS mRNA is wild type or comprises a mutation. In some instances, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of wild type KRAS DNA or RNA. In some instances, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of KRAS DNA or RNA comprising a mutation (e.g., a substitution, a deletion, or an addition).

In some embodiments, KRAS DNA or RNA comprises one or more mutations. In some embodiments, KRAS DNA or RNA comprises one or more mutations at codons 12 or 13 in exon 1. In some instances, KRAS DNA or RNA comprises one or more mutations at codons 61, 63, 117, 119, or 146. In some instances, KRAS DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues 12, 13, 18, 19, 20, 22, 24, 26, 36, 59, 61, 63, 64, 68, 110, 116, 117, 119, 146, 147, 158, 164, 176, or a combination thereof of the KRAS polypeptide. In some embodiments, KRAS DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues selected from G12V, G12D, G12C, G12A, G12S, G12F, G13C, G13D, G13V, A18D, L19F, T20R, Q22K, I24N, N26K, I36L, I36M, A59G, A59E, Q61K, Q61H, Q61L, Q61R, E63K, Y64D, Y64N, R68S, P110S, K117N, C118S, A146T, A146P, A146V, K147N, T158A, R164Q, K176Q, or a combination thereof of the KRAS polypeptide.

In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA comprising one or more mutations. In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA comprising one or more mutations at codons 12 or 13 in exon 1. In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA comprising one or more mutations at codons 61, 63, 117, 119, or 146. In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA comprising one or more mutations at positions corresponding to amino acid residues 12, 13, 18, 19, 20, 22, 24, 26, 36, 59, 61, 63, 64, 68, 110, 116, 117, 119, 146, 147, 158, 164, 176, or a combination thereof of the KRAS polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA comprising one or more mutations corresponding to amino acid residues selected from G12V, G12D, G12C, G12A, G12S, G12F, G13C, G13D, G13V, A18D, L19F, T20R, Q22K, I24N, N26K, I36L, I36M, A59G, A59E, Q61K, Q61H, Q61L, Q61R, E63K, Y64D, Y64N, R68S, P110S, K117N, C118S, A146T, A146P, A146V, K147N, T158A, R164Q, K176Q, or a combination thereof of the KRAS polypeptide.

Polynucleic Acid Molecules that Target Epidermal Growth Factor Receptor (EGFR)

Epidermal growth factor receptor (EGFR, ErbB-1, or HER1) is a transmembrane tyrosine kinase receptor and a member of the ErbB family of receptors, which also include HER2/c-neu (ErbB-2), Her3 (ErbB-3) and Her4 (ErbB-4). In some instances, EGFR mutations drive the downstream activation of RAS/RAF/MAPK, PI3K/AKT, and/or JAK/STAT pathways, leading to mitosis, cell proliferation, and suppression of apoptosis. In addition, amplification of wild-type EGFR gene has been implicated in the development of cancers such as glioblastomas and non-small cell lung cancer (Talasila, et al., “EGFR Wild-type Amplification and Activation Promote Invasion and Development of Glioblastoma Independent of Angiogenesis,” Acta Neuropathol. 125(5): 683-698 (2013); Bell et al., “Epidermal Growth Factor Receptor Mutations and Gene Amplification in Non-Small-Cell Lung Cancer: Molecular Analysis of the IDEAL/INTACT Gefitinib Trials,” J. Clinical Oncology 23(31): 8081-8092 (2005)).

In some embodiments, EGFR DNA or RNA is wild type EGFR or EGFR comprising a mutation. In some instances, EGFR is wild type EGFR. In some instances, EGFR DNA or RNA comprises a mutation. In some instances, the polynucleic acid molecule hybridizes to a target region of wild type EGFR DNA or RNA. In some instances, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising a mutation (e.g., a substitution, a deletion, or an addition).

In some instances, EGFR DNA or RNA comprises one or more mutations. In some embodiments, EGFR DNA or RNA comprises one or more mutations within one or more exons. In some instances, the one or more exons comprise exon 18, exon 19, exon 20, exon 21 or exon 22. In some instances, EGFR DNA or RNA comprises one or more mutations in exon 18, exon 19, exon 20, exon 21, exon 22 or a combination thereof.

In some instances, EGFR DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues 34, 38, 45, 62, 63, 77, 78, 108, 114, 120, 140, 148, 149, 160, 177, 178, 189, 191, 198, 220, 222, 223, 229, 237, 240, 244, 252, 254, 255, 256, 263, 270, 273, 276, 282, 288, 289, 301, 303, 304, 309, 314, 326, 331, 354, 363, 373, 337, 380, 384, 393, 427, 428, 437, 441, 447, 465, 475, 515, 526, 527, 531, 536, 541, 546, 571, 588, 589, 596, 596, 598, 602, 614, 620, 628, 636, 641, 645, 651, 671, 689, 694, 700, 709, 712, 714, 715, 716, 719, 720, 721, 731, 733, 739-744, 742, 746-750, 746-752, 746, 747, 747-749, 747-751, 747-753, 751, 752, 754, 752-759, 750, 761-762, 761, 763, 765, 767-768, 767-769, 768, 769, 769-770, 770-771, 772, 773-774, 773, 774, 774-775, 776, 779, 783, 784, 786, 790, 792, 794, 798, 803, 805, 807, 810, 826, 827, 831, 832, 833, 835, 837, 838, 839, 842, 843, 847, 850, 851, 853, 854, 856, 858, 861, 863, 894, 917, 967, 1006, 1019, 1042, 1100, 1129, 1141, 1153, 1164, 1167, or a combination thereof of the EGFR polypeptide. In some embodiments, EGFR DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues 747, 761, 790, 854, 858, or a combination thereof of the EGFR polypeptide. In some embodiments, EGFR DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues 761, 790, 858, or a combination thereof of the EGFR polypeptide. In some embodiments, EGFR DNA or RNA comprises a mutation at a position corresponding to amino acid residue 747 of the EGFR polypeptide. In some embodiments, EGFR DNA or RNA comprises a mutation at a position corresponding to amino acid residue 761 of the EGFR polypeptide. In some embodiments, EGFR DNA or RNA comprises a mutation at a position corresponding to amino acid residue 790 of the EGFR polypeptide. In some embodiments, EGFR DNA or RNA comprises a mutation at a position corresponding to amino acid residue 854 of the EGFR polypeptide. In some embodiments, EGFR DNA or RNA comprises a mutation at a position corresponding to amino acid residue 858 of the EGFR polypeptide.

In some embodiments, EGFR DNA or RNA comprises one or more mutations selected from T34M, L38V, E45Q, L62R, G63R, G63K, S77F, F78L, R108K, R108G, E114K, A120P, L140V, V148M, R149W, E160K, S177P, M178I, K189T, D191N, S198R, S220P, R222L, R222C, S223Y, S229C, A237Y, C240Y, R244G, R252C, R252P, F254I, R255 (nonsense mutation), D256Y, T263P, Y270C, T273A, Q276 (nonsense), E282K, G288 (frame shift), A289D, A289V, A289T, A289N, A289D, V301 (deletion), D303H, H304Y, R309Q, D314N, C326R, G331R, T354M, T363I, P373Q, R337S, S380 (frame shift), T384S, D393Y, R427L, G428S, S437Y, V441I, S447Y, G465R, I475V, C515S, C526S, R527L, R531 (nonsense), V536M, L541I, P546Q, C571S, G588S, P589L, P596L, P596S, P596R, P596L, G598V, G598A, E602G, G614D, C620Y, C620W, C628Y, C628F, C636Y, T638M, P641H, S645C, V651M, R671C, V689M, P694S, N700D, E709A, E709K, E709Q, E709K, F712L, K714N, I715S, K716R, G719A, G719C, G719D, G719S, S720C, S720F, G721V, W731Stop, P733L, K739-1744 (insertion), V742I, V742A, E746-A750 (deletion), E746K, L747S, L747-E749 (deletion), L747-T751 (deletion), L747-P753 (deletion), G746-S752 (deletion), T751I, S752Y, K754 (deletion), S752-1759 (deletion), A750P, D761-E762 (e.g., residues EAFQ insertion (SEQ ID NO: 2110)), D761N, D761Y, A763V, V765A, A767-S768 (e.g., residues TLA insertion), A767-V769 (e.g., residues ASV insertion), S768I, S768T, V769L, V769M, V769-D770 (e.g., residue Y insertion), 770-771 (e.g., residues GL insertion), 770-771 (e.g., residue G insertion), 770-771 (e.g., residues CV insertion), 770-771 (e.g., residues SVD insertion), P772R, 773-774 (e.g., residues NPH insertion), H773R, H773L, V774M, 774-775 (e.g., residues HV insertion), R776H, R776C, G779F, T783A, T784F, T854A, V786L, T790M, L792P, P794H, L798F, R803W, H805R, D807H, G810S, N826S, Y827 (nonsense), R831H, R832C, R832H, L833F, L833V, H835L, D837V, L838M, L838P, A839V, N842H, V843L, T847K, T847I, H850N, V851A, I853T, F856L, L858R, L858M, L861Q, L861R, G863D, Q894L, G917A, E967A, D1006Y, P1019L, S1042N, R1100S, H1129Y, T1141 S, S1153I, Q1164R, L1167M, or a combination thereof of the EGFR polypeptide.

In some instances, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising one or more mutations. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising one or more mutations in exon 18, exon 19, exon 20, exon 21, exon 22 or a combination thereof.

In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising one or more mutations at positions corresponding to amino acid residues 34, 38, 45, 62, 63, 77, 78, 108, 114, 120, 140, 148, 149, 160, 177, 178, 189, 191, 198, 220, 222, 223, 229, 237, 240, 244, 252, 254, 255, 256, 263, 270, 273, 276, 282, 288, 289, 301, 303, 304, 309, 314, 326, 331, 354, 363, 373, 337, 380, 384, 393, 427, 428, 437, 441, 447, 465, 475, 515, 526, 527, 531, 536, 541, 546, 571, 588, 589, 596, 596, 598, 602, 614, 620, 628, 636, 641, 645, 651, 671, 689, 694, 700, 709, 712, 714, 715, 716, 719, 720, 721, 731, 733, 739-744, 742, 746-750, 746-752, 746, 747, 747-749, 747-751, 747-753, 751, 752, 754, 752-759, 750, 761-762, 761, 763, 765, 767-768, 767-769, 768, 769, 769-770, 770-771, 772, 773-774, 773, 774, 774-775, 776, 779, 783, 784, 786, 790, 792, 794, 798, 803, 805, 807, 810, 826, 827, 831, 832, 833, 835, 837, 838, 839, 842, 843, 847, 850, 851, 853, 854, 856, 858, 861, 863, 894, 917, 967, 1006, 1019, 1042, 1100, 1129, 1141, 1153, 1164, 1167, or a combination thereof of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising one or more mutations at positions corresponding to amino acid residues 747, 761, 790, 854, 858, or a combination thereof of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising one or more mutations at positions corresponding to amino acid residues 761, 790, 858, or a combination thereof of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising a mutation at a position corresponding to amino acid residue 747 of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising a mutation at a position corresponding to amino acid residue 761 of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising a mutation at a position corresponding to amino acid residue 790 of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising a mutation at a position corresponding to amino acid residue 854 of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising a mutation at a position corresponding to amino acid residue 858 of the EGFR polypeptide.

In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising one or more mutations selected from T34M, L38V, E45Q, L62R, G63R, G63K, S77F, F78L, R108K, R108G, E114K, A120P, L140V, V148M, R149W, E160K, S177P, M178I, K189T, D191N, S198R, S220P, R222L, R222C, S223Y, S229C, A237Y, C240Y, R244G, R252C, R252P, F254I, R255 (nonsense mutation), D256Y, T263P, Y270C, T273A, Q276 (nonsense), E282K, G288 (frame shift), A289D, A289V, A289T, A289N, A289D, V301 (deletion), D303H, H304Y, R309Q, D314N, C326R, G331R, T354M, T363I, P373Q, R337S, S380 (frame shift), I384S, D393Y, R427L, G428S, S437Y, V441I, S447Y, G465R, I475V, C515S, C526S, R527L, R531 (nonsense), V536M, L541I, P546Q, C571S, G588S, P589L, P596L, P596S, P596R, P596L, G598V, G598A, E602G, G614D, C620Y, C620W, C628Y, C628F, C636Y, T638M, P641H, S645C, V651M, R671C, V689M, P694S, N700D, E709A, E709K, E709Q, E709K, F712L, K714N, I715S, K716R, G719A. G719C, G719D, G719S, S720C, S720F, G721V, W731Stop, P733L, K739-1744 (insertion), V742I, V742A, E746-A750 (deletion), E746K, L747S, L747-E749 (deletion), L747-T751 (deletion), L747-P753 (deletion), G746-S752 (deletion), T751I, S752Y, K754 (deletion), S752-1759 (deletion), A750P, D761-E762 (e.g., residues EAFQ insertion (SEQ ID NO: 2110)), D761N, D761Y, A763V, V765A, A767-5768 (e.g., residues TLA insertion), A767-V769 (e.g., residues ASV insertion), S768I, S768T, V769L, V769M, V769-D770 (e.g., residue Y insertion), 770-771 (e.g., residues GL insertion), 770-771 (e.g., residue G insertion), 770-771 (e.g., residues CV insertion), 770-771 (e.g., residues SVD insertion), P772R, 773-774 (e.g., residues NPH insertion), H773R, H773L, V774M, 774-775 (e.g., residues HV insertion), R776H, R776C, G779F, T783A, T784F, T854A, V786L, T790M, L792P, P794H, L798F, R803W, H805R, D807H, G810S, N826S, Y827 (nonsense), R831H, R832C, R832H, L833F, L833V, H835L, D837V, L838M, L838P, A839V, N842H, V843L, T847K, T847I, H850N, V851A, I853T, F856L, L858R, L858M, L861Q, L861R, G863D, Q894L, G917A, E967A, D1006Y, P1019L, S1042N, R1100S, H1129Y, T1141S, S1153I, Q1164R, L1167M, or a combination thereof of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising one or more mutations selected from L747S, D761Y, T790M, T854A, L858R, or a combination thereof of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising one or more mutations selected from D761Y, T790M, L858R, or a combination thereof of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising mutation L747S of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising mutation D761Y of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising mutation T790M of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising mutation T854A of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising mutation L858R of the EGFR polypeptide.

Polynucleic Acid Molecules that Target Androgen Receptor (AR)

Androgen receptor (AR) (also known as NR3C4, nuclear receptor subfamily 3, group C, gene 4) belongs to the steroid hormone group of nuclear receptor superfamily along with related members: estrogen receptor (ER), glucocorticoid receptor (GR), progesterone receptor (PR), and mineralocorticoid receptor (MR). Androgens, or steroid hormones, modulate protein synthesis and tissue remodeling through the androgen receptor. The AR protein is a ligand-inducible zinc finger transcription factor that regulates target gene expression. The presence of mutations in the AR gene has been observed in several types of cancers (e.g., prostate cancer, breast cancer, bladder cancer, or esophageal cancer), and in some instances, has been linked to metastatic progression.

In some embodiments, AR DNA or RNA is wild type or comprises one or more mutations and/or splice variants. In some instances, AR DNA or RNA comprises one or more mutations. In some instances, AR DNA or RNA comprises one or more splice variants selected from AR splice variants including but not limited to AR1/2/2b, ARV2, ARV3, ARV4, AR1/2/3/2b, ARV5, ARV6, ARV7, ARV9, ARV10, ARV11, ARV12, ARV13, ARV14, ARV15, ARV16, and ARV(v567es). In some instances, the polynucleic acid molecule hybridizes to a target region of AR DNA or RNA comprising a mutation (e.g., a substitution, a deletion, or an addition) or a splice variant.

In some embodiments, AR DNA or RNA comprises one or more mutations. In some embodiments, AR DNA or RNA comprises one or more mutations within one or more exons. In some instances, the one or more exons comprise exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or exon 8. In some embodiments, AR DNA or RNA comprises one or more mutations within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8 or a combination thereof. In some instances, AR DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues 2, 14, 16, 29, 45, 54, 57, 64, 106, 112, 176, 180, 184, 194, 198, 204, 214, 221, 222, 233, 243, 252, 255, 266, 269, 287, 288, 334, 335, 340, 363, 368, 369, 390, 403, 443, 491, 505, 513, 524, 524, 528, 533, 547, 548, 564, 567, 568, 574, 547, 559, 568, 571, 573, 575, 576, 577, 578, 579, 580, 581, 582, 585, 586, 587, 596, 597, 599, 601, 604, 607, 608, 609, 610, 611, 615, 616, 617, 619, 622, 629, 630, 638, 645, 647, 653, 662, 664, 670, 671, 672, 674, 677, 681, 682, 683, 684, 687, 688, 689, 690, 695, 700, 701, 702, 703, 705, 706, 707, 708, 710, 711, 712, 715, 717, 720, 721, 722, 723, 724, 725, 726, 727, 728, 730, 732, 733, 737, 739, 741, 742, 743, 744, 745, 746, 748, 749, 750, 751, 752, 754, 755, 756, 757, 758, 759, 762, 763, 764, 765, 766, 767, 768, 771, 772, 774, 777, 779, 786, 795, 780, 782, 784, 787, 788, 790, 791, 793, 794, 798, 802, 803, 804, 806, 807, 812, 813, 814, 819, 820, 821, 824, 827, 828, 830, 831, 834, 840, 841, 842, 846, 854, 855, 856, 863, 864, 866, 869, 870, 871, 874, 875, 877, 879, 880, 881, 886, 888, 889, 891, 892, 895, 896, 897, 898, 902, 903, 904, 907, 909, 910, 911, 913, 916, 919, or a combination thereof of the AR polypeptide. In some embodiments, AR DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues selected from E2K, P14Q, K16N, V29M, S45T, L54S, L57Q, Q64R, Y106C, Q112H, S176S, K180R, L184P, Q194R, E198G, G204S, G214R, K221N, N222D, D233K, S243L, A252V, L255P, M266T, P269S, A287D, E288K, S334P, S335T, P340L, Y363N, L368V, A369P, P390R, P390S, P390L, A403V, Q443R, G491S, G505D, P513S, G524D, G524S, D528G, P533S, L547F, P548S, D564Y, S567F, G568W, L574P, L547F, C559Y, G568W, G568V, Y571C, Y571H, A573D, T575A, C576R, C576F, G577R, S578T, C579Y, C579F, K580R, V581F, F582Y, F582S, R585K, A586V, A587S, A596T, A596S, S597G, S597I, N599Y, C601F, D604Y, R607Q, R608K, K609N, D610T, C611Y, R615H, R615P, R615G, R616C, L616R, L616P, R617P, C619Y, A622V, R629W, R629Q, K630T, L638M, A645D, S647N, E653K, S662 (nonsense), I664N, Q670L, Q670R, P671H, I672T, L674P, L677P, E681L, P682T, G683A, V684I, V684A, A687V, G688Q, H689P, D690V, D695N, D695V, D695H, L700M, L701P, L7011, H701H, S702A, S703G, N705S, N705Y, E706 (nonsense), L707R, G708A, R710T, Q711E, L712F, V715M, K717Q, K720E, A721T, L722F, P723S, G724S, G724D, G724N, F725L, R726L, N727K, L728S, L728I, V730M, D732N, D732Y, D732E, Q733H, I737T, Y739D, W741R, M742V, M742I, G743R, G743V, L744F, M745T, V746M, A748D, A748V, A748T, M749V, M749I, G750S, G750D, W751R, R752Q, F754V, F754L, T755A, N756S, N756D, V757A, N758T, S759F, S759P, L762F, Y763H, Y763C, F764L, A765T, A765V, P766A, P766S, D767E, L768P, L768M, N771H, E772G, E772A, R774H, R774C, K777T, R779W, R786Q, G795V, M780I, S782N, C784Y, M787V, R788S, L790F, S791P, E793D, F794S, Q798E, Q802R, G803L, F804L, C806Y, M807V, M807R, M8071, L812P, F813V, S814N, N819Q, G820A, L821V, Q824L, Q824R, F827L, F827V, D828H, L830V, L830P, R831Q, R831L, Y834C, R840C, R840H, I841S, I842T, R846G, R854K, R855C, R855H, F856L, L863R, D864N, D864E, D864G, V866L, V866M, V866E, I869M, A870G, A870V, R871G, H874Y, H874R, Q875K, T877S, T877A, D879T, D879G, L880Q, L881V, M886V, S888L, V889M, F891L, P892L, M895T, A896T, E897D, I898T, Q902R, V903M, P904S, P904H, L907F, G909R, G909E, K910R, V911L, P913S, F916L, Q919R, or a combination thereof of the AR polypeptide.

In some embodiments, the polynucleic acid molecule hybridizes to a target region of AR DNA or RNA comprising one or more mutations. In some embodiments the polynucleic acid hybridizes to one or more AR splice variants. In some embodiments the polynucleic acid hybridizes to AR DNA or RNA comprising one or more AR splice variants including but not limited to AR1/2/2b, ARV2, ARV3, ARV4, AR1/2/3/2b, ARV5, ARV6, ARV7, ARV9, ARV10, ARV11, ARV12, ARV13, ARV14, ARV15, ARV16, and ARV(v567es). In some embodiments, the polynucleic acid molecule hybridizes to a target region of AR DNA or RNA comprising one or more mutations within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8 or a combination thereof. In some embodiments, the polynucleic acid molecule hybridizes to a target region of AR DNA or RNA comprising one or more mutations at positions corresponding to amino acid residues 2, 14, 16, 29, 45, 54, 57, 64, 106, 112, 176, 180, 184, 194, 198, 204, 214, 221, 222, 233, 243, 252, 255, 266, 269, 287, 288, 334, 335, 340, 363, 368, 369, 390, 403, 443, 491, 505, 513, 524, 524, 528, 533, 547, 548, 564, 567, 568, 574, 547, 559, 568, 571, 573, 575, 576, 577, 578, 579, 580, 581, 582, 585, 586, 587, 596, 597, 599, 601, 604, 607, 608, 609, 610, 611, 615, 616, 617, 619, 622, 629, 630, 638, 645, 647, 653, 662, 664, 670, 671, 672, 674, 677, 681, 682, 683, 684, 687, 688, 689, 690, 695, 700, 701, 702, 703, 705, 706, 707, 708, 710, 711, 712, 715, 717, 720, 721, 722, 723, 724, 725, 726, 727, 728, 730, 732, 733, 737, 739, 741, 742, 743, 744, 745, 746, 748, 749, 750, 751, 752, 754, 755, 756, 757, 758, 759, 762, 763, 764, 765, 766, 767, 768, 771, 772, 774, 777, 779, 786, 795, 780, 782, 784, 787, 788, 790, 791, 793, 794, 798, 802, 803, 804, 806, 807, 812, 813, 814, 819, 820, 821, 824, 827, 828, 830, 831, 834, 840, 841, 842, 846, 854, 855, 856, 863, 864, 866, 869, 870, 871, 874, 875, 877, 879, 880, 881, 886, 888, 889, 891, 892, 895, 896, 897, 898, 902, 903, 904, 907, 909, 910, 911, 913, 916, 919, or a combination thereof of the AR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of AR DNA or RNA comprising one or more mutations selected from E2K, P14Q, K16N, V29M, S45T, L54S, L57Q, Q64R, Y106C, Q112H, S176S, K180R, L184P, Q194R, E198G, G204S, G214R, K221N, N222D, D233K, S243L, A252V, L255P, M266T, P269S, A287D, E288K, S334P, S335T, P340L, Y363N, L368V, A369P, P390R, P390S, P390L, A403V, Q443R, G491S, G505D, P513S, G524D, G524S, D528G, P533S, L547F, P548S, D564Y, S567F, G568W, L574P, L547F, C559Y, G568W, G568V, Y571C, Y571H, A573D, T575A, C576R, C576F, G577R, S578T, C579Y, C579F, K580R, V581F, F582Y, F582S, R585K, A586V, A587S, A596T, A596S, S597G, S597I, N599Y, C601F, D604Y, R607Q, R608K, K609N, D610T, C611Y, R615H, R615P, R615G, R616C, L616R, L616P, R617P, C619Y, A622V, R629W, R629Q, K630T, L638M, A645D, S647N, E653K, S662 (nonsense), I664N, Q670L, Q670R, P671H, I672T, L674P, L677P, E681L, P682T, G683A, V684I, V684A, A687V, G688Q, H689P, D690V, D695N, D695V, D695H, L700M, L701P, L7011, H701H, S702A, S703G, N705S, N705Y, E706 (nonsense), L707R, G708A, R710T, Q711E, L712F, V715M, K717Q, K720E, A721T, L722F, P723S, G724S, G724D, G724N, F725L, R726L, N727K, L728S, L728I, V730M, D732N, D732Y, D732E, Q733H, I737T, Y739D, W741R, M742V, M742I, G743R, G743V, L744F, M745T, V746M, A748D, A748V, A748T, M749V, M749I, G750S, G750D, W751R, R752Q, F754V, F754L, T755A, N756S, N756D, V757A, N758T, S759F, S759P, L762F, Y763H, Y763C, F764L, A765T, A765V, P766A, P766S, D767E, L768P, L768M, N771H, E772G, E772A, R774H, R774C, K777T, R779W, R786Q, G795V, M780I, S782N, C784Y, M787V, R788S, L790F, S791P, E793D, F794S, Q798E, Q802R, G803L, F804L, C806Y, M807V, M807R, M8071, L812P, F813V, S814N, N819Q, G820A, L821V, Q824L, Q824R, F827L, F827V, D828H, L830V, L830P, R831Q, R831L, Y834C, R840C, R840H, I841S, I842T, R846G, R854K, R855C, R855H, F856L, L863R, D864N, D864E, D864G, V866L, V866M, V866E, I869M, A870G, A870V, R871G, H874Y, H874R, Q875K, T877S, T877A, D879T, D879G, L880Q, L881V, M886V, S888L, V889M, F891L, P892L, M895T, A896T, E897D, I898T, Q902R, V903M, P904S, P904H, L907F, G909R, G909E, K910R, V911L, P913S, F916L, Q919R, or a combination thereof of the AR polypeptide.

Polynucleic Acid Molecules that Target B-Catenin and B-Catenin-Associated Genes

Catenin beta-1 (also known as CTNNB1, β-catenin, or beta-catenin) is a member of the catenin protein family. In humans, it is encoded by the CTNNB1 gene and is known for its dual functions—cell-cell adhesion and gene transcription. Beta-catenin is an integral structural component of cadherin-based adherens junctions and regulates cell growth and adhesion between cells and anchors the actin cytoskeleton. In some instance, beta-catenin is responsible for transmitting the contact inhibition signal that causes the cells to stop dividing once the epithelial sheet is complete. Beta-catenin is also a key nuclear effector of the Wnt signaling pathway. In some instances, imbalance in the structural and signaling properties of beta-catenin results in diseases and deregulated growth connected to malignancies such as cancer. For example, overexpression of beta-catenin has been linked to cancers such as gastric cancer (Suriano, et al., “Beta-catenin (CTNNB1) gene amplification: a new mechanism of protein overexpression in cancer,” Genes Chromosomes Cancer 42(3): 238-246 (2005)). In some cases, mutations in CTNNB1 gene have been linked to cancer development (e.g., colon cancer, melanoma, hepatocellular carcinoma, ovarian cancer, endometrial cancer, medulloblastoma pilomatricomas, or prostrate cancer), and in some instances, has been linked to metastatic progression. In additional cases, mutations in the CTNNB1 gene cause beta-catenin to translocate to the nucleus without any external stimulus and drive the transcription of its target genes continuously. In some cases, the potential of beta-catenin to change the previously epithelial phenotype of affected cells into an invasive, mesenchyme-like type contributes to metastasis formation.

In some embodiments, CTNNB1 gene is wild type CTNNB1 or CTNNB1 comprising one or more mutations. In some instances, CTNNB1 is wild type CTNNB1. In some instances, CTNNB1 is CTNNB1 comprising one or more mutations. In some instances, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of wild type CTNNB1. In some instances, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of CTNNB1 comprising a mutation (e.g., a substitution, a deletion, or an addition).

In some embodiments, CTNNB1 DNA or RNA comprises one or more mutations. In some embodiments, CTNNB1 DNA or RNA comprises one or more mutations within one or more exons. In some instances, the one or more exons comprise exon 3. In some instances, CTNNB1 DNA or RNA comprises one or more mutations at codons 32, 33, 34, 37, 41, 45, 183, 245, 287 or a combination thereof. In some instances, CTNNB1 DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues 25, 31, 32, 33, 34, 35, 36, 37, 41, 45, 140, 162, 170, 199, 213, 215, 257, 303, 322, 334, 354, 367, 373, 383, 387, 402, 426, 453, 474, 486, 515, 517, 535, 553, 555, 582, 587, 619, 623, 641, 646, 688, 703, 710, 712, 714, 724, 738, 777, or a combination thereof of the CTNNB1 polypeptide. In some embodiments, CTNNB1 DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues selected from W25 (nonsense mutation), L31M, D32A, D32N, D32Y, D32G, D32H, S33C, S33Y, S33F, S33P, G34R, G34E, G34V, I35S, H36Y, S37F, S37P, S37C, S37A, T41N, T41A, T41I, S45Y, S45F, S45C, I140T, D162E, K170M, V1991, C213F, A215T, T257I, I303M, Q322K, E334K, K354T, G367V, P373S, W383G, N387K, L402F, N426D, R453L, R453Q, 8474 (nonsense mutation), R486C, R515Q, L517F, R535 (nonsense mutation), R535Q, M553V, G555A, R582Q, R587Q, C619Y, Q623E, T641 (frame shift), S646F, M688T, Q703H, R710H, D712N, P714R, Y724H, E738K, F777S, or a combination thereof of the CTNNB1 polypeptide.

In some embodiments, the polynucleic acid molecule hybridizes to a target region of CTNNB1 DNA or RNA comprising one or more mutations. In some embodiments, the polynucleic acid molecule hybridizes to a target region of CTNNB1 DNA or RNA comprising one or more mutations within exon 3. In some embodiments, the polynucleic acid molecule hybridizes to a target region of CTNNB1 DNA or RNA comprising one or more mutations at codons 32, 33, 34, 37, 41, 45, 183, 245, 287 or a combination thereof. In some embodiments, the polynucleic acid molecule hybridizes to a target region of CTNNB1 DNA or RNA comprising one or more mutations at positions corresponding to amino acid residues 25, 31, 32, 33, 34, 35, 36, 37, 41, 45, 140, 162, 170, 199, 213, 215, 257, 303, 322, 334, 354, 367, 373, 383, 387, 402, 426, 453, 474, 486, 515, 517, 535, 553, 555, 582, 587, 619, 623, 641, 646, 688, 703, 710, 712, 714, 724, 738, 777, or a combination thereof of the CTNNB1 polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of CTNNB1 DNA or RNA comprising one or more mutations selected from W25 (nonsense mutation), L31M, D32A, D32N, D32Y, D32G, D32H, S33C, S33Y, S33F, S33P, G34R, G34E, G34V, I35S, H36Y, S37F, S37P, S37C, S37A, T41N, T41A, T41I, S45Y, S45F, S45C, I140T, D162E, K170M, V1991, C213F, A215T, T257I, I303M, Q322K, E334K, K354T, G367V, P373S, W383G, N387K, L402F, N426D, R453L, R453Q, R474 (nonsense mutation), R486C, R515Q, L517F, R535 (nonsense mutation), R535Q, M553V, G555A, R582Q, R587Q, C619Y, Q623E, T641 (frame shift), S646F, M688T, Q703H, R710H, D712N, P714R, Y724H, E738K, F777S, or a combination thereof of the CTNNB1 polypeptide.

In some embodiments, beta-catenin associated genes further comprise PIK3CA, PIK3CB, and MYC. In some embodiments, beta-catenin associated genes further comprise PIK3CA DNA or RNA. PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha or p110α protein) is a class i PI 3-kinase catalytic subunit that uses ATP to phosphorylate phosphatidylinositols. In some embodiments, PIK3CA gene is wild type PIK3CA or PIK3CA comprising one or more mutations. In some instances, PIK3CA DNA or RNA is wild type PIK3CA. In some instances, PIK3CA DNA or RNA comprises one or more mutations. In some instances, the polynucleic acid molecule hybridizes to a target region of wild type PIK3CA DNA or RNA. In some instances, the polynucleic acid molecule hybridizes to a target region of PIK3CA DNA or RNA comprising a mutation (e.g., a substitution, a deletion, or an addition).

In some embodiments, PIK3CA DNA or RNA comprises one or more mutations. In some embodiments, PIK3CA DNA or RNA comprises one or more mutation within one or more exons. In some instances, PIK3CA DNA or RNA comprises one or more mutation within exons 9 and/or 20. In some instances, PIK3CA DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues 1, 4, 10-16, 11-18, 11, 12, 38, 39, 65, 72, 75, 79, 81, 83, 88, 90, 93, 102, 103, 103-104, 103-106, 104, 105-108, 106, 106-107, 106-108, 107, 108, 109-112, 110, 111, 113, 115, 137, 170, 258, 272, 279, 320, 328, 335, 342, 344, 345, 350, 357, 359, 363, 364, 365, 366, 378, 398, 401, 417, 420, 447-455, 449, 449-457, 451, 453, 454, 455, 455-460, 463-465, 471, 495, 522, 538, 539, 542, 545, 546, 547, 576, 604, 614, 617, 629, 643, 663, 682, 725, 726, 777, 791, 818, 866, 901, 909, 939, 951, 958, 970, 971, 975, 992, 1004, 1007, 1016, 1017, 1021, 1025, 1029, 1037, 1040, 1043, 1044, 1045, 1047, 1048, 1049, 1052, 1065, 1069, or a combination thereof of the PIK3CA polypeptide. In some embodiments, PIK3CA DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues selected from M1V, R4 (nonsense mutation), L10-M16 (deletion), W11-P18 (deletion), W11L, G12D, R38L, R38H, R38C, R38S, E39K, E39G, E65K, S72G, Q75E, R79M, E81K, E81 (deletion), F83Y, R88Q, C90Y, C90G, R93Q, R93W, I102 (deletion), E103G, E103-P104 (deletion), E103-G106 (deletion), P104L, V105-R108 (deletion), G106V, G106-N107 (deletion), G106-R108 (deletion), G106R, N107S, R108L, R108H, E109-I112 (deletion), E110 (deletion), K111E, K111R, K111N, K111 (deletion), L113 (deletion), R115L, Q137L, N170S, D258N, Y272 (nonsense mutation), L2791, G320V, W328S, R335G, T342S, V344G, V344M, V344A, N345K, N345I, N345T, D350N, D350G, R357Q, G359R, G363A, G364R, E365K, E365V, P366R, C378R, C378Y, R398H, R401Q, E417K, C420R, C420G, P447-L455 (deletion), P449L, P449-N457 (deletion), G451R, G451V, E453K, E453Q, E453D, D454Y, L455 (frame shift insertion), L455-G460 (deletion), G463-N465 (deletion), P471L, P471A, H495L, H495Y, E522A, D538N, P539R, E542K, E542V, E542G, E542Q, E542A, E545K, E545A, E545G, E545Q, E545D, Q546K, Q546R, Q546P, E547D, S576Y, C604R, F614I, A617W, S629C, Q643H, I663S, Q682 (deletion), D725N, W726K, R777M, E791Q, R818C, L866W, C901F, F909L, D939G, R951C, Q958R, E970K, C971R, R975S, R992P, M1004I, G1007R, F1016C, D1017H, Y1021H, Y1021C, T1025A, T1025S, D1029H, E1037K, M1040V, M1043V, M1043I, N1044K, N1044Y, D1045V, H1047R, H1047L, H1047Y, H1047Q, H1048R, G1049R, T1052K, H1065L, 1069W (nonstop mutation), or a combination thereof of the PIK3CA polypeptide.

In some embodiments, the polynucleic acid molecule hybridizes to a target region of PIK3CA DNA or RNA comprising one or more mutations. In some embodiments, the polynucleic acid molecule hybridizes to a target region of PIK3CA DNA or RNA comprising one or more mutations within an exon. In some embodiments, the polynucleic acid molecule hybridizes to a target region of PIK3CA DNA or RNA comprising one or more mutations within exon 9 or exon 20. In some embodiments, the polynucleic acid molecule hybridizes to a target region of PIK3CA DNA or RNA comprising one or more mutations at positions corresponding to amino acid residues 1, 4, 10-16, 11-18, 11, 12, 38, 39, 65, 72, 75, 79, 81, 83, 88, 90, 93, 102, 103, 103-104, 103-106, 104, 105-108, 106, 106-107, 106-108, 107, 108, 109-112, 110, 111, 113, 115, 137, 170, 258, 272, 279, 320, 328, 335, 342, 344, 345, 350, 357, 359, 363, 364, 365, 366, 378, 398, 401, 417, 420, 447-455, 449, 449-457, 451, 453, 454, 455, 455-460, 463-465, 471, 495, 522, 538, 539, 542, 545, 546, 547, 576, 604, 614, 617, 629, 643, 663, 682, 725, 726, 777, 791, 818, 866, 901, 909, 939, 951, 958, 970, 971, 975, 992, 1004, 1007, 1016, 1017, 1021, 1025, 1029, 1037, 1040, 1043, 1044, 1045, 1047, 1048, 1049, 1052, 1065, 1069, or a combination thereof of the PIK3CA polypeptide. In some embodiments, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of PIK3CA DNA or RNA comprising one or more mutations at positions corresponding to amino acid residues selected from M1V, R4 (nonsense mutation), L10-M16 (deletion), W11-P18 (deletion), W11L, G12D, R38L, R38H, R38C, R38S, E39K, E39G, E65K, S72G, Q75E, R79M, E81K, E81 (deletion), F83Y, R88Q, C90Y, C90G, R93Q, R93W, I102 (deletion), E103G, E103-P104 (deletion), E103-G106 (deletion), P104L, V105-R108 (deletion), G106V, G106-N107 (deletion), G106-R108 (deletion), G106R, N107S, R108L, R108H, E109-I112 (deletion), E110 (deletion), K111E, K111R, K111N, K111 (deletion), L113 (deletion), R115L, Q137L, N170S, D258N, Y272 (nonsense mutation), L2791, G320V, W328S, R335G, T342S, V344G, V344M, V344A, N345K, N345I, N345T, D350N, D350G, R357Q, G359R, G363A, G364R, E365K, E365V, P366R, C378R, C378Y, R398H, R401Q, E417K, C420R, C420G, P447-L455 (deletion), P449L, P449-N457 (deletion), G451R, G451V, E453K, E453Q, E453D, D454Y, L455 (frame shift insertion), L455-G460 (deletion), G463-N465 (deletion), P471L, P471A, H495L, H495Y, E522A, D538N, P539R, E542K, E542V, E542G, E542Q, E542A, E545K, E545A, E545G, E545Q, E545D, Q546K, Q546R, Q546P, E547D, S576Y, C604R, F614I, A617W, S629C, Q643H, I663S, Q682 (deletion), D725N, W726K, R777M, E791Q, R818C, L866W, C901F, F909L, D939G, R951C, Q958R, E970K, C971R, R975S, R992P, M1004I, G1007R, F1016C, D1017H, Y1021H, Y1021C, T1025A, T1025S, D1029H, E1037K, M1040V, M1043V, M1043I, N1044K, N1044Y, D1045V, H1047R, H1047L, H1047Y, H1047Q, H1048R, G1049R, T1052K, H1065L, 1069W (nonstop mutation), or a combination thereof of the PIK3CB polypeptide.

In some embodiments, beta-catenin associated genes further comprise PIK3CB. In some embodiments, PIK3CB gene is wild type or comprises one or more mutations. In some instances, PIK3CB DNA or RNA is wild type PIK3CB DNA or RNA. In some instances, PIK3CB DNA or RNA comprises one or more mutations. In some instances, the polynucleic acid molecule hybridizes to a target region of wild type PIK3CB DNA or RNA. In some instances, the polynucleic acid molecule hybridizes to a target region of PIK3CB DNA or RNA comprising a mutation (e.g., a substitution, a deletion, or an addition).

In some embodiments, PIK3CB DNA or RNA comprises one or more mutations. In some embodiments, PIK3CB DNA or RNA comprises one or more mutations within one or more exons. In some instances, PIK3CB DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues 18, 19, 21, 28, 50, 61, 68, 103, 135, 140, 167, 252, 270, 290, 301, 304, 321, 369, 417, 442, 470, 497, 507, 512, 540, 551, 552, 554, 562, 567, 593, 595, 619, 628, 668, 768, 805, 824, 830, 887, 967, 992, 1005, 1020, 1036, 1046, 1047, 1048, 1049, 1051, 1055, 1067, or a combination thereof of the PIK3CB polypeptide. In some embodiments, PIK3CB DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues selected from W18 (nonsense mutation), A19V, D21H, G28S, A50P, K61T, M68I, R103K, H135N, L140S, S167C, G252W, R270W, K290N, E301V, I304R, R321Q, V369I, T417M, N442K, E470K, E497D, P507S, I512M, E540 (nonsense mutation), C551R, E552K, E554K, R562 (nonsense mutation), E567D, A593V, L595P, V619A, R628 (nonsense mutation), R668W, L768F, K805E, D824E, A830T, E887 (nonsense mutation), V967A, I992T, A1005V, D1020H, E1036K, D1046N, E1047K, A1048V, L1049R, E1051K, T1055A, D1067V, D1067A, or a combination thereof of the PIK3CB polypeptide.

In some embodiments, the polynucleic acid molecule hybridizes to a target region of PIK3CB DNA or RNA comprising one or more mutations. In some embodiments, the polynucleic acid molecule hybridizes to a target region of PIK3CB DNA or RNA comprising one or more mutations within an exon. In some embodiments, the polynucleic acid molecule hybridizes to a target region of PIK3CB DNA or RNA comprising one or more mutations at positions corresponding to amino acid residues 18, 19, 21, 28, 50, 61, 68, 103, 135, 140, 167, 252, 270, 290, 301, 304, 321, 369, 417, 442, 470, 497, 507, 512, 540, 551, 552, 554, 562, 567, 593, 595, 619, 628, 668, 768, 805, 824, 830, 887, 967, 992, 1005, 1020, 1036, 1046, 1047, 1048, 1049, 1051, 1055, 1067, or a combination thereof of the PIK3CB polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of PIK3CB DNA or RNA comprising one or more mutations at positions corresponding to amino acid residues selected from W18 (nonsense mutation), A19V, D21H, G28S, A50P, K61T, M68I, R103K, H135N, L140S, S167C, G252W, R270W, K290N, E301V, I304R, R321Q, V369I, T417M, N442K, E470K, E497D, P507S, I512M, E540 (nonsense mutation), C551R, E552K, E554K, R562 (nonsense mutation), E567D, A593V, L595P, V619A, R628 (nonsense mutation), R668W, L768F, K805E, D824E, A830T, E887 (nonsense mutation), V967A, I992T, A1005V, D1020H, E1036K, D1046N, E1047K, A1048V, L1049R, E1051K, T1055A, D1067V, D1067A, or a combination thereof of the PIK3CB polypeptide.

In some embodiments, beta-catenin associated genes further comprise MYC. In some embodiments, MYC gene is wild type MYC or MYC comprising one or more mutations. In some instances, MYC is wild type MYC DNA or RNA. In some instances, MYC DNA or RNA comprises one or more mutations. In some instances, the polynucleic acid molecule hybridizes to a target region of wild type MYC DNA or RNA. In some instances, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of MYC DNA or RNA comprising a mutation (e.g., a substitution, a deletion, or an addition).

In some embodiments, MYC DNA or RNA comprises one or more mutations. In some embodiments, MYC DNA or RNA comprises one or more mutation within one or more exons. In some instances, MYC DNA or RNA comprises one or more mutations within exon 2 or exon 3. In some instances, MYC DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues 2, 7, 17, 20, 32, 44, 58, 59, 76, 115, 138, 141, 145, 146, 169, 175, 188, 200, 202, 203, 248, 251, 298, 321, 340, 369, 373, 374, 389, 395, 404, 419, 431, 439, or a combination thereof. In some embodiments, MYC DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues selected from P2L, F7L, D17N, Q20E, Y32N, A44V, A44T, T581, P59L, A76V, F115L, F138S, A141S, V1451, S146L, S169C, S175N, C188F, N200S, S202N, S203T, T248S, D251E, S298Y, Q321E, V340D, V369D, T373K, H374R, F389L, Q395H, K404N, L419M, E431K, R439Q, or a combination thereof of the MYC polypeptide.

In some embodiments, the polynucleic acid molecule hybridizes to a target region of MYC DNA or RNA comprising one or more mutations. In some embodiments, the polynucleic acid molecule hybridizes to a target region of MYC DNA or RNA comprising one or more mutations within an exon. In some embodiments, the polynucleic acid molecule hybridizes to a target region of MYC DNA or RNA comprising one or more mutations within exon 2 or exon 3. In some embodiments, the polynucleic acid molecule hybridizes to a target region of MYC DNA or RNA comprising one or more mutations at positions corresponding to amino acid residues 2, 7, 17, 20, 32, 44, 58, 59, 76, 115, 138, 141, 145, 146, 169, 175, 188, 200, 202, 203, 248, 251, 298, 321, 340, 369, 373, 374, 389, 395, 404, 419, 431, 439, or a combination thereof of the MYC polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of MYC DNA or RNA comprising one or more mutations at positions corresponding to amino acid residues selected from P2L, F7L, D17N, Q20E, Y32N, A44V, A44T, T58I, P59L, A76V, F115L, F138S, A141S, V1451, S146L, S169C, S175N, C188F, N200S, S202N, S203T, T248S, D251E, S298Y, Q321E, V340D, V369D, T373K, H374R, F389L, Q395H, K404N, L419M, E431K, R439Q, or a combination thereof of the MYC polypeptide.

Polynucleic Acid Molecules that Target Hypoxanthine Phosphoribosyltransferase 1 (HPRT1)

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a transferase that catalyzes the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. HGPRT is encoded by the hypoxanthine Phosphoribosyltransferase 1 (HPRT1) gene.

In some embodiments, HPRT1 DNA or RNA is wild type or comprises one or more mutations. In some instances, HPRT1 DNA or RNA comprises one or more mutations within one or more exons. In some instances, the one or more exons comprise exon 2, exon 3, exon 4, exon 6, exon 8, or exon 9. In some instances, HPRT1 DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues 35, 48, 56, 74, 87, 129, 154, 162, 195, 200, 210, or a combination thereof of the HPRT1 polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of HPRT1 DNA or RNA comprising one or more mutations selected from V35M, R48H, E56D, F74L, R87I, N129 (splice-site mutation), N154H, S162 (splice-site mutation), Y195C, Y195N, R200M, E210K, or a combination thereof of the HPRT1 polypeptide.

In some embodiments, the polynucleic acid molecule hybridizes to a target region of HPRT1 DNA or RNA comprising one or more mutations. In some embodiments, the polynucleic acid molecule hybridizes to a target region of HPRT1 DNA or RNA comprising one or more mutations within exon 2, exon 3, exon 4, exon 6, exon 8, or exon 9. In some embodiments, the polynucleic acid molecule hybridizes to a target region of HPRT1 DNA or RNA comprising one or more mutations at positions corresponding to amino acid residues 35, 48, 56, 74, 87, 129, 154, 162, 195, 200, 210, or a combination thereof of the HPRT1 polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of HPRT1 DNA or RNA comprising one or more mutations selected from V35M, R48H, E56D, F74L, R87I, N129 (splice-site mutation), N154H, S162 (splice-site mutation), Y195C, Y195N, R200M, E210K, or a combination thereof of the HPRT1 polypeptide.

Polynucleic Acid Molecule Sequences

In some embodiments, the polynucleic acid molecule comprises a sequence that hybridizes to a target sequence illustrated in Tables 1, 4, 7, 8, or 10. In some instances, the polynucleic acid molecule is B. In some instances, the polynucleic acid molecule B comprises a sequence that hybridizes to a target sequence illustrated in Table 1 (KRAS target sequences). In some instances, the polynucleic acid molecule B comprises a sequence that hybridizes to a target sequence illustrated in Table 4 (EGFR target sequences). In some cases, the polynucleic acid molecule B comprises a sequence that hybridizes to a target sequence illustrated in Table 7 (AR target sequences). In some cases, the polynucleic acid molecule B comprises a sequence that hybridizes to a target sequence illustrated in Table 8 (β-catenin target sequences). In additional cases, the polynucleic acid molecule B comprises a sequence that hybridizes to a target sequence illustrated in Table 10 (PIK3CA and PIK3CB target sequences).

In some embodiments, the polynucleic acid molecule B comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listed in Table 2 or Table 3. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs: 16-75. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NOs: 16-75. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NOs: 16-75. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NOs: 16-75. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NOs: 16-75. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NOs: 16-75. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NOs: 16-75. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NOs: 16-75. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs: 16-75. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs: 16-75. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NOs: 16-75. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NOs: 16-75. In some embodiments, the polynucleic acid molecule consists of SEQ ID NOs: 16-75.

In some embodiments, the polynucleic acid molecule B comprises a first polynucleotide and a second polynucleotide. In some instances, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75. In some cases, the second polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75. In some cases, the polynucleic acid molecule comprises a first polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75 and a second polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75.

In some embodiments, the polynucleic acid molecule B comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listed in Table 5 or Table 6. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid molecule consists of SEQ ID NOs: 452-1955.

In some embodiments, the polynucleic acid molecule B comprises a first polynucleotide and a second polynucleotide. In some instances, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 452-1955. In some cases, the second polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 452-1955. In some cases, the polynucleic acid molecule comprises a first polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 452-1955 and a second polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 452-1955.

In some embodiments, the polynucleic acid molecule B comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listed in Table 7. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NOs: 1956-1962. In some embodiments, the polynucleic acid molecule consists of SEQ ID NOs: 1956-1962.

In some embodiments, the polynucleic acid molecule B comprises a first polynucleotide and a second polynucleotide. In some instances, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1956-1962. In some cases, the second polynucleotide comprises a sequence that is complementary to a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1956-1962. In some instances, the polynucleic acid molecule comprises a first polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1956-1962, and a second polynucleotide that is complementary to a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1956-1962.

In some embodiments, the polynucleic acid molecule B comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listed in Table 9. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NOs: 1967-2002. In some embodiments, the polynucleic acid molecule consists of SEQ ID NOs: 1967-2002.

In some embodiments, the polynucleic acid molecule B comprises a first polynucleotide and a second polynucleotide. In some instances, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1967-2002. In some cases, the second polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1967-2002. In some cases, the polynucleic acid molecule comprises a first polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1967-2002 and a second polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1967-2002.

In some embodiments, the polynucleic acid molecule B comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listed in Table 11. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NOs: 2013-2032. In some embodiments, the polynucleic acid molecule consists of SEQ ID NOs: 2013-2032.

In some embodiments, the polynucleic acid molecule B comprises a first polynucleotide and a second polynucleotide. In some instances, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2013-2032. In some cases, the second polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2013-2032. In some cases, the polynucleic acid molecule comprises a first polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2013-2032 and a second polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2013-2032.

In some embodiments, the polynucleic acid molecule B comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listed in Table 12.

In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some embodiments, the polynucleic acid molecule consists of SEQ ID NOs: 2082-2109 or 2117.

In some embodiments, the polynucleic acid molecule B comprises a first polynucleotide and a second polynucleotide. In some instances, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some cases, the second polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2082-2109 or 2117. In some cases, the polynucleic acid molecule comprises a first polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2082-2109 or 2117 and a second polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2082-2109 or 2117.

Polynucleic Acid Molecules

In some embodiments, the polynucleic acid molecule described herein comprises RNA or DNA. In some cases, the polynucleic acid molecule comprises RNA. In some instances, RNA comprises short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterogeneous nuclear RNA (hnRNA). In some instances, RNA comprises shRNA. In some instances, RNA comprises miRNA. In some instances, RNA comprises dsRNA. In some instances, RNA comprises tRNA. In some instances, RNA comprises rRNA. In some instances, RNA comprises hnRNA. In some instances, the RNA comprises siRNA. In some instances, the polynucleic acid molecule comprises siRNA. In some cases, B comprises siRNA.

In some embodiments, the polynucleic acid molecule is from about 10 to about 50 nucleotides in length. In some instances, the polynucleic acid molecule is from about 10 to about 30, from about 15 to about 30, from about 18 to about 25, from about 18 to about 24, from about 19 to about 23, or from about 20 to about 22 nucleotides in length.

In some embodiments, the polynucleic acid molecule is about 50 nucleotides in length. In some instances, the polynucleic acid molecule is about 45 nucleotides in length. In some instances, the polynucleic acid molecule is about 40 nucleotides in length. In some instances, the polynucleic acid molecule is about 35 nucleotides in length. In some instances, the polynucleic acid molecule is about 30 nucleotides in length. In some instances, the polynucleic acid molecule is about 25 nucleotides in length. In some instances, the polynucleic acid molecule is about 20 nucleotides in length. In some instances, the polynucleic acid molecule is about 19 nucleotides in length. In some instances, the polynucleic acid molecule is about 18 nucleotides in length. In some instances, the polynucleic acid molecule is about 17 nucleotides in length. In some instances, the polynucleic acid molecule is about 16 nucleotides in length. In some instances, the polynucleic acid molecule is about 15 nucleotides in length. In some instances, the polynucleic acid molecule is about 14 nucleotides in length. In some instances, the polynucleic acid molecule is about 13 nucleotides in length. In some instances, the polynucleic acid molecule is about 12 nucleotides in length. In some instances, the polynucleic acid molecule is about 11 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 nucleotides in length. In some instances, the polynucleic acid molecule is from about 10 to about 50 nucleotides in length. In some instances, the polynucleic acid molecule is from about 10 to about 45 nucleotides in length. In some instances, the polynucleic acid molecule is from about 10 to about 40 nucleotides in length. In some instances, the polynucleic acid molecule is from about 10 to about 35 nucleotides in length. In some instances, the polynucleic acid molecule is from about 10 to about 30 nucleotides in length. In some instances, the polynucleic acid molecule is from about 10 to about 25 nucleotides in length. In some instances, the polynucleic acid molecule is from about 10 to about 20 nucleotides in length. In some instances, the polynucleic acid molecule is from about 15 to about 25 nucleotides in length. In some instances, the polynucleic acid molecule is from about 15 to about 30 nucleotides in length. In some instances, the polynucleic acid molecule is from about 12 to about 30 nucleotides in length.

In some embodiments, the polynucleic acid molecule comprises a first polynucleotide. In some instances, the polynucleic acid molecule comprises a second polynucleotide. In some instances, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some instances, the first polynucleotide is a sense strand or passenger strand. In some instances, the second polynucleotide is an antisense strand or guide strand.

In some embodiments, the polynucleic acid molecule is a first polynucleotide. In some embodiments, the first polynucleotide is from about 10 to about 50 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 30, from about 15 to about 30, from about 18 to about 25, from about 18 to about 24, from about 19 to about 23, or from about 20 to about 22 nucleotides in length.

In some instances, the first polynucleotide is about 50 nucleotides in length. In some instances, the first polynucleotide is about 45 nucleotides in length. In some instances, the first polynucleotide is about 40 nucleotides in length. In some instances, the first polynucleotide is about 35 nucleotides in length. In some instances, the first polynucleotide is about 30 nucleotides in length. In some instances, the first polynucleotide is about 25 nucleotides in length. In some instances, the first polynucleotide is about 20 nucleotides in length. In some instances, the first polynucleotide is about 19 nucleotides in length. In some instances, the first polynucleotide is about 18 nucleotides in length. In some instances, the first polynucleotide is about 17 nucleotides in length. In some instances, the first polynucleotide is about 16 nucleotides in length. In some instances, the first polynucleotide is about 15 nucleotides in length. In some instances, the first polynucleotide is about 14 nucleotides in length. In some instances, the first polynucleotide is about 13 nucleotides in length. In some instances, the first polynucleotide is about 12 nucleotides in length. In some instances, the first polynucleotide is about 11 nucleotides in length. In some instances, the first polynucleotide is about 10 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 50 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 45 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 40 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 35 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 30 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 25 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 20 nucleotides in length. In some instances, the first polynucleotide is from about 15 to about 25 nucleotides in length. In some instances, the first polynucleotide is from about 15 to about 30 nucleotides in length. In some instances, the first polynucleotide is from about 12 to about 30 nucleotides in length.

In some embodiments, the polynucleic acid molecule is a second polynucleotide. In some embodiments, the second polynucleotide is from about 10 to about 50 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 30, from about 15 to about 30, from about 18 to about 25, from about 18 to about 24, from about 19 to about 23, or from about 20 to about 22 nucleotides in length.

In some instances, the second polynucleotide is about 50 nucleotides in length. In some instances, the second polynucleotide is about 45 nucleotides in length. In some instances, the second polynucleotide is about 40 nucleotides in length. In some instances, the second polynucleotide is about 35 nucleotides in length. In some instances, the second polynucleotide is about 30 nucleotides in length. In some instances, the second polynucleotide is about 25 nucleotides in length. In some instances, the second polynucleotide is about 20 nucleotides in length. In some instances, the second polynucleotide is about 19 nucleotides in length. In some instances, the second polynucleotide is about 18 nucleotides in length. In some instances, the second polynucleotide is about 17 nucleotides in length. In some instances, the second polynucleotide is about 16 nucleotides in length. In some instances, the second polynucleotide is about 15 nucleotides in length. In some instances, the second polynucleotide is about 14 nucleotides in length. In some instances, the second polynucleotide is about 13 nucleotides in length. In some instances, the second polynucleotide is about 12 nucleotides in length. In some instances, the second polynucleotide is about 11 nucleotides in length. In some instances, the second polynucleotide is about 10 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 50 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 45 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 40 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 35 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 30 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 25 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 20 nucleotides in length. In some instances, the second polynucleotide is from about 15 to about 25 nucleotides in length. In some instances, the second polynucleotide is from about 15 to about 30 nucleotides in length. In some instances, the second polynucleotide is from about 12 to about 30 nucleotides in length.

In some embodiments, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some instances, the polynucleic acid molecule further comprises a blunt terminus, an overhang, or a combination thereof. In some instances, the blunt terminus is a 5′ blunt terminus, a 3′ blunt terminus, or both. In some cases, the overhang is a 5′ overhang, 3′ overhang, or both. In some cases, the overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-base pairing nucleotides. In some cases, the overhang comprises 1, 2, 3, 4, 5, or 6 non-base pairing nucleotides. In some cases, the overhang comprises 1, 2, 3, or 4 non-base pairing nucleotides. In some cases, the overhang comprises 1 non-base pairing nucleotide. In some cases, the overhang comprises 2 non-base pairing nucleotides. In some cases, the overhang comprises 3 non-base pairing nucleotides. In some cases, the overhang comprises 4 non-base pairing nucleotides.

In some embodiments, the sequence of the polynucleic acid molecule is at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 50% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 60% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 70% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 80% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 90% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 95% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 99% complementary to a target sequence described herein. In some instances, the sequence of the polynucleic acid molecule is 100% complementary to a target sequence described herein.

In some embodiments, the sequence of the polynucleic acid molecule has 5 or less mismatches to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule has 4 or less mismatches to a target sequence described herein. In some instances, the sequence of the polynucleic acid molecule may has 3 or less mismatches to a target sequence described herein. In some cases, the sequence of the polynucleic acid molecule may has 2 or less mismatches to a target sequence described herein. In some cases, the sequence of the polynucleic acid molecule may has 1 or less mismatches to a target sequence described herein.

In some embodiments, the specificity of the polynucleic acid molecule that hybridizes to a target sequence described herein is a 95%, 98%, 99%, 99.5%, or 100% sequence complementarity of the polynucleic acid molecule to a target sequence. In some instances, the hybridization is a high stringent hybridization condition.

In some embodiments, the polynucleic acid molecule hybridizes to at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 8 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 9 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 10 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 11 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 12 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 13 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 14 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 15 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 16 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 17 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 18 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 19 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 20 contiguous bases of a target sequence described herein.

In some embodiments, the polynucleic acid molecule has reduced off-target effect. In some instances, “off-target” or “off-target effects” refer to any instance in which a polynucleic acid polymer directed against a given target causes an unintended effect by interacting either directly or indirectly with another mRNA sequence, a DNA sequence or a cellular protein or other moiety. In some instances, an “off-target effect” occurs when there is a simultaneous degradation of other transcripts due to partial homology or complementarity between that other transcript and the sense and/or antisense strand of the polynucleic acid molecule.

In some embodiments, the polynucleic acid molecule comprises natural, synthetic, or artificial nucleotide analogues or bases. In some cases, the polynucleic acid molecule comprises combinations of DNA, RNA and/or nucleotide analogues. In some instances, the synthetic or artificial nucleotide analogues or bases comprise modifications at one or more of ribose moiety, phosphate moiety, nucleoside moiety, or a combination thereof.

In some embodiments, a nucleotide analogue or artificial nucleotide base described above comprises a nucleic acid with a modification at a 2′ hydroxyl group of the ribose moiety. In some instances, the modification includes an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. Exemplary alkyl moiety includes, but is not limited to, halogens, sulfurs, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols and oxygen. In some instances, the alkyl moiety further comprises a modification. In some instances, the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso, group, a nitrile group, a heterocycle (e.g., imidazole, hydrazino or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some instances, the alkyl moiety further comprises a hetero substitution. In some instances, the carbon of the heterocyclic group is substituted by a nitrogen, oxygen or sulfur. In some instances, the heterocyclic substitution includes but is not limited to, morpholino, imidazole, and pyrrolidino.

In some instances, the modification at the 2′ hydroxyl group is a 2′-O-methyl modification or a 2′-O-methoxyethyl (2′-O-MOE) modification. In some cases, the 2′-O-methyl modification adds a methyl group to the 2′ hydroxyl group of the ribose moiety whereas the 2′O-methoxyethyl modification adds a methoxyethyl group to the 2′ hydroxyl group of the ribose moiety. Exemplary chemical structures of a 2′-O-methyl modification of an adenosine molecule and 2′O-methoxyethyl modification of an uridine are illustrated below.

In some instances, the modification at the 2′ hydroxyl group is a 2′-O-aminopropyl modification in which an extended amine group comprising a propyl linker binds the amine group to the 2′ oxygen. In some instances, this modification neutralizes the phosphate-derived overall negative charge of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar and thereby improves cellular uptake properties due to its zwitterionic properties. An exemplary chemical structure of a 2′-O-aminopropyl nucleoside phosphoramidite is illustrated below.

In some instances, the modification at the 2′ hydroxyl group is a locked or bridged ribose modification (e.g., locked nucleic acid or LNA) in which the oxygen molecule bound at the 2′ carbon is linked to the 4′ carbon by a methylene group, thus forming a 2′-C,4′-C-oxy-methylene-linked bicyclic ribonucleotide monomer. Exemplary representations of the chemical structure of LNA are illustrated below. The representation shown to the left highlights the chemical connectivities of an LNA monomer. The representation shown to the right highlights the locked 3′-endo (³E) conformation of the furanose ring of an LNA monomer.

In some instances, the modification at the 2′ hydroxyl group comprises ethylene nucleic acids (ENA) such as for example 2′-4′-ethylene-bridged nucleic acid, which locks the sugar conformation into a C₃′-endo sugar puckering conformation. ENA are part of the bridged nucleic acids class of modified nucleic acids that also comprises LNA. Exemplary chemical structures of the ENA and bridged nucleic acids are illustrated below.

In some embodiments, additional modifications at the 2′ hydroxyl group include 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA).

In some embodiments, a nucleotide analogue comprises a modified base such as, but not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N, N, -dimethyladenine, 2-propyladenine, 2propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5-(2-amino) propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2, 2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides (such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, or 6-azothymidine), 5-methyl-2-thiouridine, other thio bases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines (such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, or pyridine-2-one), phenyl and modified phenyl groups such as aminophenol or 2,4, 6-trimethoxy benzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyi nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moieties, in some cases are or are based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4′-thioribose, and other sugars, heterocycles, or carbocycles. The term nucleotide also includes what are known in the art as universal bases. By way of example, universal bases include but are not limited to 3-nitropyrrole, 5-nitroindole, or nebularine.

In some embodiments, a nucleotide analogue further comprises a morpholino, a peptide nucleic acid (PNA), a methylphosphonate nucleotide, a thiolphosphonate nucleotide, a 2′-fluoro N3-P5′-phosphoramidite, or a 1′, 5′-anhydrohexitol nucleic acid (HNA). Morpholino or phosphorodiamidate morpholino oligo (PMO) comprises synthetic molecules whose structure mimics natural nucleic acid structure but deviates from the normal sugar and phosphate structures. In some instances, the five member ribose ring is substituted with a six member morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, the ribose monomers are linked by a phosphordiamidate group instead of a phosphate group. In such cases, the backbone alterations remove all positive and negative charges making morpholinos neutral molecules capable of crossing cellular membranes without the aid of cellular delivery agents such as those used by charged oligonucleotides.

In some embodiments, a morpholino or PMO described above is a PMO comprising a positive or cationic charge. In some instances, the PMO is PMOplus (Sarepta). PMOplus refers to phosphorodiamidate morpholino oligomers comprising any number of (1-piperazino)phosphinylideneoxy, (1-(4-(omega-guanidino-alkanoyl))-piperazino)phosphinylideneoxy linkages (e.g., as such those described in PCT Publication No. WO2008/036127. In some cases, the PMO is a PMO described in U.S. Pat. No. 7,943,762.

In some embodiments, a morpholino or PMO described above is a PMO-X (Sarepta). In some cases, PMO-X refers to phosphorodiamidate morpholino oligomers comprising at least one linkage or at least one of the disclosed terminal modifications, such as those disclosed in PCT Publication No. WO2011/150408 and U.S. Publication No. 2012/0065169.

In some embodiments, a morpholino or PMO described above is a PMO as described in Table 5 of U.S. Publication No. 2014/0296321.

In some embodiments, peptide nucleic acid (PNA) does not contain sugar ring or phosphate linkage and the bases are attached and appropriately spaced by oligoglycine-like molecules, therefore, eliminating a backbone charge.

In some embodiments, one or more modifications optionally occur at the internucleotide linkage. In some instances, modified internucleotide linkage includes, but is not limited to, phosphorothioates; phosphorodithioates; methylphosphonates; 5′-alkylenephosphonates; 5′-methylphosphonate; 3′-alkylene phosphonates; borontrifluoridates; borano phosphate esters and selenophosphates of 3′-5′linkage or 2′-5′linkage; phosphotriesters; thionoalkylphosphotriesters; hydrogen phosphonate linkages; alkyl phosphonates; alkylphosphonothioates; arylphosphonothioates; phosphoroselenoates; phosphorodiselenoates; phosphinates; phosphoramidates; 3′-alkylphosphoramidates; aminoalkylphosphoramidates; thionophosphoramidates; phosphoropiperazidates; phosphoroanilothioates; phosphoroanilidates; ketones; sulfones; sulfonamides; carbonates; carbamates; methylenehydrazos; methylenedimethylhydrazos; formacetals; thioformacetals; oximes; methyleneiminos; methylenemethyliminos; thioamidates; linkages with riboacetyl groups; aminoethyl glycine; silyl or siloxane linkages; alkyl or cycloalkyl linkages with or without heteroatoms of, for example, 1 to 10 carbons that are saturated or unsaturated and/or substituted and/or contain heteroatoms; linkages with morpholino structures, amides, or polyamides wherein the bases are attached to the aza nitrogens of the backbone directly or indirectly; and combinations thereof.

In some instances, the modification is a methyl or thiol modification such as methylphosphonate or thiolphosphonate modification. Exemplary thiolphosphonate nucleotide (left) and methylphosphonate nucleotide (right) are illustrated below.

In some instances, a modified nucleotide includes, but is not limited to, 2′-fluoro N3-P5′-phosphoramidites illustrated as:

In some instances, a modified nucleotide includes, but is not limited to, hexitol nucleic acid (or 1′, 5′-anhydrohexitol nucleic acids (HNA)) illustrated as:

In some embodiments, one or more modifications comprise a modified phosphate backbone in which the modification generates a neutral or uncharged backbone. In some instances, the phosphate backbone is modified by alkylation to generate an uncharged or neutral phosphate backbone. As used herein, alkylation includes methylation, ethylation, and propylation. In some cases, an alkyl group, as used herein in the context of alkylation, refers to a linear or branched saturated hydrocarbon group containing from 1 to 6 carbon atoms. In some instances, exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, isohexyl, 1, 1-dimethylbutyl, 2,2-dimethylbutyl, 3.3-dimethylbutyl, and 2-ethylbutyl groups. In some cases, a modified phosphate is a phosphate group as described in U.S. Pat. No. 9,481,905.

In some embodiments, additional modified phosphate backbones comprise methylphosphonate, ethylphosphonate, methylthiophosphonate, or methoxyphosphonate. In some cases, the modified phosphate is methylphosphonate. In some cases, the modified phosphate is ethylphosphonate. In some cases, the modified phosphate is methylthiophosphonate. In some cases, the modified phosphate is methoxyphosphonate.

In some embodiments, one or more modifications further optionally include modifications of the ribose moiety, phosphate backbone and the nucleoside, or modifications of the nucleotide analogues at the 3′ or the 5′ terminus. For example, the 3′ terminus optionally include a 3′ cationic group, or by inverting the nucleoside at the 3′-terminus with a 3′-3′ linkage. In another alternative, the 3′-terminus is optionally conjugated with an aminoalkyl group, e.g., a 3′ C5-aminoalkyl dT. In an additional alternative, the 3′-terminus is optionally conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site. In some instances, the 5′-terminus is conjugated with an aminoalkyl group, e.g., a 5′-O-alkylamino substituent. In some cases, the 5′-terminus is conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site.

In some embodiments, the polynucleic acid molecule comprises one or more of the artificial nucleotide analogues described herein. In some instances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of the artificial nucleotide analogues described herein. In some embodiments, the artificial nucleotide analogues include 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2′-fluoro N3-P5′-phosphoramidites, or a combination thereof. In some instances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of the artificial nucleotide analogues selected from 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2′-fluoro N3-P5′-phosphoramidites, or a combination thereof. In some instances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of 2′-O-methyl modified nucleotides. In some instances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of 2′-O-methoxyethyl (2′-O-MOE) modified nucleotides. In some instances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of thiolphosphonate nucleotides.

In some embodiments, the polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, or more modifications. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117.

In some instances, the polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, or more modified nucleotides. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117.

In some instances, the polynucleic acid molecule comprises at least one of: from about 5% to about 100% modification, from about 10% to about 100% modification, from about 20% to about 100% modification, from about 30% to about 100% modification, from about 40% to about 100% modification, from about 50% to about 100% modification, from about 60% to about 100% modification, from about 70% to about 100% modification, from about 80% to about 100% modification, and from about 90% to about 100% modification. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117.

In some instances, about 5 to about 100% of the polynucleic acid molecule comprise the artificial nucleotide analogues described herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the polynucleic acid molecule comprise the artificial nucleotide analogues described herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a polynucleic acid molecule of SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117 comprise the artificial nucleotide analogues described herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a polynucleic acid molecule of SEQ ID NOs: 16-45 comprise the artificial nucleotide analogues described herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a polynucleic acid molecule of SEQ ID NOs: 452-1203 comprise the artificial nucleotide analogues described herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a polynucleic acid molecule of SEQ ID NOs: 1956-1962 comprise the artificial nucleotide analogues described herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a polynucleic acid molecule of SEQ ID NOs: 1967-2002 comprise the artificial nucleotide analogues described herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a polynucleic acid molecule of SEQ ID NOs: 2013-2032 comprise the artificial nucleotide analogues described herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a polynucleic acid molecule of SEQ ID NOs: 2082-2109 or 2117 comprise the artificial nucleotide analogues described herein. In some embodiments, the artificial nucleotide analogues include 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (T-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2′-fluoro N3-P5′-phosphoramidites, or a combination thereof.

In some instances, the polynucleic acid molecule that comprises an artificial nucleotide analogue comprises SEQ ID NOs: 46-75. In some instances, the polynucleic acid molecule that comprises an artificial nucleotide analogue comprises SEQ ID NOs: 1204-1955. In some instances, the polynucleic acid molecule that comprises an artificial nucleotide analogue comprises SEQ ID NOs: 1967-2002. In some instances, the polynucleic acid molecule that comprises an artificial nucleotide analogue comprises SEQ ID NOs: 2013-2032. In some instances, the polynucleic acid molecule that comprises an artificial nucleotide analogue comprises SEQ ID NOs: 2082-2109 or 2117.

In some cases, one or more of the artificial nucleotide analogues described herein are resistant toward nucleases such as for example ribonuclease such as RNase H, deoxyribunuclease such as DNase, or exonuclease such as 5′-3′ exonuclease and 3′-5′ exonuclease when compared to natural polynucleic acid molecules. In some instances, artificial nucleotide analogues comprising 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2′-fluoro N3-P5′-phosphoramidites, or combinations thereof are resistant toward nucleases such as for example ribonuclease such as RNase H, deoxyribunuclease such as DNase, or exonuclease such as 5′-3′ exonuclease and 3′-5′ exonuclease. In some instances, 2′-O-methyl modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, 2′O-methoxyethyl (2′-O-MOE) modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, 2′-O-aminopropyl modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, 2′-deoxy modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, T-deoxy-2′-fluoro modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, 2′-O-aminopropyl (2′-O-AP) modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, 2′-O-dimethylaminoethyl (2′-O-DMAOE) modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, 2′-O-dimethylaminopropyl (2′-O-DMAP) modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE) modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, 2′-O—N-methylacetamido (2′-O-NMA) modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, LNA-modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, ENA-modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, HNA-modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). Morpholinos may be nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, PNA-modified polynucleic acid molecule is resistant to nucleases (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, methylphosphonate nucleotide-modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, thiolphosphonate nucleotide-modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, polynucleic acid molecule comprising 2′-fluoro N3-P5′-phosphoramidites is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, the 5′ conjugates described herein inhibit 5′-3′ exonucleolytic cleavage. In some instances, the 3′ conjugates described herein inhibit 3′-5′ exonucleolytic cleavage.

In some embodiments, one or more of the artificial nucleotide analogues described herein have increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. The one or more of the artificial nucleotide analogues comprising 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, or 2% fluoro N3-P5′-phosphoramidites can have increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2′-O-methyl modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2′-O-methoxyethyl (2′-O-MOE) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2′-O-aminopropyl modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2′-deoxy modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, T-deoxy-2′-fluoro modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2′-O-aminopropyl (2′-O-AP) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2′-O-dimethylaminoethyl (2′-O-DMAOE) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2′-O-dimethylaminopropyl (2′-O-DMAP) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2′-O—N-methylacetamido (2′-O-NMA) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, LNA-modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, ENA-modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, PNA-modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, HNA-modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, morpholino-modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, methylphosphonate nucleotide-modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, thiolphosphonate nucleotide-modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, polynucleic acid molecule comprising 2′-fluoro N3-P5′-phosphoramidites has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some cases, the increased affinity is illustrated with a lower Kd, a higher melt temperature (Tm), or a combination thereof.

In some embodiments, a polynucleic acid molecule described herein is a chirally pure (or stereo pure) polynucleic acid molecule, or a polynucleic acid molecule comprising a single enantiomer. In some instances, the polynucleic acid molecule comprises L-nucleotide. In some instances, the polynucleic acid molecule comprises D-nucleotides. In some instance, a polynucleic acid molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its mirror enantiomer. In some cases, a polynucleic acid molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic mixture. In some instances, the polynucleic acid molecule is a polynucleic acid molecule described in: U.S. Patent Publication Nos: 2014/194610 and 2015/211006; and PCT Publication No.: WO2015107425.

In some embodiments, a polynucleic acid molecule described herein is further modified to include an aptamer-conjugating moiety. In some instances, the aptamer conjugating moiety is a DNA aptamer-conjugating moiety. In some instances, the aptamer-conjugating moiety is Alphamer (Centauri Therapeutics), which comprises an aptamer portion that recognizes a specific cell-surface target and a portion that presents a specific epitopes for attaching to circulating antibodies. In some instance, a polynucleic acid molecule described herein is further modified to include an aptamer-conjugating moiety as described in: U.S. Pat. Nos. 8,604,184, 8,591,910, and 7,850,975.

In additional embodiments, a polynucleic acid molecule described herein is modified to increase its stability. In some embodiment, the polynucleic acid molecule is RNA (e.g., siRNA), the polynucleic acid molecule is modified to increase its stability. In some instances, the polynucleic acid molecule is modified by one or more of the modifications described above to increase its stability. In some cases, the polynucleic acid molecule is modified at the 2′ hydroxyl position, such as by 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modification or by a locked or bridged ribose conformation (e.g., LNA or ENA). In some cases, the polynucleic acid molecule is modified by 2′-O-methyl and/or 2′-O-methoxyethyl ribose. In some cases, the polynucleic acid molecule also includes morpholinos, PNAs, HNA, methylphosphonate nucleotides, thiolphosphonate nucleotides, and/or 2′-fluoro N3-P5′-phosphoramidites to increase its stability. In some instances, the polynucleic acid molecule is a chirally pure (or stereo pure) polynucleic acid molecule. In some instances, the chirally pure (or stereo pure) polynucleic acid molecule is modified to increase its stability. Suitable modifications to the RNA to increase stability for delivery will be apparent to the skilled person.

In some embodiments, a polynucleic acid molecule described herein has RNAi activity that modulates expression of RNA encoded by a gene described supra. In some instances, a polynucleic acid molecule described herein is a double-stranded siRNA molecule that down-regulates expression of a gene, wherein one of the strands of the double-stranded siRNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of the gene or RNA encoded by the gene or a portion thereof, and wherein the second strand of the double-stranded siRNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of the gene or RNA encoded by the gene or a portion thereof. In some cases, a polynucleic acid molecule described herein is a double-stranded siRNA molecule that down-regulates expression of a gene, wherein each strand of the siRNA molecule comprises about 15 to 25, 18 to 24, or 19 to about 23 nucleotides, and wherein each strand comprises at least about 14, 17, or 19 nucleotides that are complementary to the nucleotides of the other strand. In some cases, a polynucleic acid molecule described herein is a double-stranded siRNA molecule that down-regulates expression of a gene, wherein each strand of the siRNA molecule comprises about 19 to about 23 nucleotides, and wherein each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand. In some instances, the gene is KRAS, EGFR, AR, HPRT1, CNNTB1 (β-catenin), or β-catenin associated genes.

In some embodiments, a polynucleic acid molecule described herein is constructed using chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. For example, a polynucleic acid molecule is chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the polynucleic acid molecule and target nucleic acids. Exemplary methods include those described in: U.S. Pat. Nos. 5,142,047; 5,185,444; 5,889,136; 6,008,400; and 6,111,086; PCT Publication No. WO2009099942; or European Publication No. 1579015. Additional exemplary methods include those described in: Griffey et al., “2′-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides,” J. Med. Chem. 39(26):5100-5109 (1997)); Obika, et al. “Synthesis of 2′-O,4′-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering”. Tetrahedron Letters 38 (50): 8735 (1997); Koizumi, M. “ENA oligonucleotides as therapeutics”. Current opinion in molecular therapeutics 8 (2): 144-149 (2006); and Abramova et al., “Novel oligonucleotide analogues based on morpholino nucleoside subunits-antisense technologies: new chemical possibilities,” Indian Journal of Chemistry 48B:1721-1726 (2009). Alternatively, the polynucleic acid molecule is produced biologically using an expression vector into which a polynucleic acid molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted polynucleic acid molecule will be of an antisense orientation to a target polynucleic acid molecule of interest).

Conjugation Chemistry

In some embodiments, a polynucleic acid molecule is conjugated to a binding moiety. In some instances, the binding moiety comprises amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, as well as analogs or derivatives of all of these classes of substances. Additional examples of binding moiety also include steroids, such as cholesterol, phospholipids, di- and triacylglycerols, fatty acids, hydrocarbons (e.g., saturated, unsaturated, or contains substitutions), enzyme substrates, biotin, digoxigenin, and polysaccharides. In some instances, the binding moiety is an antibody or binding fragment thereof. In some instances, the polynucleic acid molecule is further conjugated to a polymer, and optionally an endosomolytic moiety.

In some embodiments, the polynucleic acid molecule is conjugated to the binding moiety by a chemical ligation process. In some instances, the polynucleic acid molecule is conjugated to the binding moiety by a native ligation. In some instances, the conjugation is as described in: Dawson, et al. “Synthesis of proteins by native chemical ligation,” Science 1994, 266, 776-779; Dawson, et al. “Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives,” J. Am. Chem. Soc. 1997, 119, 4325-4329; Hackeng, et al. “Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology.,” Proc. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or Wu, et al. “Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol,” Angew. Chem. Int. Ed. 2006, 45, 4116-4125. In some instances, the conjugation is as described in U.S. Pat. No. 8,936,910. In some embodiments, the polynucleic acid molecule is conjugated to the binding moiety either site-specifically or non-specifically via native ligation chemistry.

In some instances, the polynucleic acid molecule is conjugated to the binding moiety by a site-directed method utilizing a “traceless” coupling technology (Philochem). In some instances, the “traceless” coupling technology utilizes an N-terminal 1,2-aminothiol group on the binding moiety which is then conjugate with a polynucleic acid molecule containing an aldehyde group. (see Casi et al., “Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery,” JACS 134(13): 5887-5892 (2012))

In some instances, the polynucleic acid molecule is conjugated to the binding moiety by a site-directed method utilizing an unnatural amino acid incorporated into the binding moiety. In some instances, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some instances, the keto group of pAcPhe is selectively coupled to an alkoxy-amine derivatived conjugating moiety to form an oxime bond. (see Axup et al., “Synthesis of site-specific antibody-drug conjugates using unnatural amino acids,” PNAS 109(40): 16101-16106 (2012)).

In some instances, the polynucleic acid molecule is conjugated to the binding moiety by a site-directed method utilizing an enzyme-catalyzed process. In some instances, the site-directed method utilizes SMARTag™ technology (Redwood). In some instances, the SMARTag™ technology comprises generation of a formylglycine (FGly) residue from cysteine by formylglycine-generating enzyme (FGE) through an oxidation process under the presence of an aldehyde tag and the subsequent conjugation of FGly to an alkylhydraine-functionalized polynucleic acid molecule via hydrazino-Pictet-Spengler (HIPS) ligation. (see Wu et al., “Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag,” PNAS 106(9): 3000-3005 (2009); Agarwal, et al., “A Pictet-Spengler ligation for protein chemical modification,” PNAS 110(1): 46-51 (2013))

In some instances, the enzyme-catalyzed process comprises microbial transglutaminase (mTG). In some cases, the polynucleic acid molecule is conjugated to the binding moiety utilizing a microbial transglutaminze catalyzed process. In some instances, mTG catalyzes the formation of a covalent bond between the amide side chain of a glutamine within the recognition sequence and a primary amine of a functionalized polynucleic acid molecule. In some instances, mTG is produced from Streptomyces mobarensis. (see Strop et al., “Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates,” Chemistry and Biology 20(2) 161-167 (2013))

In some instances, the polynucleic acid molecule is conjugated to the binding moiety by a method as described in PCT Publication No. WO2014/140317, which utilizes a sequence-specific transpeptidase.

In some instances, the polynucleic acid molecule is conjugated to the binding moiety by a method as described in U.S. Patent Publication Nos. 2015/0105539 and 2015/0105540.

Binding Moiety

In some embodiments, the binding moiety A is a polypeptide. In some instances, the polypeptide is an antibody or its fragment thereof. In some cases, the fragment is a binding fragment. In some instances, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, murine antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab₂, F(ab)′₃ fragments, single-chain variable fragment (scFv), bis-scFv, (scFv)₂, diabody, minibody, nanobody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof.

In some instances, A is an antibody or binding fragment thereof. In some instances, A is a humanized antibody or binding fragment thereof, murine antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab₂, F(ab)′₃ fragments, single-chain variable fragment (scFv), bis-scFv, (scFv)₂, diabody, minibody, nanobody, triabody, tetrabody, disulfide stabilized Fv protein (“dsFv”), single-domain antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof. In some instances, A is a humanized antibody or binding fragment thereof. In some instances, A is a murine antibody or binding fragment thereof. In some instances, A is a chimeric antibody or binding fragment thereof. In some instances, A is a monoclonal antibody or binding fragment thereof. In some instances, A is a monovalent Fab′. In some instances, A is a diavalent Fab₂. In some instances, A is a single-chain variable fragment (scFv).

In some embodiments, the binding moiety A is a bispecific antibody or binding fragment thereof. In some instances, the bispecific antibody is a trifunctional antibody or a bispecific mini-antibody. In some cases, the bispecific antibody is a trifunctional antibody. In some instances, the trifunctional antibody is a full length monoclonal antibody comprising binding sites for two different antigens. Exemplary trifunctional antibodies include catumaxomab (which targets EpCAM and CD3; Fresenius Biotech/Trion Pharma), ertumaxomab (targets HER2/neu/CD3; Fresenius Biotech/Trion Pharma), lymphomun FBTA05 (targets CD20/CD3; Fresenius Biotech/Trion Pharma), RG7221 (RO5520985; targets Angiopoietin 2/VEGF; Roche), RG7597 (targets Her1/Her3; Genentech/Roche), MM141 (targets IGF1R/Her3; Merrimack), ABT122 (targets TNFα/IL17; Abbvie), ABT981 (targets IL1α/IL1β; Abbott), LY3164530 (targets Her1/cMET; Eli Lilly), and TRBS07 (Ektomab; targets GD2/CD3; Trion Research Gmbh). Additional exemplary trifunctional antibodies include mAb² from F-star Biotechnology Ltd. In some instances, A is a bispecific trifunctional antibody. In some embodiments, A is a bispecific trifunctional antibody selected from: catumaxomab (which targets EpCAM and CD3; Fresenius Biotech/Trion Pharma), ertumaxomab (targets HER2/neu/CD3; Fresenius Biotech/Trion Pharma), lymphomun FBTA05 (targets CD20/CD3; Fresenius Biotech/Trion Pharma), RG7221 (RO5520985; targets Angiopoietin 2/VEGF; Roche), RG7597 (targets Her1/Her3; Genentech/Roche), MM141 (targets IGF1R/Her3; Merrimack), ABT122 (targets TNFα/IL17; Abbvie), ABT981 (targets IL1α/IL1β; Abbott), LY3164530 (targets Her1/cMET; Eli Lilly), TRBS07 (Ektomab; targets GD2/CD3; Trion Research Gmbh), and a mAb² from F-star Biotechnology Ltd.

In some cases, the bispecific antibody is a bispecific mini-antibody. In some instances, the bispecific mini-antibody comprises divalent Fab₂, F(ab)′₃ fragments, bis-scFv, (scFv)₂, diabody, minibody, triabody, tetrabody or a bi-specific T-cell engager (BiTE). In some embodiments, the bi-specific T-cell engager is a fusion protein that contains two single-chain variable fragments (scFvs) in which the two scFvs target epitopes of two different antigens. Exemplary bispecific mini-antibodies include, but are not limited to, DART (dual-affinity re-targeting platform; MacroGenics), blinatumomab (MT103 or AMG103; which targets CD19/CD3; Micromet), MT111 (targets CEA/CD3; Micromet/Amegen), MT112 (BAY2010112; targets PSMA/CD3; Micromet/Bayer), MT110 (AMG 110; targets EPCAM/CD3; Amgen/Micromet), MGD006 (targets CD123/CD3; MacroGenics), MGD007 (targets GPA33/CD3; MacroGenics), BI1034020 (targets two different epitopes on β-amyloid; Ablynx), ALX0761 (targets IL17A/IL17F; Ablynx), TF2 (targets CEA/hepten; Immunomedics), IL-17/IL-34 biAb (BMS), AFM13 (targets CD30/CD16; Affimed), AFM11 (targets CD19/CD3; Affimed), and domain antibodies (dAbs from Domantis/GSK).

In some embodiments, the binding moiety A is a bispecific mini-antibody. In some instances, A is a bispecific Fab₂. In some instances, A is a bispecific F(ab)′₃ fragment. In some cases, A is a bispecific bis-scFv. In some cases, A is a bispecific (scFv)₂. In some embodiments, A is a bispecific diabody. In some embodiments, A is a bispecific minibody. In some embodiments, A is a bispecific triabody. In other embodiments, A is a bispecific tetrabody. In other embodiments, A is a bi-specific T-cell engager (BiTE). In additional embodiments, A is a bispecific mini-antibody selected from: DART (dual-affinity re-targeting platform; MacroGenics), blinatumomab (MT103 or AMG103; which targets CD19/CD3; Micromet), MT111 (targets CEA/CD3; Micromet/Amegen), MT112 (BAY2010112; targets PSMA/CD3; Micromet/Bayer), MT110 (AMG 110; targets EPCAM/CD3; Amgen/Micromet), MGD006 (targets CD123/CD3; MacroGenics), MGD007 (targets GPA33/CD3; MacroGenics), BI1034020 (targets two different epitopes on β-amyloid; Ablynx), ALX0761 (targets IL17A/IL17F; Ablynx), TF2 (targets CEA/hepten; Immunomedics), IL-17/IL-34 biAb (BMS), AFM13 (targets CD30/CD16; Affimed), AFM11 (targets CD19/CD3; Affimed), and domain antibodies (dAbs from Domantis/GSK).

In some embodiments, the binding moiety A is a trispecific antibody. In some instances, the trispecific antibody comprises F(ab)′₃ fragments or a triabody. In some instances, A is a trispecific F(ab)′₃ fragment. In some cases, A is a triabody. In some embodiments, A is a trispecific antibody as described in Dimas, et al., “Development of a trispecific antibody designed to simultaneously and efficiently target three different antigens on tumor cells,” Mol. Pharmaceutics, 12(9): 3490-3501 (2015).

In some embodiments, the binding moiety A is an antibody or binding fragment thereof that recognizes a cell surface protein. In some instances, the cell surface protein is an antigen expressed by a cancerous cell. Exemplary cancer antigens include, but are not limited to, alpha fetoprotein, ASLG659, B7-H3, BAFF-R, Brevican, CA125 (MUC16), CA15-3, CA19-9, carcinoembryonic antigen (CEA), CA242, CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor), CTLA-4, CXCR5, E16 (LAT1, SLC7A5), FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C), epidermal growth factor, ETBR, Fc receptor-like protein 1 (FCRH1), GEDA, HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen), human chorionic gonadotropin, ICOS, IL-2 receptor, IL20Rα, Immunoglobulin superfamily receptor translocation associated 2 (IRTA2), L6, Lewis Y, Lewis X, MAGE-1, MAGE-2, MAGE-3, MAGE 4, MART1, mesothelin, MDP, MPF (SMR, MSLN), MCP1 (CCL2), macrophage inhibitory factor (MIF), MPG, MSG783, mucin, MUC1-KLH, Napi3b (SLC34A2), nectin-4, Neu oncogene product, NCA, placental alkaline phosphatase, prostate specific membrane antigen (PMSA), prostatic acid phosphatase, PSCA hlg, p97, Purinergic receptor P2X ligand-gated ion channel 5 (P2X5), LY64 (Lymphocyte antigen 64 (RP105), gp100, P21, six transmembrane epithelial antigen of prostate (STEAP1), STEAP2, Sema 5b, tumor-associated glycoprotein 72 (TAG-72), TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4) and the like.

In some instances, the cell surface protein comprises clusters of differentiation (CD) cell surface markers. Exemplary CD cell surface markers include, but are not limited to, CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L (L-selectin), CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD79 (e.g., CD79a, CD79b), CD90, CD95 (Fas), CD103, CD104, CD125 (IL5RA), CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD221, CD274, CD279 (PD-1), CD319 (SLAMF7), CD326 (EpCAM), and the like.

In some instances, the binding moiety A is an antibody or binding fragment thereof that recognizes a cancer antigen. In some instances, the binding moiety A is an antibody or binding fragment thereof that recognizes alpha fetoprotein, ASLG659, B7-H3, BAFF-R, Brevican, CA125 (MUC16), CA15-3, CA19-9, carcinoembryonic antigen (CEA), CA242, CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor), CTLA-4, CXCR5, E16 (LAT1, SLC7A5), FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C), epidermal growth factor, ETBR, Fc receptor-like protein 1 (FCRH1), GEDA, HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen), human chorionic gonadotropin, ICOS, IL-2 receptor, IL20Rα, Immunoglobulin superfamily receptor translocation associated 2 (IRTA2), L6, Lewis Y, Lewis X, MAGE-1, MAGE-2, MAGE-3, MAGE 4, MART1, mesothelin, MCP1 (CCL2), MDP, macrophage inhibitory factor (MIF), MPF (SMR, MSLN), MPG, MSG783, mucin, MUC1-KLH, Napi3b (SLC34A2), nectin-4, Neu oncogene product, NCA, placental alkaline phosphatase, prostate specific membrane antigen (PMSA), prostatic acid phosphatase, PSCA hlg, p97, Purinergic receptor P2X ligand-gated ion channel 5 (P2X5), LY64 (Lymphocyte antigen 64 (RP105), gp100, P21, six transmembrane epithelial antigen of prostate (STEAP1), STEAP2, Sema 5b, tumor-associated glycoprotein 72 (TAG-72), TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4) or a combination thereof.

In some instances, the binding moiety A is an antibody or binding fragment thereof that recognizes a CD cell surface marker. In some instances, the binding moiety A is an antibody or binding fragment thereof that recognizes CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L (L-selectin), CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD79 (e.g., CD79a, CD79b), CD90, CD95 (Fas), CD103, CD104, CD125 (IL5RA), CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD221, CD274, CD279 (PD-1), CD319 (SLAMF7), CD326 (EpCAM), or a combination thereof.

In some embodiments, the antibody or binding fragment thereof comprises zalutumumab (HuMax-EFGr, Genmab), abagovomab (Menarini), abituzumab (Merck), adecatumumab (MT201), alacizumab pegol, alemtuzumab (Campath®, MabCampath, or Campath-1H; Leukosite), AlloMune (BioTransplant), amatuximab (Morphotek, Inc.), anti-VEGF (Genetech), anatumomab mafenatox, apolizumab (hu1D10), ascrinvacumab (Pfizer Inc.), atezolizumab (MPDL3280A; Genentech/Roche), B43.13 (OvaRex, AltaRex Corporation), basiliximab (Simulect®, Novartis), belimumab (Benlysta®, GlaxoSmithKline), bevacizumab (Avastin®, Genentech), blinatumomab (Blincyto, AMG103; Amgen), BEC2 (ImGlone Systems Inc.), carlumab (Janssen Biotech), catumaxomab (Removab, Trion Pharma), CEAcide (Immunomedics), Cetuximab (Erbitux®, ImClone), citatuzumab bogatox (VB6-845), cixutumumab (IMC-A12, ImClone Systems Inc.), conatumumab (AMG 655, Amgen), dacetuzumab (SGN-40, huS2C6; Seattle Genetics, Inc.), daratumumab (Darzalex®, Janssen Biotech), detumomab, drozitumab (Genentech), durvalumab (MedImmune), dusigitumab (MedImmune), edrecolomab (MAb17-1A, Panorex, Glaxo Wellcome), elotuzumab (Empliciti™, Bristol-Myers Squibb), emibetuzumab (Eli Lilly), enavatuzumab (Facet Biotech Corp.), enfortumab vedotin (Seattle Genetics, Inc.), enoblituzumab (MGA271, MacroGenics, Inc.), ensituxumab (Neogenix Oncology, Inc.), epratuzumab (LymphoCide, Immunomedics, Inc.), ertumaxomab (Rexomun®, Trion Pharma), etaracizumab (Abegrin, MedImmune), farletuzumab (MORAb-003, Morphotek, Inc), FBTA05 (Lymphomun, Trion Pharma), ficlatuzumab (AVEO Pharmaceuticals), figitumumab (CP-751871, Pfizer), flanvotumab (ImClone Systems), fresolimumab (GC1008, Aanofi-Aventis), futuximab, glaximab, ganitumab (Amgen), girentuximab (Rencarex®, Wilex AG), IMAB362 (Claudiximab, Ganymed Pharmaceuticals AG), imalumab (Baxalta), IMC-1C11 (ImClone Systems), IMC-C225 (Imclone Systems Inc.), imgatuzumab (Genentech/Roche), intetumumab (Centocor, Inc.), ipilimumab (Yervoy®, Bristol-Myers Squibb), iratumumab (Medarex, Inc.), isatuximab (SAR650984, Sanofi-Aventis), labetuzumab (CEA-CIDE, Immunomedics), lexatumumab (ETR2-ST01, Cambridge Antibody Technology), lintuzumab (SGN-33, Seattle Genetics), lucatumumab (Novartis), lumiliximab, mapatumumab (HGS-ETR1, Human Genome Sciences), matuzumab (EMD 72000, Merck), milatuzumab (hLL1, Immunomedics, Inc.), mitumomab (BEC-2, ImClone Systems), narnatumab (ImClone Systems), necitumumab (Portrazza™, Eli Lilly), nesvacumab (Regeneron Pharmaceuticals), nimotuzumab (h-R3, BIOMAb EGFR, TheraCIM, Theraloc, or CIMAher; Biotech Pharmaceutical Co.), nivolumab (Opdivo®, Bristol-Myers Squibb), obinutuzumab (Gazyva or Gazyvaro; Hoffmann-La Roche), ocaratuzumab (AME-133v, LY2469298; Mentrik Biotech, LLC), ofatumumab (Arzerra®, Genmab), onartuzumab (Genentech), Ontuxizumab (Morphotek, Inc.), oregovomab (OvaRex®, AltaRex Corp.), otlertuzumab (Emergent BioSolutions), panitumumab (ABX-EGF, Amgen), pankomab (Glycotope GMBH), parsatuzumab (Genentech), patritumab, pembrolizumab (Keytruda®, Merck), pemtumomab (Theragyn, Antisoma), pertuzumab (Perjeta, Genentech), pidilizumab (CT-011, Medivation), polatuzumab vedotin (Genentech/Roche), pritumumab, racotumomab (Vaxira®, Recombio), ramucirumab (Cyramza®, ImClone Systems Inc.), rituximab (Rituxan®, Genentech), robatumumab (Schering-Plough), Seribantumab (Sanofi/Merrimack Pharmaceuticals, Inc.), sibrotuzumab, siltuximab (Sylvant™, Janssen Biotech), Smart MI95 (Protein Design Labs, Inc.), Smart ID10 (Protein Design Labs, Inc.), tabalumab (LY2127399, Eli Lilly), taplitumomab paptox, tenatumomab, teprotumumab (Roche), tetulomab, TGN1412 (CD28-SuperMAB or TAB08), tigatuzumab (CD-1008, Daiichi Sankyo), tositumomab, trastuzumab (Herceptin®), tremelimumab (CP-672,206; Pfizer), tucotuzumab celmoleukin (EMD Pharmaceuticals), ublituximab, urelumab (BMS-663513, Bristol-Myers Squibb), volociximab (M200, Biogen Idec), zatuximab, and the like.

In some embodiments, the binding moiety A comprises zalutumumab (HuMax-EFGr, Genmab), abagovomab (Menarini), abituzumab (Merck), adecatumumab (MT201), alacizumab pegol, alemtuzumab (Campath®, MabCampath, or Campath-1H; Leukosite), AlloMune (BioTransplant), amatuximab (Morphotek, Inc.), anti-VEGF (Genetech), anatumomab mafenatox, apolizumab (hu1D10), ascrinvacumab (Pfizer Inc.), atezolizumab (MPDL3280A; Genentech/Roche), B43.13 (OvaRex, AltaRex Corporation), basiliximab (Simulect®, Novartis), belimumab (Benlysta®, GlaxoSmithKline), bevacizumab (Avastin®, Genentech), blinatumomab (Blincyto, AMG103; Amgen), BEC2 (ImGlone Systems Inc.), carlumab (Janssen Biotech), catumaxomab (Removab, Trion Pharma), CEAcide (Immunomedics), Cetuximab (Erbitux®, ImClone), citatuzumab bogatox (VB6-845), cixutumumab (IMC-A12, ImClone Systems Inc.), conatumumab (AMG 655, Amgen), dacetuzumab (SGN-40, huS2C6; Seattle Genetics, Inc.), daratumumab (Darzalex®, Janssen Biotech), detumomab, drozitumab (Genentech), durvalumab (MedImmune), dusigitumab (MedImmune), edrecolomab (MAb17-1A, Panorex, Glaxo Wellcome), elotuzumab (Empliciti™, Bristol-Myers Squibb), emibetuzumab (Eli Lilly), enavatuzumab (Facet Biotech Corp.), enfortumab vedotin (Seattle Genetics, Inc.), enoblituzumab (MGA271, MacroGenics, Inc.), ensituxumab (Neogenix Oncology, Inc.), epratuzumab (LymphoCide, Immunomedics, Inc.), ertumaxomab (Rexomun®, Trion Pharma), etaracizumab (Abegrin, MedImmune), farletuzumab (MORAb-003, Morphotek, Inc), FBTA05 (Lymphomun, Trion Pharma), ficlatuzumab (AVEO Pharmaceuticals), figitumumab (CP-751871, Pfizer), flanvotumab (ImClone Systems), fresolimumab (GC1008, Aanofi-Aventis), futuximab, glaximab, ganitumab (Amgen), girentuximab (Rencarex®, Wilex AG), IMAB362 (Claudiximab, Ganymed Pharmaceuticals AG), imalumab (Baxalta), IMC-1C11 (ImClone Systems), IMC-C225 (Imclone Systems Inc.), imgatuzumab (Genentech/Roche), intetumumab (Centocor, Inc.), ipilimumab (Yervoy®, Bristol-Myers Squibb), iratumumab (Medarex, Inc.), isatuximab (SAR650984, Sanofi-Aventis), labetuzumab (CEA-CIDE, Immunomedics), lexatumumab (ETR2-ST01, Cambridge Antibody Technology), lintuzumab (SGN-33, Seattle Genetics), lucatumumab (Novartis), lumiliximab, mapatumumab (HGS-ETR1, Human Genome Sciences), matuzumab (EMD 72000, Merck), milatuzumab (hLL1, Immunomedics, Inc.), mitumomab (BEC-2, ImClone Systems), narnatumab (ImClone Systems), necitumumab (Portrazza™, Eli Lilly), nesvacumab (Regeneron Pharmaceuticals), nimotuzumab (h-R3, BIOMAb EGFR, TheraCIM, Theraloc, or CIMAher; Biotech Pharmaceutical Co.), nivolumab (Opdivo®, Bristol-Myers Squibb), obinutuzumab (Gazyva or Gazyvaro; Hoffmann-La Roche), ocaratuzumab (AME-133v, LY2469298; Mentrik Biotech, LLC), ofatumumab (Arzerra®, Genmab), onartuzumab (Genentech), Ontuxizumab (Morphotek, Inc.), oregovomab (OvaRex®, AltaRex Corp.), otlertuzumab (Emergent BioSolutions), panitumumab (ABX-EGF, Amgen), pankomab (Glycotope GMBH), parsatuzumab (Genentech), patritumab, pembrolizumab (Keytruda®, Merck), pemtumomab (Theragyn, Antisoma), pertuzumab (Perjeta, Genentech), pidilizumab (CT-011, Medivation), polatuzumab vedotin (Genentech/Roche), pritumumab, racotumomab (Vaxira®, Recombio), ramucirumab (Cyramza®, ImClone Systems Inc.), rituximab (Rituxan®, Genentech), robatumumab (Schering-Plough), Seribantumab (Sanofi/Merrimack Pharmaceuticals, Inc.), sibrotuzumab, siltuximab (Sylvant™, Janssen Biotech), Smart MI95 (Protein Design Labs, Inc.), Smart ID10 (Protein Design Labs, Inc.), tabalumab (LY2127399, Eli Lilly), taplitumomab paptox, tenatumomab, teprotumumab (Roche), tetulomab, TGN1412 (CD28-SuperMAB or TAB08), tigatuzumab (CD-1008, Daiichi Sankyo), tositumomab, trastuzumab (Herceptin®), tremelimumab (CP-672,206; Pfizer), tucotuzumab celmoleukin (EMD Pharmaceuticals), ublituximab, urelumab (BMS-663513, Bristol-Myers Squibb), volociximab (M200, Biogen Idec), or zatuximab. In some embodiments, the binding moiety A is zalutumumab (HuMax-EFGr, by Genmab).

In some embodiments, the binding moiety A is conjugated according to Formula (I) to a polynucleic acid molecule (B), and a polymer (C), and optionally an endosomolytic moiety (D) according to Formula (II) described herein. In some instances, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listed in Tables 2, 3, 5, 6, 7, 9, or 11. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117. In some instances, the polynucleic acid molecule comprises a sequence selected from SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117. In some instances, the polymer C comprises polyalkylen oxide (e.g., polyethylene glycol). In some embodiments, the endosomolytic moiety D comprises INF7 or melittin, or their respective derivatives.

In some embodiments, the binding moiety A is conjugated to a polynucleic acid molecule (B), and a polymer (C), and optionally an endosomolytic moiety (D) as illustrated in FIGS. 1A-IV. In some instances, the binding moiety A is an antibody or binding fragment thereof.

In some embodiments, the binding moiety A is conjugated to a polynucleic acid molecule (B) non-specifically. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) via a lysine residue or a cysteine residue, in a non-site specific manner. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) via a lysine residue in a non-site specific manner. In some cases, the binding moiety A is conjugated to a polynucleic acid molecule (B) via a cysteine residue in a non-site specific manner. In some instances, the binding moiety A is an antibody or binding fragment thereof.

In some embodiments, the binding moiety A is conjugated to a polynucleic acid molecule (B) in a site-specific manner. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) through a lysine residue, a cysteine residue, at the 5′-terminus, at the 3′-terminus, an unnatural amino acid, or an enzyme-modified or enzyme-catalyzed residue, via a site-specific manner. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) through a lysine residue via a site-specific manner. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) through a cysteine residue via a site-specific manner. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) at the 5′-terminus via a site-specific manner. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) at the 3′-terminus via a site-specific manner. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) through an unnatural amino acid via a site-specific manner. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) through an enzyme-modified or enzyme-catalyzed residue via a site-specific manner. In some instances, the binding moiety A is an antibody or binding fragment thereof.

In some embodiments, one or more regions of a binding moiety A (e.g., an antibody or binding fragment thereof) is conjugated to a polynucleic acid molecule (B). In some instances, the one or more regions of a binding moiety A comprise the N-terminus, the C-terminus, in the constant region, at the hinge region, or the Fc region of the binding moiety A. In some instances, the polynucleic acid molecule (B) is conjugated to the N-terminus of the binding moiety A (e.g., the N-terminus of an antibody or binding fragment thereof). In some instances, the polynucleic acid molecule (B) is conjugated to the C-terminus of the binding moiety A (e.g., the N-terminus of an antibody or binding fragment thereof). In some instances, the polynucleic acid molecule (B) is conjugated to the constant region of the binding moiety A (e.g., the constant region of an antibody or binding fragment thereof). In some instances, the polynucleic acid molecule (B) is conjugated to the hinge region of the binding moiety A (e.g., the constant region of an antibody or binding fragment thereof). In some instances, the polynucleic acid molecule (B) is conjugated to the Fc region of the binding moiety A (e.g., the constant region of an antibody or binding fragment thereof).

In some embodiments, one or more polynucleic acid molecule (B) is conjugated to a binding moiety A. In some instances, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 1 polynucleic acid molecule is conjugated to one binding moiety A. In some instances, about 2 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 3 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 4 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 5 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 6 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 7 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 8 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 9 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 10 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 11 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 12 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 13 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 14 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 15 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 16 polynucleic acid molecules are conjugated to one binding moiety A. In some cases, the one or more polynucleic acid molecules are the same. In other cases, the one or more polynucleic acid molecules are different. In some instances, the binding moiety A is an antibody or binding fragment thereof.

In some embodiments, the number of polynucleic acid molecule (B) conjugated to a binding moiety A (e.g., an antibody or binding fragment thereof) forms a ratio. In some instances, the ratio is referred to as a DAR (drug-to-antibody) ratio, in which the drug as referred to herein is the polynucleic acid molecule (B). In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 1 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 2 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 3 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 4 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 5 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 6 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 7 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 8 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 9 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 10 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 11 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 12 or greater.

In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A (e.g., an antibody or binding fragment thereof) is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 1. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 2. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 3. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 4. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 5. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 6. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 7. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 8. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 9. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 10. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 11. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 12. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 13. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 14. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 15. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 16.

In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is 1. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is 2. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is 4. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is 6. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is 8. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is 12.

In some embodiments, an antibody or its binding fragment is further modified using conventional techniques known in the art, for example, by using amino acid deletion, insertion, substitution, addition, and/or by recombination and/or any other modification (e.g. posttranslational and chemical modifications, such as glycosylation and phosphorylation) known in the art either alone or in combination. In some instances, the modification further comprises a modification for modulating interaction with Fc receptors. In some instances, the one or more modifications include those described in, for example, International Publication No. WO97/34631, which discloses amino acid residues involved in the interaction between the Fc domain and the FcRn receptor. Methods for introducing such modifications in the nucleic acid sequence underlying the amino acid sequence of an antibody or its binding fragment is well known to the person skilled in the art.

In some instances, an antibody binding fragment further encompasses its derivatives and includes polypeptide sequences containing at least one CDR.

In some instances, the term “single-chain” as used herein means that the first and second domains of a bi-specific single chain construct are covalently linked, preferably in the form of a co-linear amino acid sequence encodable by a single nucleic acid molecule.

In some instances, a bispecific single chain antibody construct relates to a construct comprising two antibody derived binding domains. In such embodiments, bi-specific single chain antibody construct is tandem bi-scFv or diabody. In some instances, a scFv contains a VH and VL domain connected by a linker peptide. In some instances, linkers are of a length and sequence sufficient to ensure that each of the first and second domains can, independently from one another, retain their differential binding specificities.

In some embodiments, binding to or interacting with as used herein defines a binding/interaction of at least two antigen-interaction-sites with each other. In some instances, antigen-interaction-site defines a motif of a polypeptide that shows the capacity of specific interaction with a specific antigen or a specific group of antigens. In some cases, the binding/interaction is also understood to define a specific recognition. In such cases, specific recognition refers to that the antibody or its binding fragment is capable of specifically interacting with and/or binding to at least two amino acids of each of a target molecule. For example, specific recognition relates to the specificity of the antibody molecule, or to its ability to discriminate between the specific regions of a target molecule. In additional instances, the specific interaction of the antigen-interaction-site with its specific antigen results in an initiation of a signal, e.g. due to the induction of a change of the conformation of the antigen, an oligomerization of the antigen, etc. In further embodiments, the binding is exemplified by the specificity of a “key-lock-principle”. Thus in some instances, specific motifs in the amino acid sequence of the antigen-interaction-site and the antigen bind to each other as a result of their primary, secondary or tertiary structure as well as the result of secondary modifications of said structure. In such cases, the specific interaction of the antigen-interaction-site with its specific antigen results as well in a simple binding of the site to the antigen.

In some instances, specific interaction further refers to a reduced cross-reactivity of the antibody or its binding fragment or a reduced off-target effect. For example, the antibody or its binding fragment that bind to the polypeptide/protein of interest but do not or do not essentially bind to any of the other polypeptides are considered as specific for the polypeptide/protein of interest. Examples for the specific interaction of an antigen-interaction-site with a specific antigen comprise the specificity of a ligand for its receptor, for example, the interaction of an antigenic determinant (epitope) with the antigenic binding site of an antibody.

Additional Binding Moieties

In some embodiments, the binding moiety is a plasma protein. In some instances, the plasma protein comprises albumin. In some instances, the binding moiety A is albumin. In some instances, albumin is conjugated by one or more of a conjugation chemistry described herein to a polynucleic acid molecule. In some instances, albumin is conjugated by native ligation chemistry to a polynucleic acid molecule. In some instances, albumin is conjugated by lysine conjugation to a polynucleic acid molecule.

In some instances, the binding moiety is a steroid. Exemplary steroids include cholesterol, phospholipids, di- and triacylglycerols, fatty acids, hydrocarbons that are saturated, unsaturated, comprise substitutions, or combinations thereof. In some instances, the steroid is cholesterol. In some instances, the binding moiety is cholesterol. In some instances, cholesterol is conjugated by one or more of a conjugation chemistry described herein to a polynucleic acid molecule. In some instances, cholesterol is conjugated by native ligation chemistry to a polynucleic acid molecule. In some instances, cholesterol is conjugated by lysine conjugation to a polynucleic acid molecule.

In some instances, the binding moiety is a polymer, including but not limited to poly nucleic acid molecule aptamers that bind to specific surface markers on cells. In this instance the binding moiety is a polynucleic acid that does not hybridize to a target gene or mRNA, but instead is capable of selectively binding to a cell surface marker similarly to an antibody binding to its specific epitope of a cell surface marker.

In some cases, the binding moiety is a peptide. In some cases, the peptide comprises between about 1 and about 3 kDa. In some cases, the peptide comprises between about 1.2 and about 2.8 kDa, about 1.5 and about 2.5 kDa, or about 1.5 and about 2 kDa. In some instances, the peptide is a bicyclic peptide. In some cases, the bicyclic peptide is a constrained bicyclic peptide. In some instances, the binding moiety is a bicyclic peptide (e.g., bicycles from Bicycle Therapeutics).

In additional cases, the binding moiety is a small molecule. In some instances, the small molecule is an antibody-recruiting small molecule. In some cases, the antibody-recruiting small molecule comprises a target-binding terminus and an antibody-binding terminus, in which the target-binding terminus is capable of recognizing and interacting with a cell surface receptor. For example, in some instances, the target-binding terminus comprising a glutamate urea compound enables interaction with PSMA, thereby, enhances an antibody interaction with a cell (e.g., a cancerous cell) that expresses PSMA. In some instances, a binding moiety is a small molecule described in Zhang et al., “A remote arene-binding site on prostate specific membrane antigen revealed by antibody-recruiting small molecules,” J Am Chem Soc. 132(36): 12711-12716 (2010); or McEnaney, et al., “Antibody-recruiting molecules: an emerging paradigm for engaging immune function in treating human disease,” ACS Chem Biol. 7(7): 1139-1151 (2012).

Production of Antibodies or Binding Fragments Thereof

In some embodiments, polypeptides described herein (e.g., antibodies and its binding fragments) are produced using any method known in the art to be useful for the synthesis of polypeptides (e.g., antibodies), in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.

In some instances, an antibody or its binding fragment thereof is expressed recombinantly, and the nucleic acid encoding the antibody or its binding fragment is assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a nucleic acid molecule encoding an antibody is optionally generated from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.

In some instances, an antibody or its binding is optionally generated by immunizing an animal, such as a rabbit, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, a clone encoding at least the Fab portion of the antibody is optionally obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

In some embodiments, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity are used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g., humanized antibodies.

In some embodiments, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242:1038-1041).

In some embodiments, an expression vector comprising the nucleotide sequence of an antibody or the nucleotide sequence of an antibody is transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In specific embodiments, the expression of the antibody is regulated by a constitutive, an inducible or a tissue, specific promoter.

In some embodiments, a variety of host-expression vector systems is utilized to express an antibody or its binding fragment described herein. Such host-expression systems represent vehicles by which the coding sequences of the antibody is produced and subsequently purified, but also represent cells that are, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody or its binding fragment in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing an antibody or its binding fragment coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing an antibody or its binding fragment coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an antibody or its binding fragment coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an antibody or its binding fragment coding sequences; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5K promoter).

For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some instances, cell lines that stably express an antibody are optionally engineered. Rather than using expression vectors that contain viral origins of replication, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells are then allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn are cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the antibody or its binding fragments.

In some instances, a number of selection systems are used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes are employed in tk−, hgprt− or aprt− cells, respectively. Also, antimetabolite resistance are used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215) and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).

In some instances, the expression levels of an antibody are increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing an antibody is amplifiable, an increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

In some instances, any method known in the art for purification of an antibody is used, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

Polymer Conjugating Moiety

In some embodiments, a polymer moiety C is further conjugated to a polynucleic acid molecule described herein, a binding moiety described herein, or in combinations thereof. In some instances, a polymer moiety C is conjugated a polynucleic acid molecule. In some cases, a polymer moiety C is conjugated to a binding moiety. In other cases, a polymer moiety C is conjugated to a polynucleic acid molecule-binding moiety molecule. In additional cases, a polymer moiety C is conjugated, as illustrated in FIG. 1, and as discussed under the Therapeutic Molecule Platform section.

In some instances, the polymer moiety C is a natural or synthetic polymer, consisting of long chains of branched or unbranched monomers, and/or cross-linked network of monomers in two or three dimensions. In some instances, the polymer moiety C includes a polysaccharide, lignin, rubber, or polyalkylen oxide (e.g., polyethylene glycol). In some instances, the at least one polymer moiety C includes, but is not limited to, alpha-, omega-dihydroxylpolyethyleneglycol, biodegradable lactone-based polymer, e.g. polyacrylic acid, polylactide acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin, polyamide, polycyanoacrylate, polyimide, polyethylenterephthalat (PET, PETG), polyethylene terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethane as well as mixtures thereof. As used herein, a mixture refers to the use of different polymers within the same compound as well as in reference to block copolymers. In some cases, block copolymers are polymers wherein at least one section of a polymer is build up from monomers of another polymer. In some instances, the polymer moiety C comprises polyalkylene oxide. In some instances, the polymer moiety C comprises PEG. In some instances, the polymer moiety C comprises polyethylene imide (PEI) or hydroxy ethyl starch (HES).

In some instances, C is a PEG moiety. In some instances, the PEG moiety is conjugated at the 5′ terminus of the polynucleic acid molecule while the binding moiety is conjugated at the 3′ terminus of the polynucleic acid molecule. In some instances, the PEG moiety is conjugated at the 3′ terminus of the polynucleic acid molecule while the binding moiety is conjugated at the 5′ terminus of the polynucleic acid molecule. In some instances, the PEG moiety is conjugated to an internal site of the polynucleic acid molecule. In some instances, the PEG moiety, the binding moiety, or a combination thereof, are conjugated to an internal site of the polynucleic acid molecule. In some instances, the conjugation is a direct conjugation. In some instances, the conjugation is via native ligation.

In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydispers or monodispers compound. In some instances, polydispers material comprises disperse distribution of different molecular weight of the material, characterized by mean weight (weight average) size and dispersity. In some instances, the monodisperse PEG comprises one size of molecules. In some embodiments, C is poly- or monodispersed polyalkylene oxide (e.g., PEG) and the indicated molecular weight represents an average of the molecular weight of the polyalkylene oxide, e.g., PEG, molecules.

In some embodiments, the molecular weight of the polyalkylene oxide (e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.

In some embodiments, C is polyalkylene oxide (e.g., PEG) and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some embodiments, C is PEG and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some instances, the molecular weight of C is about 200 Da. In some instances, the molecular weight of C is about 300 Da. In some instances, the molecular weight of C is about 400 Da. In some instances, the molecular weight of C is about 500 Da. In some instances, the molecular weight of C is about 600 Da. In some instances, the molecular weight of C is about 700 Da. In some instances, the molecular weight of C is about 800 Da. In some instances, the molecular weight of C is about 900 Da. In some instances, the molecular weight of C is about 1000 Da. In some instances, the molecular weight of C is about 1100 Da. In some instances, the molecular weight of C is about 1200 Da. In some instances, the molecular weight of C is about 1300 Da. In some instances, the molecular weight of C is about 1400 Da. In some instances, the molecular weight of C is about 1450 Da. In some instances, the molecular weight of C is about 1500 Da. In some instances, the molecular weight of C is about 1600 Da. In some instances, the molecular weight of C is about 1700 Da. In some instances, the molecular weight of C is about 1800 Da. In some instances, the molecular weight of C is about 1900 Da. In some instances, the molecular weight of C is about 2000 Da. In some instances, the molecular weight of C is about 2100 Da. In some instances, the molecular weight of C is about 2200 Da. In some instances, the molecular weight of C is about 2300 Da. In some instances, the molecular weight of C is about 2400 Da. In some instances, the molecular weight of C is about 2500 Da. In some instances, the molecular weight of C is about 2600 Da. In some instances, the molecular weight of C is about 2700 Da. In some instances, the molecular weight of C is about 2800 Da. In some instances, the molecular weight of C is about 2900 Da. In some instances, the molecular weight of C is about 3000 Da. In some instances, the molecular weight of C is about 3250 Da. In some instances, the molecular weight of C is about 3350 Da. In some instances, the molecular weight of C is about 3500 Da. In some instances, the molecular weight of C is about 3750 Da. In some instances, the molecular weight of C is about 4000 Da. In some instances, the molecular weight of C is about 4250 Da. In some instances, the molecular weight of C is about 4500 Da. In some instances, the molecular weight of C is about 4600 Da. In some instances, the molecular weight of C is about 4750 Da. In some instances, the molecular weight of C is about 5000 Da. In some instances, the molecular weight of C is about 5500 Da. In some instances, the molecular weight of C is about 6000 Da. In some instances, the molecular weight of C is about 6500 Da. In some instances, the molecular weight of C is about 7000 Da. In some instances, the molecular weight of C is about 7500 Da. In some instances, the molecular weight of C is about 8000 Da. In some instances, the molecular weight of C is about 10,000 Da. In some instances, the molecular weight of C is about 12,000 Da. In some instances, the molecular weight of C is about 20,000 Da. In some instances, the molecular weight of C is about 35,000 Da. In some instances, the molecular weight of C is about 40,000 Da. In some instances, the molecular weight of C is about 50,000 Da. In some instances, the molecular weight of C is about 60,000 Da. In some instances, the molecular weight of C is about 100,000 Da.

In some embodiments, the polyalkylene oxide (e.g., PEG) is a discrete PEG, in which the discrete PEG is a polymeric PEG comprising more than one repeating ethylene oxide units. In some instances, a discrete PEG (dPEG) comprises from 2 to 60, from 2 to 50, or from 2 to 48 repeating ethylene oxide units. In some instances, a dPEG comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 2 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 3 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 4 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 5 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 6 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 7 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 8 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 9 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 10 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 11 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 12 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 13 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 14 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 15 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 16 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 17 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 18 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 19 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 20 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 22 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 24 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 26 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 28 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 30 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 35 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 40 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 42 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 48 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 50 or more repeating ethylene oxide units. In some cases, a dPEG is synthesized as a single molecular weight compound from pure (e.g., about 95%, 98%, 99%, or 99.5%) staring material in a step-wise fashion. In some cases, a dPEG has a specific molecular weight, rather than an average molecular weight. In some cases, a dPEG described herein is a dPEG from Quanta Biodesign, LMD.

In some embodiments, the polymer moiety C comprises a cationic mucic acid-based polymer (cMAP). In some instances, cMPA comprises one or more subunit of at least one repeating subunit, and the subunit structure is represented as Formula (III):

wherein m is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4-6 or 5; and n is independently at each occurrence 1, 2, 3, 4, or 5. In some embodiments, in and n are, for example, about 10.

In some instances, cMAP is further conjugated to a PEG moiety, generating a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some instances, the PEG moiety is in a range of from about 500 Da to about 50,000 Da. In some instances, the PEG moiety is in a range of from about 500 Da to about 1000 Da, greater than 1000 Da to about 5000 Da, greater than 5000 Da to about 10,000 Da, greater than 10,000 to about 25,000 Da, greater than 25,000 Da to about 50,000 Da, or any combination of two or more of these ranges.

In some instances, the polymer moiety C is cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some cases, the polymer moiety C is cMAP-PEG copolymer. In other cases, the polymer moiety C is an mPEG-cMAP-PEGm triblock polymer. In additional cases, the polymer moiety C is a cMAP-PEG-cMAP triblock polymer.

In some embodiments, the polymer moiety C is conjugated to the polynucleic acid molecule, the binding moiety, and optionally to the endosomolytic moiety as illustrated in FIG. 1A-1V.

Endosomolytic Moiety

In some embodiments, a molecule of Formula (I): A-X—B—Y—C, further comprises an additional conjugating moiety. In some instances, the additional conjugating moiety is an endosomolytic moiety. In some cases, the endosomolytic moiety is a cellular compartmental release component, such as a compound capable of releasing from any of the cellular compartments known in the art, such as the endosome, lysosome, endoplasmic reticulum (ER), golgi apparatus, microtubule, peroxisome, or other vesicular bodies with the cell. In some cases, the endosomolytic moiety comprises an endosomolytic polypeptide, an endosomolytic polymer, an endosomolytic lipid, or an endosomolytic small molecule. In some cases, the endosomolytic moiety comprises an endosomolytic polypeptide. In other cases, the endosomolytic moiety comprises an endosomolytic polymer.

Endosomolytic Polypeptides

In some embodiments, a molecule of Formula (I): A-X—B—Y—C, is further conjugated with an endosomolytic polypeptide. In some cases, the endosomolytic polypeptide is a pH-dependent membrane active peptide. In some cases, the endosomolytic polypeptide is an amphipathic polypeptide. In additional cases, the endosomolytic polypeptide is a peptidomimetic. In some instances, the endosomolytic polypeptide comprises INF, melittin, meucin, or their respective derivatives thereof. In some instances, the endosomolytic polypeptide comprises INF or its derivatives thereof. In other cases, the endosomolytic polypeptide comprises melittin or its derivatives thereof. In additional cases, the endosomolytic polypeptide comprises meucin or its derivatives thereof.

In some instances, INF7 is a 24 residue polypeptide those sequence comprises CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO: 2055), or GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 2056). In some instances, INF7 or its derivatives comprise a sequence of: GLFEAIEGFIENGWEGMIWDYGSGSCG (SEQ ID NO: 2057), GLFEAIEGFIENGWEGMIDG WYG-(PEG)6-NH2 (SEQ ID NO: 2058), or GLFEAIEGFIENGWEGMIWDYG-SGSC-K(GalNAc)2 (SEQ ID NO: 2059).

In some cases, melittin is a 26 residue polypeptide those sequence comprises CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 2060), or GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 2061). In some instances, melittin comprises a polypeptide sequence as described in U.S. Pat. No. 8,501,930.

In some instances, meucin is an antimicrobial peptide (AMP) derived from the venom gland of the scorpion Mesobuthus eupeus. In some instances, meucin comprises of meucin-13 those sequence comprises IFGAIAGLLKNIF-NH₂ (SEQ ID NO: 2062) and meucin-18 those sequence comprises FFGHLFKLATKIIPSLFQ (SEQ ID NO: 2063).

In some instances, the endosomolytic polypeptide comprises a polypeptide in which its sequence is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence identity to INF7 or its derivatives thereof, melittin or its derivatives thereof, or meucin or its derivatives thereof. In some instances, the endosomolytic moiety comprises INF7 or its derivatives thereof, melittin or its derivatives thereof, or meucin or its derivatives thereof.

In some instances, the endosomolytic moiety is INF7 or its derivatives thereof. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2055-2059. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2055. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2056-2059. In some cases, the endosomolytic moiety comprises SEQ ID NO: 2055. In some cases, the endosomolytic moiety comprises SEQ ID NO: 2056-2059. In some cases, the endosomolytic moiety consists of SEQ ID NO: 2055. In some cases, the endosomolytic moiety consists of SEQ ID NO: 2056-2059.

In some instances, the endosomolytic moiety is melittin or its derivatives thereof. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2060 or 2061. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2060. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2061. In some cases, the endosomolytic moiety comprises SEQ ID NO: 2060. In some cases, the endosomolytic moiety comprises SEQ ID NO: 2061. In some cases, the endosomolytic moiety consists of SEQ ID NO: 2060. In some cases, the endosomolytic moiety consists of SEQ ID NO: 2061.

In some instances, the endosomolytic moiety is meucin or its derivatives thereof. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2062 or 2063. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2062. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2063. In some cases, the endosomolytic moiety comprises SEQ ID NO: 2062. In some cases, the endosomolytic moiety comprises SEQ ID NO: 2063. In some cases, the endosomolytic moiety consists of SEQ ID NO: 2062. In some cases, the endosomolytic moiety consists of SEQ ID NO: 2063.

In some instances, the endosomolytic moiety comprises a sequence as illustrated in Table 62.

TABLE 62
			SEQ
			ID
Name	Origin	Amino Acid Sequence	NO:	Type
Pep-1	NLS from Simian Virus	KETWWETWWTEWSQPKKKRKV	2064	Primary
	40 large antigen and			amphipathic
	Reverse transcriptase of
	HIV
pVEC	VE-cadherin	LLIILRRRRIRKQAHAHSK	2065	Primary
				amphipathic
VT5	Synthetic peptide	DPKGDPKGVTVTVTVTVTGKGDP	2066	β-sheet
		KPD		amphipathic
C105Y	1-antitrypsin	CSIPPEVKFNKPFVYLI	2067	—
Transportan	Galanin and mastoparan	GWTLNSAGYLLGKINLKALAALA	2068	Primary
		KKIL		amphipathic
TP10	Galanin and mastoparan	AGYLLGKINLKALAALAKKIL	2069	Primary
				amphipathic
MPG	A hydrofobic domain	GALFLGFLGAAGSTMGA	2070	β-sheet
	from the fusion			amphipathic
	sequence of HIV gp41
	and NLS of SV40 T
	antigen
gH625	Glycoprotein gH of	HGLASTLTRWAHYNALIRAF	2071	Secondary
	HSV type I			amphipathic
				α-helical
CADY	PPTG1 peptide	GLWRALWRLLRSLWRLLWRA	2072	Secondary
				amphipathic
				α-helical
GALA	Synthetic peptide	WEAALAEALAEALAEHLAEALAE	2073	Secondary
		ALEALAA		amphipathic
				α-helical
INF	Influenza HA2 fusion	GLFEAIEGFIENGWEGMIDGWYGC	2074	Secondary
	peptide			amphipathic
				α-helical/
				pH-
				dependent
				membrane
				active
				peptide
HA2E5-	Influenza HA2 subunit	GLFGAIAGFIENGWEGMIDGWYG	2075	Secondary
TAT	of influenza virus X31			amphipathic
	strain fusion peptide			α-helical/
				pH-
				dependent
				membrane
				active
				peptide
HA2-	Influenza HA2 subunit	GLFGAIAGFIENGWEGMIDGRQIKI	2076	pH-
penetratin	of influenza virus X31	WFQNRRMKW		dependent
	strain fusion peptide	KK-amide		membrane
				active
				peptide
HA-K4	Influenza HA2 subunit	GLFGAIAGFIENGWEGMIDG-	2077	pH-
	of influenza virus X31	SSKKKK		dependent
	strain fusion peptide			membrane
				active
				peptide
HA2E4	Influenza HA2 subunit	GLFEAIAGFIENGWEGMIDGGGYC	2078	pH-
	of influenza virus X31			dependent
	strain fusion peptide			membrane
				active
				peptide
H5WYG	HA2 analogue	GLFHAIAHFIHGGWH	2079	pH-
		GLIHGWYG		dependent
				membrane
				active
				peptide
GALA-	INF3 fusion peptide	GLFEAIEGFIENGWEGLAEALAEAL	2080	pH-
INF3-		EALAA-		dependent
(PEG)6-NH		(PEG)6-NH2		membrane
				active
				peptide
CM18-	Cecropin-A-Melittin2-12	KWKLFKKIGAVLKVLTTG-	2081	pH-
TAT11	(CM18) fusion peptide	YGRKKRRQRRR		dependent
				membrane
				active
				peptide

In some cases, the endosomolytic moiety comprises a Bak BH3 polypeptide which induces apoptosis through antagonization of suppressor targets such as Bcl-2 and/or Bcl-x_L. In some instances, the endosomolytic moiety comprises a Bak BH3 polypeptide described in Albarran, et al., “Efficient intracellular delivery of a pro-apoptotic peptide with a pH-responsive carrier,” Reactive & Functional Polymers 71: 261-265 (2011).

In some instances, the endosomolytic moiety comprises a polypeptide (e.g., a cell-penetrating polypeptide) as described in PCT Publication Nos. WO2013/166155 or WO2015/069587.

Endosomolytic Polymers

In some embodiments, a molecule of Formula (I): A-X—B—Y—C, is further conjugated with an endosomolytic polymer. As used herein, an endosomolytic polymer comprises a linear, a branched network, a star, a comb, or a ladder type of polymer. In some instances, an endosomolytic polymer is a homopolymer or a copolymer comprising two or more different types of monomers. In some cases, an endosomolytic polymer is a polycation polymer. In other cases, an endosomolytic polymer is a polyanion polymer.

In some instances, a polycation polymer comprises monomer units that are charge positive, charge neutral, or charge negative, with a net charge being positive. In other cases, a polycation polymer comprises a non-polymeric molecule that contains two or more positive charges. Exemplary cationic polymers include, but are not limited to, poly(L-lysine) (PLL), poly(L-arginine) (PLA), polyethyleneimine (PEI), poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), or N,N-Diethylaminoethyl Methacrylate (DEAEMA).

In some cases, a polyanion polymer comprises monomer units that are charge positive, charge neutral, or charge negative, with a net charge being negative. In other cases, a polyanion polymer comprises a non-polymeric molecule that contains two or more negative charges. Exemplary anionic polymers include p(alkylacrylates) (e.g., poly(propyl acrylic acid) (PPAA)) or poly(N-isopropylacrylamide) (NIPAM). Additional examples include PP75, a L-phenylalanine-poly(L-lysine isophthalamide) polymer described in Khormaee, et al., “Edosomolytic anionic polymer for the cytoplasmic delivery of siRNAs in localized in vivo applications,” Advanced Functional Materials 23: 565-574 (2013).

In some embodiments, an endosomolytic polymer described herein is a pH-responsive endosomolytic polymer. A pH-responsive polymer comprises a polymer that increases in size (swell) or collapses depending on the pH of the environment. Polyacrylic acid and chitosan are examples of pH-responsive polymers.

In some instances, an endosomolytic moiety described herein is a membrane-disruptive polymer. In some cases, the membrane-disruptive polymer comprises a cationic polymer, a neutral or hydrophobic polymer, or an anionic polymer. In some instances, the membrane-disruptive polymer is a hydrophilic polymer.

In some instances, an endosomolytic moiety described herein is a pH-responsive membrane-disruptive polymer. Exemplary pH-responsive membrane-disruptive polymers include p(alkylacrylic acids), poly(N-isopropylacrylamide) (NIPAM) copolymers, succinylated p(glycidols), and p(β-malic acid) polymers.

In some instances, p(alkylacrylic acids) include poly(propylacrylic acid) (polyPAA), poly(methacrylic acid) (PMAA), poly(ethylacrylic acid) (PEAA), and poly(propyl acrylic acid) (PPAA). In some instances, a p(alkylacrylic acid) include a p(alkylacrylic acid) described in Jones, et al., Biochemistry Journal 372: 65-75 (2003).

In some embodiments, a pH-responsive membrane-disruptive polymer comprises p(butyl acrylate-co-methacrylic acid). (see Bulmus, et al., Journal of Controlled Release 93: 105-120 (2003); and Yessine, et al., Biochimica et Biophysica Acta 1613: 28-38 (2003))

In some embodiments, a pH-responsive membrane-disruptive polymer comprises p(styrene-alt-maleic anhydride). (see Henry, et al., Biomacromolecules 7: 2407-2414 (2006))

In some embodiments, a pH-responsive membrane-disruptive polymer comprises pyridyldisulfide acrylate (PDSA) polymers such as poly(MAA-co-PDSA), poly(EAA-co-PDSA), poly(PAA-co-PDSA), poly(MAA-co-BA-co-PDSA), poly(EAA-co-BA-co-PDSA), or poly(PAA-co-BA-co-PDSA) polymers. (see El-Sayed, et al., “Rational design of composition and activity correlations for pH-responsive and glutathione-reactive polymer therapeutics,” Journal of Controlled Release 104: 417-427 (2005); or Flanary et al., “Antigen delivery with poly(propylacrylic acid) conjugation enhanced MHC-1 presentation and T-cell activation,” Bioconjugate Chem. 20: 241-248 (2009))

In some embodiments, a pH-responsive membrane-disruptive polymer comprises a lytic polymer comprising the base structure of:

In some instances, an endosomolytic moiety described herein is further conjugated to an additional conjugate, e.g., a polymer (e.g., PEG), or a modified polymer (e.g., cholesterol-modified polymer).

In some instances, the additional conjugate comprises a detergent (e.g., Triton X-100). In some instances, an endosomolytic moiety described herein comprises a polymer (e.g., a poly(amidoamine)) conjugated with a detergent (e.g., Triton X-100). In some instances, an endosomolytic moiety described herein comprises poly(amidoamine)-Triton X-100 conjugate (Duncan, et al., “A polymer-Triton X-100 conjugate capable of pH-dependent red blood cell lysis: a model system illustrating the possibility of drug delivery within acidic intracellular compartments,” Journal of Drug Targeting 2: 341-347 (1994)).

Endosomolytic Lipids

In some embodiments, the endosomolytic moiety is a lipid (e.g., a fusogenic lipid). In some embodiments, a molecule of Formula (I): A-X—B—Y—C, is further conjugated with an endosomolytic lipid (e.g., fusogenic lipid). Exemplary fusogenic lipids include 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k-DMA) and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (XTC).

In some instances, an endosomolytic moiety is a lipid (e.g., a fusogenic lipid) described in PCT Publication No. WO09/126,933.

Endosomolytic Small Molecules

In some embodiments, the endosomolytic moiety is a small molecule. In some embodiments, a molecule of Formula (I): A-X—B—Y—C, is further conjugated with an endosomolytic small molecule. Exemplary small molecules suitable as endosomolytic moieties include, but are not limited to, quinine, chloroquine, hydroxychloroquines, amodiaquine (carnoquines), amopyroquines, primaquines, mefloquines, nivaquines, halofantrines, quinone imines, or a combination thereof. In some instances, quinoline endosomolytic moieties include, but are not limited to, 7-chloro-4-(4-diethylamino-1-methylbutyl-amino)quinoline (chloroquine); 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyl-amino)quinoline (hydroxychloroquine); 7-fluoro-4-(4-diethylamino-1-methylbutyl-amino)quinoline; 4-(4-diethylamino-1-methylbutylamino) quinoline; 7-hydroxy-4-(4-diethyl-amino-1-methylbutylamino)quinoline; 7-chloro-4-(4-diethylamino-1-butylamino)quinoline (desmethylchloroquine); 7-fluoro-4-(4-diethylamino-1-butylamino)quinoline); 4-(4-diethyl-amino-1-butylamino)quinoline; 7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline; 7-fluoro-4-(1-carboxy-4-diethyl-amino-1-butylamino)quinoline; 4-(1-carboxy-4-diethylamino-1-butylamino) quinoline; 7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline; 7-fluoro-4-(1-carboxy-4-diethyl-amino-1-methylbutylamino)quinoline; 4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline; 7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 4-(4-ethyl-(2-hydroxy-ethyl)-amino-1-methylbutylamino-)quinoline; 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; hydroxychloroquine phosphate; 7-chloro-4-(4-ethyl-(2-hydroxyethyl-1)-amino-1-butylamino)quinoline (desmethylhydroxychloroquine); 7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino) quinoline; 7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 8-[(4-aminopentyl)amino-6-methoxydihydrochloride quinoline; 1-acetyl-1,2,3,4-tetrahydroquinoline; 8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride; 1-butyryl-1,2,3,4-tetrahydroquinoline; 3-chloro-4-(4-hydroxy-alpha,alpha′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethyl-amino)-1-methylbutyl-amino]-6-methoxyquinoline; 3-fluoro-4-(4-hydroxy-alpha,alpha′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline; 4-(4-hydroxy-alpha,alpha′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline; 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline; 3,4-dihydro-1-(2H)-quinolinecarboxyaldehyde; 1,1′-pentamethylene diquinoleinium diiodide; 8-quinolinol sulfate and amino, aldehyde, carboxylic, hydroxyl, halogen, keto, sulfhydryl and vinyl derivatives or analogs thereof. In some instances, an endosomolytic moiety is a small molecule described in Naisbitt et al (1997, J Pharmacol Exp Therapy 280:884-893) and in U.S. Pat. No. 5,736,557.

Formula (I) Molecule-Endosomolytic Moiety Conjugates

In some embodiments, one or more endosomolytic moieties are conjugated to a molecule comprising at least one binding moiety, at least one polynucleotide, at least one polymer, or any combinations thereof. In some instances, the endosomolytic moiety is conjugated according to Formula (II):(A-X—B—Y—C_c)-L-D   Formula IIwherein,

A is a binding moiety;

B is a polynucleotide;

C is a polymer;

X is a bond or first linker;

Y is a bond or second linker;

L is a bond or third linker;

D is an endosomolytic moiety; and

c is an integer between 0 and 1; and

wherein the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety; and D is conjugated anywhere on A, B, or C.

In some embodiments, A and C are not attached to B at the same terminus.

In some embodiments, the at least one 2′ modified nucleotide comprises 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified nucleotide. In some instances, the at least one 2′ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA). In some cases, the at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some instances, the second polynucleotide comprises at least one modification. In some cases, the first polynucleotide and the second polynucleotide are RNA molecules. In some cases, the first polynucleotide and the second polynucleotide are siRNA molecules. In some embodiments, X, Y, and L are independently a bond or a non-polymeric linker group. In some instances, A is an antibody or binding fragment thereof. In some instances, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or binding fragment thereof. In some cases, C is polyethylene glycol.

In some instances, the endosomolytic moiety comprises a polypeptide, a polymer, a lipid, or a small molecule. In some instances, the endosomolytic moiety is an endosomolytic polypeptide. In some cases, the endosomolytic moiety is an endosomolytic polymer. In other cases, the endosomolytic moiety is an endosomolytic lipid. In additional cases, the endosomolytic moiety is an endosomolytic small molecule.

In some instances, the endosomolytic moiety is INF7 or its derivatives thereof. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ 1D NOs: 2055. In some cases, the endosomolytic moiety comprises SEQ ID NO: 2055. In some cases, the endosomolytic moiety consists of SEQ ID NO: 2055.

In some instances, the endosomolytic moiety is melittin or its derivatives thereof. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2060. In some cases, the endosomolytic moiety comprises SEQ ID NO: 2060. In some cases, the endosomolytic moiety consists of SEQ ID NO: 2060.

In some instances, the endosomolytic moiety is a sequence as illustrated in Table 62.

In additional cases, the endosomolytic moiety is an endosomolytic polymer, such as for example, a pH-responsive endosomolytic polymer, a membrane-disruptive polymer, a polycation polymer, a polyanion polymer, a pH-responsive membrane-disruptive polymer, or a combination thereof. In additional cases, the endosomolytic moiety comprises a p(alkylacrylic acid) polymer, a p(butyl acrylate-co-methacrylic acid) polymer, a p(styrene-alt-maleic anhydride) polymer, a pyridyldisulfide acrylate (PDSA) polymer, a polymer-PEG conjugate, a polymer-detergent conjugate, or a combination thereof.

In some embodiments, the endosomolytic moiety conjugate is according to Formula (IIa):D-L-A-X—B—Y—C_c   Formula IIawherein,

A is a binding moiety;

B is a polynucleotide;

C is a polymer;

X is a bond or first linker;

Y is a bond or second linker;

L is a bond or third linker;

D is an endosomolytic moiety; and

c is an integer of 1; and

wherein the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety.

In some embodiments, A and C are not attached to B at the same terminus.

In some embodiments, the at least one 2′ modified nucleotide comprises 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (T-O-NMA) modified nucleotide. In some instances, the at least one 2′ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA). In some cases, the at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some instances, the second polynucleotide comprises at least one modification. In some cases, the first polynucleotide and the second polynucleotide are RNA molecules. In some cases, the first polynucleotide and the second polynucleotide are siRNA molecules. In some embodiments, X, Y, and L are independently a bond or a non-polymeric linker group. In some instances, A is an antibody or binding fragment thereof. In some instances, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or binding fragment thereof. In some cases, C is polyethylene glycol.

In some instances, the endosomolytic moiety comprises a polypeptide, a polymer, a lipid, or a small molecule. In some instances, the endosomolytic moiety is an endosomolytic polypeptide. In some cases, the endosomolytic moiety is an endosomolytic polymer. In other cases, the endosomolytic moiety is an endosomolytic lipid. In additional cases, the endosomolytic moiety is an endosomolytic small molecule.

In some instances, the endosomolytic moiety is INF7 or its derivatives thereof. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2055. In some cases, the endosomolytic moiety comprises SEQ ID NO: 2055. In some cases, the endosomolytic moiety consists of SEQ ID NO: 2055.

In some instances, the endosomolytic moiety is melittin or its derivatives thereof. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2060. In some cases, the endosomolytic moiety comprises SEQ ID NO: 2060. In some cases, the endosomolytic moiety consists of SEQ ID NO: 2060.

In some instances, the endosomolytic moiety is a sequence as illustrated in Table 62.

In additional cases, the endosomolytic moiety is an endosomolytic polymer, such as for example, a pH-responsive endosomolytic polymer, a membrane-disruptive polymer, a polycation polymer, a polyanion polymer, a pH-responsive membrane-disruptive polymer, or a combination thereof. In additional cases, the endosomolytic moiety comprises a p(alkylacrylic acid) polymer, a p(butyl acrylate-co-methacrylic acid) polymer, a p(styrene-alt-maleic anhydride) polymer, a pyridyldisulfide acrylate (PDSA) polymer, a polymer-PEG conjugate, a polymer-detergent conjugate, or a combination thereof.

In some instances, the endosomolytic moiety conjugate is according to Formula (IIb):A-X—B-L-D   Formula IIbwherein,

A is a binding moiety;

B is a polynucleotide;

X is a bond or first linker;

L is a bond or third linker; and

D is an endosomolytic moiety; and

wherein the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety.

In some embodiments, A and C are not attached to B at the same terminus.

In some embodiments, the at least one 2′ modified nucleotide comprises 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified nucleotide. In some instances, the at least one 2′ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA). In some cases, the at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some instances, the second polynucleotide comprises at least one modification. In some cases, the first polynucleotide and the second polynucleotide are RNA molecules. In some cases, the first polynucleotide and the second polynucleotide are siRNA molecules. In some embodiments, X and L are independently a bond or a non-polymeric linker group. In some instances, A is an antibody or binding fragment thereof. In some instances, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or binding fragment thereof. In some cases, C is polyethylene glycol.

In some instances, the endosomolytic moiety comprises a polypeptide, a polymer, a lipid, or a small molecule. In some instances, the endosomolytic moiety is an endosomolytic polypeptide. In some cases, the endosomolytic moiety is an endosomolytic polymer. In other cases, the endosomolytic moiety is an endosomolytic lipid. In additional cases, the endosomolytic moiety is an endosomolytic small molecule.

In some instances, the endosomolytic moiety is INF7 or its derivatives thereof. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2055. In some cases, the endosomolytic moiety comprises SEQ ID NO: 2055. In some cases, the endosomolytic moiety consists of SEQ ID NO: 2055.

In some instances, the endosomolytic moiety is melittin or its derivatives thereof. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2060. In some cases, the endosomolytic moiety comprises SEQ ID NO: 2060. In some cases, the endosomolytic moiety consists of SEQ ID NO: 2060.

In some instances, the endosomolytic moiety is a sequence as illustrated in Table 62.

In additional cases, the endosomolytic moiety is an endosomolytic polymer, such as for example, a pH-responsive endosomolytic polymer, a membrane-disruptive polymer, a polycation polymer, a polyanion polymer, a pH-responsive membrane-disruptive polymer, or a combination thereof. In additional cases, the endosomolytic moiety comprises a p(alkylacrylic acid) polymer, a p(butyl acrylate-co-methacrylic acid) polymer, a p(styrene-alt-maleic anhydride) polymer, a pyridyldisulfide acrylate (PDSA) polymer, a polymer-PEG conjugate, a polymer-detergent conjugate, or a combination thereof.

In some instances, the endosomolytic moiety conjugate is according to Formula (IIc):A-X—B—Y—C_c-L-D   Formula IIcwherein,

A is a binding moiety;

B is a polynucleotide;

C is a polymer;

X is a bond or first linker;

Y is a bond or second linker;

L is a bond or third linker;

D is an endosomolytic moiety; and

c is an integer of 1; and

wherein the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety.

In some embodiments, A and C are not attached to B at the same terminus.

In some embodiments, the at least one 2′ modified nucleotide comprises 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified nucleotide. In some instances, the at least one 2′ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA). In some cases, the at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some instances, the second polynucleotide comprises at least one modification. In some cases, the first polynucleotide and the second polynucleotide are RNA molecules. In some cases, the first polynucleotide and the second polynucleotide are siRNA molecules. In some embodiments, X, Y, and L are independently a bond or a non-polymeric linker group. In some instances, A is an antibody or binding fragment thereof. In some instances, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or binding fragment thereof. In some cases, C is polyethylene glycol.

In some instances, the endosomolytic moiety comprises a polypeptide, a polymer, a lipid, or a small molecule. In some instances, the endosomolytic moiety is an endosomolytic polypeptide. In some cases, the endosomolytic moiety is an endosomolytic polymer. In other cases, the endosomolytic moiety is an endosomolytic lipid. In additional cases, the endosomolytic moiety is an endosomolytic small molecule.

In some instances, the endosomolytic moiety is INF7 or its derivatives thereof. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2055. In some cases, the endosomolytic moiety comprises SEQ ID NO: 2055. In some cases, the endosomolytic moiety consists of SEQ ID NO: 2055.

In some instances, the endosomolytic moiety is melittin or its derivatives thereof. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2060. In some cases, the endosomolytic moiety comprises SEQ ID NO: 2060. In some cases, the endosomolytic moiety consists of SEQ ID NO: 2060.

In some instances, the endosomolytic moiety is a sequence as illustrated in Table 62.

In additional cases, the endosomolytic moiety is an endosomolytic polymer, such as for example, a pH-responsive endosomolytic polymer, a membrane-disruptive polymer, a polycation polymer, a polyanion polymer, a pH-responsive membrane-disruptive polymer, or a combination thereof. In additional cases, the endosomolytic moiety comprises a p(alkylacrylic acid) polymer, a p(butyl acrylate-co-methacrylic acid) polymer, a p(styrene-alt-maleic anhydride) polymer, a pyridyldisulfide acrylate (PDSA) polymer, a polymer-PEG conjugate, a polymer-detergent conjugate, or a combination thereof.

In some instances, the endosomolytic moiety conjugate is according to Formula (IId):A-L-D-X—B—Y—C_c   Formula IIdwherein,

A is a binding moiety;

B is a polynucleotide;

C is a polymer;

X is a bond or first linker;

Y is a bond or second linker;

L is a bond or third linker;

D is an endosomolytic moiety; and

c is an integer of 1; and

wherein the polynucleotide comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety.

In some embodiments, A and C are not attached to B at the same terminus.

In some embodiments, the at least one 2′ modified nucleotide comprises 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified nucleotide. In some instances, the at least one 2′ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA). In some cases, the at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some instances, the second polynucleotide comprises at least one modification. In some cases, the first polynucleotide and the second polynucleotide are RNA molecules. In some cases, the first polynucleotide and the second polynucleotide are siRNA molecules. In some embodiments, X, Y, and L are independently a bond or a non-polymeric linker group. In some instances, A is an antibody or binding fragment thereof. In some instances, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or binding fragment thereof. In some cases, C is polyethylene glycol.

In some instances, the endosomolytic moiety comprises a polypeptide, a polymer, a lipid, or a small molecule. In some instances, the endosomolytic moiety is an endosomolytic polypeptide. In some cases, the endosomolytic moiety is an endosomolytic polymer. In other cases, the endosomolytic moiety is an endosomolytic lipid. In additional cases, the endosomolytic moiety is an endosomolytic small molecule.

In some instances, the endosomolytic moiety is INF7 or its derivatives thereof. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2055. In some cases, the endosomolytic moiety comprises SEQ ID NO: 2055. In some cases, the endosomolytic moiety consists of SEQ ID NO: 2055.

In some instances, the endosomolytic moiety is melittin or its derivatives thereof. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2060. In some cases, the endosomolytic moiety comprises SEQ ID NO: 2060. In some cases, the endosomolytic moiety consists of SEQ ID NO: 2060.

In some instances, the endosomolytic moiety is a sequence as illustrated in Table 62.

In additional cases, the endosomolytic moiety is an endosomolytic polymer, such as for example, a pH-responsive endosomolytic polymer, a membrane-disruptive polymer, a polycation polymer, a polyanion polymer, a pH-responsive membrane-disruptive polymer, or a combination thereof. In additional cases, the endosomolytic moiety comprises a p(alkylacrylic acid) polymer, a p(butyl acrylate-co-methacrylic acid) polymer, a p(styrene-alt-maleic anhydride) polymer, a pyridyldisulfide acrylate (PDSA) polymer, a polymer-PEG conjugate, a polymer-detergent conjugate, or a combination thereof.

Linkers

In some embodiments, a linker described herein is a cleavable linker or a non-cleavable linker. In some instances, the linker is a cleavable linker. In some instances, the linker is an acid cleavable linker. In some instances, the linker is a non-cleavable linker. In some instances, the linker includes a C₁-C₆ alkyl group (e.g., a C₅, C₄, C₃, C₂, or C₁ alkyl group). In some instances, the linker includes homobifunctional cross linkers, heterobifunctional cross linkers, and the like. In some instances, the linker is a traceless linker (or a zero-length linker). In some instances, the linker is a non-polymeric linker. In some cases, the linker is a non-peptide linker or a linker that does not contain an amino acid residue.

In some instances, the linker comprises a homobifuctional linker. Exemplary homobifuctional linkers include, but are not limited to, Lomant's reagent dithiobis (succinimidylpropionate) DSP, 3′3′-dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG), N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3′-dithiobispropionimidate (DTBP), 1,4-di-3′-(2′-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), such as e.g. 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4′-difluoro-3,3′-dinitrophenylsulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3′-dimethylbenzidine, benzidine, α,α′-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N′-ethylene-bis(iodoacetamide), or N,N′-hexamethylene-bis(iodoacetamide).

In some embodiments, the linker comprises a heterobifunctional linker. Exemplary heterobifunctional linker include, but are not limited to, amine-reactive and sulfhydryl cross-linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chain N-succinimidyl 3-(2-pyridyldithio) propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs), N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB), sulfosuccinimidyl(4-iodoacteyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMBs), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-((((4-iodoacetyl)amino)methy 1)cyclohexane-1-carbonyl)amino) hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive cross-linkers such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M₂C₂H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive and photoreactive cross-linkers such as N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(ρ-azidosalicylamido)ethyl-1,3′-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3′-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3′-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate (sulfo-sAMCA), ρ-nitrophenyl diazopyruvate (pNPDP), ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive cross-linkers such as 1-(ρ-Azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimide carbonyl-reactive and photoreactive cross-linkers such as p-azidobenzoyl hydrazide (ABH), carboxylate-reactive and photoreactive cross-linkers such as 4-(ρ-azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive cross-linkers such as p-azidophenyl glyoxal (APG).

In some instances, the linker comprises a reactive functional group. In some cases, the reactive functional group comprises a nucleophilic group that is reactive to an electrophilic group present on a binding moiety. Exemplary electrophilic groups include carbonyl groups—such as aldehyde, ketone, carboxylic acid, ester, amide, enone, acyl halide or acid anhydride. In some embodiments, the reactive functional group is aldehyde. Exemplary nucleophilic groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

In some embodiments, the linker comprises a maleimide group. In some instances, the maleimide group is also referred to as a maleimide spacer. In some instances, the maleimide group further encompasses a caproic acid, forming maleimidocaproyl (mc). In some cases, the linker comprises maleimidocaproyl (mc). In some cases, the linker is maleimidocaproyl (mc). In other instances, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC) described above.

In some embodiments, the maleimide group is a self-stabilizing maleimide. In some instances, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis of tiosuccinimide ring hydrolysis, thereby eliminating maleimide from undergoing an elimination reaction through a retro-Michael reaction. In some instances, the self-stabilizing maleimide is a maleimide group described in Lyon, et al., “Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates,” Nat. Biotechnol. 32(10):1059-1062 (2014). In some instances, the linker comprises a self-stabilizing maleimide. In some instances, the linker is a self-stabilizing maleimide.

In some embodiments, the linker comprises a peptide moiety. In some instances, the peptide moiety comprises at least 2, 3, 4, 5, 6, 7, 8, or more amino acid residues. In some instances, the peptide moiety is a cleavable peptide moiety (e.g., either enzymatically or chemically). In some instances, the peptide moiety is a non-cleavable peptide moiety. In some instances, the peptide moiety comprises Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 2111), Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 2112), or Gly-Phe-Leu-Gly (SEQ ID NO: 2113). In some instances, the linker comprises a peptide moiety such as: Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 2111), Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 2112), or Gly-Phe-Leu-Gly (SEQ ID NO: 2113). In some cases, the linker comprises Val-Cit. In some cases, the linker is Val-Cit.

In some embodiments, the linker comprises a benzoic acid group, or its derivatives thereof. In some instances, the benzoic acid group or its derivatives thereof comprise paraaminobenzoic acid (PABA). In some instances, the benzoic acid group or its derivatives thereof comprise gamma-aminobutyric acid (GABA).

In some embodiments, the linker comprises one or more of a maleimide group, a peptide moiety, and/or a benzoic acid group, in any combination. In some embodiments, the linker comprises a combination of a maleimide group, a peptide moiety, and/or a benzoic acid group. In some instances, the maleimide group is maleimidocaproyl (mc). In some instances, the peptide group is val-cit. In some instances, the benzoic acid group is PABA. In some instances, the linker comprises a mc-val-cit group. In some cases, the linker comprises a val-cit-PABA group. In additional cases, the linker comprises a mc-val-cit-PABA group.

In some embodiments, the linker is a self-immolative linker or a self-elimination linker. In some cases, the linker is a self-immolative linker. In other cases, the linker is a self-elimination linker (e.g., a cyclization self-elimination linker). In some instances, the linker comprises a linker described in U.S. Pat. No. 9,089,614 or PCT Publication No. WO2015038426.

In some embodiments, the linker is a dendritic type linker. In some instances, the dendritic type linker comprises a branching, multifunctional linker moiety. In some instances, the dendritic type linker is used to increase the molar ratio of polynucleotide B to the binding moiety A. In some instances, the dendritic type linker comprises PAMAM dendrimers.

In some embodiments, the linker is a traceless linker or a linker in which after cleavage does not leave behind a linker moiety (e.g., an atom or a linker group) to a binding moiety A, a polynucleotide B, a polymer C, or an endosomolytic moiety D. Exemplary traceless linkers include, but are not limited to, germanium linkers, silicium linkers, sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers, boron linkers, chromium linkers, or phenylhydrazide linker. In some cases, the linker is a traceless aryl-triazene linker as described in Hejesen, et al., “A traceless aryl-triazene linker for DNA-directed chemistry,” Org Biomol Chem 11(15): 2493-2497 (2013). In some instances, the linker is a traceless linker described in Blaney, et al., “Traceless solid-phase organic synthesis,” Chem. Rev. 102: 2607-2024 (2002). In some instances, a linker is a traceless linker as described in U.S. Pat. No. 6,821,783.

In some instances, the linker comprises a functional group that exerts steric hinderance at the site of bonding between the linker and a conjugating moiety (e.g., A, B, C, or D described herein). In some instances, the steric hinderance is a steric hindrance around a disulfide bond. Exemplary linkers that exhibit steric hinderance comprises a heterobifuctional linker, such as a heterobifuctional linker described above. In some cases, a linker that exhibits steric hinderance comprises SMCC and SPDB.

In some instances, the linker is an acid cleavable linker. In some instances, the acid cleavable linker comprises a hydrazone linkage, which is susceptible to hydrolytic cleavage. In some cases, the acid cleavable linker comprises a thiomaleamic acid linker. In some cases, the acid cleavable linker is a thiomaleamic acid linker as described in Castaneda, et al, “Acid-cleavable thiomaleamic acid linker for homogeneous antibody-drug conjugation,” Chem. Commun. 49: 8187-8189 (2013).

In some instances, the linker is a linker described in U.S. Pat. Nos. 6,884,869; 7,498,298; 8,288,352; 8,609,105; or 8,697,688; U.S. Patent Publication Nos. 2014/0127239; 2013/028919; 2014/286970; 2013/0309256; 2015/037360; or 2014/0294851; or PCT Publication Nos. WO2015057699; WO2014080251; WO2014197854; WO2014145090; or WO2014177042.

In some embodiments, X, Y, and L are independently a bond or a linker. In some instances, X, Y, and L are independently a bond. In some cases, X, Y, and L are independently a linker.

In some instances, X is a bond or a linker. In some instances, X is a bond. In some instances, X is a linker. In some instances, the linker is a C₁-C₆ alkyl group. In some cases, X is a C₁-C₆ alkyl group, such as for example, a C₅, C₄, C₃, C₂, or C₁ alkyl group. In some cases, the C₁-C₆ alkyl group is an unsubstituted C₁-C₆ alkyl group. As used in the context of a linker, and in particular in the context of X, alkyl means a saturated straight or branched hydrocarbon radical containing up to six carbon atoms. In some instances, X is a non-polymeric linker. In some instances, X includes a homobifuctional linker or a heterobifuctional linker described supra. In some cases, X includes a heterobifunctional linker. In some cases, X includes sMCC. In other instances, X includes a heterobifuctional linker optionally conjugated to a C₁-C₆ alkyl group. In other instances, X includes sMCC optionally conjugated to a C₁-C₆ alkyl group. In additional instances, X does not include a homobifuctional linker or a heterobifunctional linker described supra.

In some instances, Y is a bond or a linker. In some instances, Y is a bond. In other cases, Y is a linker. In some embodiments, Y is a C₁-C₆ alkyl group. In some instances, Y is a homobifuctional linker or a heterobifunctional linker described supra. In some instances, Y is a homobifuctional linker described supra. In some instances, Y is a heterobifunctional linker described supra. In some instances, Y comprises a maleimide group, such as maleimidocaproyl (mc) or a self-stabilizing maleimide group described above. In some instances, Y comprises a peptide moiety, such as Val-Cit. In some instances, Y comprises a benzoic acid group, such as PABA. In additional instances, Y comprises a combination of a maleimide group, a peptide moiety, and/or a benzoic acid group. In additional instances, Y comprises a mc group. In additional instances, Y comprises a mc-val-cit group. In additional instances, Y comprises a val-cit-PABA group. In additional instances, Y comprises a mc-val-cit-PABA group.

In some instances, L is a bond or a linker. In some cases, L is a bond. In other cases, L is a linker. In some embodiments, L is a C₁-C₆ alkyl group. In some instances, L is a homobifuctional linker or a heterobifunctional linker described supra. In some instances, L is a homobifuctional linker described supra. In some instances, L is a heterobifunctional linker described supra. In some instances, L comprises a maleimide group, such as maleimidocaproyl (mc) or a self-stabilizing maleimide group described above. In some instances, L comprises a peptide moiety, such as Val-Cit. In some instances, L comprises a benzoic acid group, such as PABA. In additional instances, L comprises a combination of a maleimide group, a peptide moiety, and/or a benzoic acid group. In additional instances, L comprises a mc group. In additional instances, L comprises a mc-val-cit group. In additional instances, L comprises a val-cit-PABA group. In additional instances, L comprises a mc-val-cit-PABA group.

Methods of Use

In some embodiments, a composition or a pharmaceutical formulation described herein comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer is used for the treatment of a disease or disorder. In some instances, the disease or disorder is a cancer. In some embodiments, a composition or a pharmaceutical formulation described herein is used as an immunotherapy for the treatment of a disease or disorder. In some instances, the immunotherapy is an immuno-oncology therapy.

Cancer

In some embodiments, a composition or a pharmaceutical formulation described herein is used for the treatment of cancer. In some instances, the cancer is a solid tumor. In some instances, the cancer is a hematologic malignancy. In some instances, the cancer is a relapsed or refractory cancer, or a metastatic cancer. In some instances, the solid tumor is a relapsed or refractory solid tumor, or a metastatic solid tumor. In some cases, the hematologic malignancy is a relapsed or refractory hematologic malignancy, or a metastatic hematologic malignancy.

In some embodiments, the cancer is a solid tumor. Exemplary solid tumor includes, but is not limited to, anal cancer, appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, cancer of Unknown Primary (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastroenterological cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.

In some instances, a composition or a pharmaceutical formulation described herein comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer is used for the treatment of a solid tumor. In some instances, a composition or a pharmaceutical formulation described herein comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer is used for the treatment of anal cancer, appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, cancer of Unknown Primary (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastroenterological cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer. In some instances, the solid tumor is a relapsed or refractory solid tumor, or a metastatic solid tumor.

In some instances, the cancer is a hematologic malignancy. In some instances, the hematologic malignancy is a leukemia, a lymphoma, a myeloma, a non-Hodgkin's lymphoma, or a Hodgkin's lymphoma. In some instances, the hematologic malignancy comprises chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high risk CLL, a non-CLL/SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

In some instances, a composition or a pharmaceutical formulation described herein comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer is used for the treatment of a hematologic malignancy. In some instances, a composition or a pharmaceutical formulation described herein comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer is used for the treatment of a leukemia, a lymphoma, a myeloma, a non-Hodgkin's lymphoma, or a Hodgkin's lymphoma. In some instances, the hematologic malignancy comprises chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high risk CLL, a non-CLL/SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. In some cases, the hematologic malignancy is a relapsed or refractory hematologic malignancy, or a metastatic hematologic malignancy.

In some instances, the cancer is a KRAS-associated, EGFR-associated, AR-associated cancer, HPRT1-associated cancer, or β-catenin associated cancer. In some instances, a composition or a pharmaceutical formulation described herein comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer is used for the treatment of a KRAS-associated, EGFR-associated, AR-associated cancer, HPRT1-associated cancer, or β-catenin associated cancer. In some instances, a composition or a pharmaceutical formulation described herein comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer is used for the treatment of a KRAS-associated cancer. In some instances, a composition or a pharmaceutical formulation described herein comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer is used for the treatment of an EGFR-associated cancer. In some instances, a composition or a pharmaceutical formulation described herein comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer is used for the treatment of an AR-associated cancer. In some instances, a composition or a pharmaceutical formulation described herein comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer is used for the treatment of an HPRT1-associated cancer. In some instances, a composition or a pharmaceutical formulation described herein comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer is used for the treatment of a β-catenin associated cancer. In some instances, the cancer is a solid tumor. In some instances, the cancer is a hematologic malignancy. In some instances, the solid tumor is a relapsed or refractory solid tumor, or a metastatic solid tumor. In some cases, the hematologic malignancy is a relapsed or refractory hematologic malignancy, or a metastatic hematologic malignancy. In some instances, the cancer comprises bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, glioblastoma multiforme, head and neck cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, acute myeloid leukemia, CLL, DLBCL, or multiple myeloma. In some instances, the β-catenin associated cancer further comprises PIK3C-associated cancer and/or MYC-associated cancer.

Immunotherapy

In some embodiments, a composition or a pharmaceutical formulation described herein is used as an immunotherapy for the treatment of a disease or disorder. In some instances, the immunotherapy is an immuno-oncology therapy. In some instances, immuno-oncology therapy is categorized into active, passive, or combinatory (active and passive) methods. In active immuno-oncology therapy method, for example, tumor-associated antigens (TAAs) are presented to the immune system to trigger an attack on cancer cells presenting these TAAs. In some instances, the active immune-oncology therapy method includes tumor-targeting and/or immune-targeting agents (e.g., checkpoint inhibitor agents such as monoclonal antibodies), and/or vaccines, such as in situ vaccination and/or cell-based or non-cell based (e.g., dendritic cell-based, tumor cell-based, antigen, anti-idiotype, DNA, or vector-based) vaccines. In some instances, the cell-based vaccines are vaccines which are generated using activated immune cells obtained from a patient's own immune system which are then activated by the patient's own cancer. In some instances, the active immune-oncology therapy is further subdivided into non-specific active immunotherapy and specific active immunotherapy. In some instances, non-specific active immunotherapy utilizes cytokines and/or other cell signaling components to induce a general immune system response. In some cases, specific active immunotherapy utilizes specific TAAs to elicite an immune response.

In some embodiments, a composition or a pharmaceutical formulation described herein is used as an active immuno-oncology therapy method for the treatment of a disease or disorder (e.g., cancer). In some embodiments, the composition or a pharmaceutical formulation described herein comprises a tumor-targeting agent. In some instances, the tumor-targeting agent is encompassed by a binding moiety A. In other instances, the tumor-targeting agent is an additional agent used in combination with a molecule of Formula (I). In some instances, the tumor-targeting agent is a tumor-directed polypeptide (e.g., a tumor-directed antibody). In some instances, the tumor-targeting agent is a tumor-directed antibody, which exerts its antitumor activity through mechanisms such as direct killing (e.g., signaling-induced apoptosis), complement-dependent cytotoxicity (CDC), and/or antibody-dependent cell-mediated cytotoxicity (ADCC). In additional instances, the tumor-targeting agent elicits an adaptive immune response, with the induction of antitumor T cells.

In some embodiments, the binding moiety A is a tumor-directed polypeptide (e.g., a tumor-directed antibody). In some instances, the binding moiety A is a tumor-directed antibody, which exerts its antitumor activity through mechanisms such as direct killing (e.g., signaling-induced apoptosis), complement-dependent cytotoxicity (CDC), and/or antibody-dependent cell-mediated cytotoxicity (ADCC). In additional instances, the binding moiety A elicits an adaptive immune response, with the induction of antitumor T cells.

In some embodiments, the composition or a pharmaceutical formulation described herein comprises an immune-targeting agent. In some instances, the immune-targeting agent is encompassed by a binding moiety A. In other instances, the immune-targeting agent is an additional agent used in combination with a molecule of Formula (I). In some instances, the immune-targeting agent comprises cytokines, checkpoint inhibitors, or a combination thereof.

In some embodiments, the immune-targeting agent is a checkpoint inhibitor. In some cases, an immune checkpoint molecule is a molecule presented on the cell surface of CD4 and/or CD8 T cells. Exemplary immune checkpoint molecules include, but are not limited to, Programmed Death-Ligand 1 (PD-L1, also known as B7-H1, CD274), Programmed Death 1 (PD-1), CTLA-4, B7H1, B7H4, OX-40, CD137, CD40, 2B4, IDO1, IDO2, VISTA, CD27, CD28, PD-L2 (B7-DC, CD273), LAG3, CD80, CD86, PDL2, B7H3, HVEM, BTLA, KIR, GAL9, TIM3, A2aR, MARCO (macrophage receptor with collageneous structure), PS (phosphatidylserine), ICOS (inducible T cell costimulator), HAVCR2, CD276, VTCN1, CD70, and CD160.

In some instances, an immune checkpoint inhibitor refers to any molecule that modulates or inhibits the activity of an immune checkpoint molecule. In some instances, immune checkpoint inhibitors include antibodies, antibody-derivatives (e.g., Fab fragments, scFvs, minobodies, diabodies), antisense oligonucleotides, siRNA, aptamers, or peptides. In some embodiments, an immune checkpoint inhibitor is an inhibitor of Programmed Death-Ligand 1 (PD-L1, also known as B7-H1, CD274), Programmed Death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulator), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collageneous structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combinations thereof.

In some embodiments, exemplary checkpoint inhibitors include:

PD-L1 inhibitors such as Genentech's MPDL3280A (RG7446), Anti-mouse PD-L1 antibody Clone 10F.9G2 (Cat # BE0101) from BioXcell, anti-PD-L1 monoclonal antibody MDX-1105 (BMS-936559) and BMS-935559 from Bristol-Meyer's Squibb, MSB0010718C, mouse anti-PD-L1 Clone 29E.2A3, and AstraZeneca's MEDI4736;

PD-L2 inhibitors such as GlaxoSmithKline's AMP-224 (Amplimmune), and rHIgM1287;

PD-1 inhibitors such as anti-mouse PD-1 antibody Clone J43 (Cat # BE0033-2) from BioXcell, anti-mouse PD-1 antibody Clone RMP1-14 (Cat # BE0146) from BioXcell, mouse anti-PD-1 antibody Clone EH12, Merck's MK-3475 anti-mouse PD-1 antibody (Keytruda, pembrolizumab, lambrolizumab), AnaptysBio's anti-PD-1 antibody known as ANB011, antibody MDX-1 106 (ONO-4538), Bristol-Myers Squibb's human IgG4 monoclonal antibody nivolumab (Opdivo®, BMS-936558, MDX1106), AstraZeneca's AMP-514 and AMP-224, and Pidilizumab (CT-011) from CureTech Ltd;

CTLA-4 inhibitors such as Bristol Meyers Squibb's anti-CTLA-4 antibody ipilimumab (also known as Yervoy®, MDX-010, BMS-734016 and MDX-101), anti-CTLA4 Antibody, clone 9H10 from Millipore, Pfizer's tremelimumab (CP-675,206, ticilimumab), and anti-CTLA4 antibody clone BNI3 from Abcam;

LAG3 inhibitors such as anti-Lag-3 antibody clone eBioC9B7W (C9B7W) from eBioscience, anti-Lag3 antibody LS-B2237 from LifeSpan Biosciences, IMP321 (ImmuFact) from Immutep, anti-Lag3 antibody BMS-986016, and the LAG-3 chimeric antibody A9H12;

B7-H3 inhibitors such as MGA271;

KIR inhibitors such as Lirilumab (IPH2101);

CD137 (41BB) inhibitors such as urelumab (BMS-663513, Bristol-Myers Squibb), PF-05082566 (anti-4-1BB, PF-2566, Pfizer), or XmAb-5592 (Xencor);

PS inhibitors such as Bavituximab;

and inhibitors such as an antibody or fragments (e.g., a monoclonal antibody, a human, humanized, or chimeric antibody) thereof, RNAi molecules, or small molecules to TIM3, CD52, CD30, CD20, CD33, CD27, OX40 (CD134), GITR, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM.

In some embodiments, a binding moiety A comprising an immune checkpoint inhibitor is used for the treatment of a disease or disorder (e.g., cancer). In some instances, the binding moiety A is a bispecific antibody or a binding fragment thereof that comprises an immune checkpoint inhibitor. In some cases, a binding moiety A comprising an inhibitor of Programmed Death-Ligand 1 (PD-L1, also known as B7-H1, CD274), Programmed Death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulator), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collageneous structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combinations thereof, is used for the treatment of a disease or disorder (e.g., cancer).

In some embodiments, a molecule of Formula (I) in combination with an immune checkpoint inhibitor is used for the treatment of a disease or disorder (e.g., cancer). In some instances, the immune checkpoint inhibitor comprises an inhibitor of Programmed Death-Ligand 1 (PD-L1, also known as B7-H1, CD274), Programmed Death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulator), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collageneous structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combinations thereof. In some cases, a molecule of Formula (I) is used in combination with ipilimumab, tremelimumab, nivolumab, pemrolizumab, pidilizumab, MPDL3280A, MEDI4736, MSB0010718C, MK-3475, or BMS-936559, for the treatment of a disease or disorder (e.g., cancer).

In some embodiments, the immune-targeting agent is a cytokine. In some cases, cytokine is further subgrouped into chemokine, interferon, interleukin, and tumor necrosis factor. In some embodiments, chemokine plays a role as a chemoattractant to guide the migration of cells, and is classified into four subfamilies: CXC, CC, CX3C, and XC. Exemplary chemokines include chemokines from the CC subfamily: CCL1, CCL2 (MCP-1), CCL3, CCL4, CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (or CCL10), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, and CCL28; the CXC subfamily: CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, and CXCL17; the XC subfamily: XCL1 and XCL2; and the CX3C subfamily CX3CL1.

Interferon (IFNs) comprises interferon type I (e.g. IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω), interferon type II (e.g. IFN-γ), and interferon type III. In some embodiments, IFN-α is further classified into about 13 subtypes which include IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21.

Interleukin is expressed by leukocyte or white blood cell and promote the development and differentiation of T and B lymphocytes and hematopoietic cells. Exemplary interleukins include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, and IL-36.

Tumor necrosis factors (TNFs) are a group of cytokines that modulate apoptosis. In some instances, there are about 19 members within the TNF family, including, not limited to, TNFα, lymphotoxin-alpha (LT-alpha), lymphotoxin-beta (LT-beta), T cell antigen gp39 (CD40L), CD27L, CD30L, FASL, 4-1BBL, OX40L, and TNF-related apoptosis inducing ligand (TRAIL).

In some embodiments, a molecule of Formula (I) in combination with a cytokine is used for the treatment of a disease or disorder (e.g., cancer). In some cases, a molecule of Formula (I) in combination with a chemokine is used for the treatment of a disease or disorder (e.g., cancer). In some cases, a molecule of Formula (I) in combination with an interferon is used for the treatment of a disease or disorder (e.g., cancer). In some cases, a molecule of Formula (I) in combination with an interleukin is used for the treatment of a disease or disorder (e.g., cancer). In some cases, a molecule of Formula (I) in combination with a tumor necrosis factor is used for the treatment of a disease or disorder (e.g., cancer). In some instances, a molecule of Formula (I) in combination with IL-1β, IL-2, IL-7, IL-8, IL-15, MCP-1 (CCL2), MIP-1α, RANTES, MCP-3, MIP5, CCL19, CCL21, CXCL2, CXCL9, CXCL10, or CXCL11 is used for the treatment of a disease or disorder (e.g., cancer).

In some embodiments, the composition or a pharmaceutical formulation described herein comprises a vaccine. In some instances, the vaccine is an in situ vaccination. In some instances, the vaccine is a cell-based vaccine. In some instances, the vaccine is a non-cell based vaccine. In some instances, a molecule of Formula (I) in combination with dendritic cell-based vaccine is used for the treatment of a disease or disorder (e.g., cancer). In some instances, a molecule of Formula (I) in combination with tumor cell-based vaccine is used for the treatment of a disease or disorder (e.g., cancer). In some instances, a molecule of Formula (I) in combination with antigen vaccine is used for the treatment of a disease or disorder (e.g., cancer). In some instances, a molecule of Formula (I) in combination with anti-idiotype vaccine is used for the treatment of a disease or disorder (e.g., cancer). In some instances, a molecule of Formula (I) in combination with DNA vaccine is used for the treatment of a disease or disorder (e.g., cancer). In some instances, a molecule of Formula (I) in combination with vector-based vaccine is used for the treatment of a disease or disorder (e.g., cancer).

In some embodiments, a composition or a pharmaceutical formulation described herein is used as a passive immuno-oncology therapy method for the treatment of a disease or disorder (e.g., cancer). The passive method, in some instances, utilizes adoptive immune system components such as T cells, natural killer (NK) T cells, and/or chimeric antigen receptor (CAR) T cells generated exogenously to attack cancer cells.

In some embodiments, a molecule of Formula (I) in combination with a T-cell based therapeutic agent is used for the treatment of a disease or disorder (e.g., cancer). In some cases, the T-cell based therapeutic agent is an activated T-cell agent that recognizes one or more of a CD cell surface marker described above. In some instances, the T-cell based therapeutic agent comprises an activated T-cell agent that recognizes one or more of CD2, CD3, CD4, CD5, CD8, CD27, CD28, CD80, CD134, CD137, CD152, CD154, CD160, CD200R, CD223, CD226, CD244, CD258, CD267, CD272, CD274, CD278, CD279, or CD357. In some instances, a molecule of Formula (I) in combination with an activated T-cell agent recognizing one or more of CD2, CD3, CD4, CD5, CD8, CD27, CD28, CD80, CD134, CD137, CD152, CD154, CD160, CD200R, CD223, CD226, CD244, CD258, CD267, CD272, CD274, CD278, CD279, or CD357 is used for the treatment of a disease or disorder (e.g., cancer).

In some embodiments, a molecule of Formula (I) in combination with natural killer (NK) T cell-based therapeutic agent is used for the treatment of a disease or disorder (e.g., cancer). In some instances, the NK-based therapeutic agent is an activated NK agent that recognizes one or more of a CD cell surface marker described above. In some cases, the NK-based therapeutic agent is an activated NK agent that recognizes one or more of CD2, CD11a, CD11b, CD16, CD56, CD58, CD62L, CD85j, CD158a/b, CD158c, CD158e/f/k, CD158h/j, CD159a, CD162, CD226, CD314, CD335, CD337, CD244, or CD319. In some instances, a molecule of Formula (I) in combination with an activated NK agent recognizing one or more of CD2, CD11a, CD11b, CD16, CD56, CD58, CD62L, CD85j, CD158a/b, CD158c, CD158e/f/k, CD158h/j, CD159a, CD162, CD226, CD314, CD335, CD337, CD244, or CD319 is used for the treatment of a disease or disorder (e.g., cancer).

In some embodiments, a molecule of Formula (I) in combination with CAR-T cell-based therapeutic agent is used for the treatment of a disease or disorder (e.g., cancer).

In some embodiments, a molecule of Formula (I) in combination with an additional agent that destabilizes the endosomal membrane (or disrupts the endosomal-lysosomal membrane trafficking) is used for the treatment of a disease or disorder (e.g., cancer). In some instances, the additional agent comprises an antimitotic agent. Exemplary antimitotic agents include, but are not limited to, taxanes such as paclitaxel and docetaxel; vinca alkaloids such as vinblastine, vincristine, vindesine, and vinorelbine; cabazitaxel; colchicine; eribulin; estramustine; etoposide; ixabepilone; podophyllotoxin; teniposide; or griseofulvin. In some instances, the additional agent comprises paclitaxel, docetaxel, vinblastine, vincristine, vindesine, vinorelbine, cabazitaxel, colchicine, eribulin, estramustine, etoposide, ixabepilone, podophyllotoxin, teniposide, or griseofulvin. In some instances, the additional agent comprises taxol. In some instances, the additional agent comprises paclitaxel. In some instances, the additional agent comprises etoposide. In other instances, the additional agent comprises vitamin K3.

In some embodiments, a composition or a pharmaceutical formulation described herein is used as a combinatory method (including for both active and passive methods) in the treatment of a disease or disorder (e.g., cancer).

Pharmaceutical Formulation

In some embodiments, the pharmaceutical formulations described herein are administered to a subject by multiple administration routes, including but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal administration routes. In some instances, the pharmaceutical composition describe herein is formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular) administration. In other instances, the pharmaceutical composition describe herein is formulated for oral administration. In still other instances, the pharmaceutical composition describe herein is formulated for intranasal administration.

In some embodiments, the pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate-release formulations, controlled-release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate and controlled release formulations.

In some instances, the pharmaceutical formulation includes multiparticulate formulations. In some instances, the pharmaceutical formulation includes nanoparticle formulations. In some instances, nanoparticles comprise cMAP, cyclodextrin, or lipids. In some cases, nanoparticles comprise solid lipid nanoparticles, polymeric nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micellar solutions. Additional exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as with covalently attached metal chelates), nanofibers, nanohorns, nano-onions, nanorods, nanoropes and quantum dots. In some instances, a nanoparticle is a metal nanoparticle, e.g., a nanoparticle of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium, lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium, potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, and combinations, alloys or oxides thereof.

In some instances, a nanoparticle includes a core or a core and a shell, as in a core-shell nanoparticle.

In some instances, a nanoparticle is further coated with molecules for attachment of functional elements (e.g., with one or more of a polynucleic acid molecule or binding moiety described herein). In some instances, a coating comprises chondroitin sulfate, dextran sulfate, carboxymethyl dextran, alginic acid, pectin, carragheenan, fucoidan, agaropectin, porphyran, karaya gum, gellan gum, xanthan gum, hyaluronic acids, glucosamine, galactosamine, chitin (or chitosan), polyglutamic acid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, α-chymotrypsin, polylysine, polyarginine, histone, protamine, ovalbumin, dextrin, or cyclodextrin. In some instances, a nanoparticle comprises a graphene-coated nanoparticle.

In some cases, a nanoparticle has at least one dimension of less than about 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm.

In some instances, the nanoparticle formulation comprises paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as with covalently attached metal chelates), nanofibers, nanohorns, nano-onions, nanorods, nanoropes or quantum dots. In some instances, a polynucleic acid molecule or a binding moiety described herein is conjugated either directly or indirectly to the nanoparticle. In some instances, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more polynucleic acid molecules or binding moieties described herein are conjugated either directly or indirectly to a nanoparticle.

In some embodiments, the pharmaceutical formulations include a carrier or carrier materials selected on the basis of compatibility with the composition disclosed herein, and the release profile properties of the desired dosage form. Exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

In some instances, the pharmaceutical formulations further include pH-adjusting agents or buffering agents which include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

In some instances, the pharmaceutical formulation includes one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

In some instances, the pharmaceutical formulations further include diluent which are used to stabilize compounds because they can provide a more stable environment. Salts dissolved in buffered solutions (which also can provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution. In certain instances, diluents increase bulk of the composition to facilitate compression or create sufficient bulk for homogenous blend for capsule filling. Such compounds can include e.g., lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®; dibasic calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugar, such as Di-Pac® (Amstar); mannitol, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents, confectioner's sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

In some cases, the pharmaceutical formulations include disintegration agents or disintegrants to facilitate the breakup or disintegration of a substance. The term “disintegrate” include both the dissolution and dispersion of the dosage form when contacted with gastrointestinal fluid. Examples of disintegration agents include a starch, e.g., a natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551 or Amijel®, or sodium starch glycolate such as Promogel® or Explotab®, a cellulose such as a wood product, methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel®PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linked croscarmellose, a cross-linked starch such as sodium starch glycolate, a cross-linked polymer such as crospovidone, a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate, a clay such as Veegum® HV (magnesium aluminum silicate), a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, a natural sponge, a surfactant, a resin such as a cation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination starch, and the like.

In some instances, the pharmaceutical formulations include filling agents such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

Lubricants and glidants are also optionally included in the pharmaceutical formulations described herein for preventing, reducing or inhibiting adhesion or friction of materials. Exemplary lubricants include, e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a methoxypolyethylene glycol such as Carbowax™, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil®, a starch such as corn starch, silicone oil, a surfactant, and the like.

Plasticizers include compounds used to soften the microencapsulation material or film coatings to make them less brittle. Suitable plasticizers include, e.g., polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethyl cellulose and triacetin. Plasticizers can also function as dispersing agents or wetting agents.

Solubilizers include compounds such as triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, dimethyl isosorbide, and the like.

Stabilizers include compounds such as any antioxidation agents, buffers, acids, preservatives and the like.

Suspending agents include compounds such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like.

Surfactants include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic® (BASF), and the like. Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40. Sometimes, surfactants is included to enhance physical stability or for other purposes.

Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof.

Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts and the like.

Therapeutic Regimens

In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic applications. In some embodiments, the pharmaceutical composition is administered once per day, twice per day, three times per day or more. The pharmaceutical composition is administered daily, every day, every alternate day, five days a week, once a week, every other week, two weeks per month, three weeks per month, once a month, twice a month, three times per month, or more. The pharmaceutical composition is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.

In some embodiments, one or more pharmaceutical compositions are administered simultaneously, sequentially, or at an interval period of time. In some embodiments, one or more pharmaceutical compositions are administered simultaneously. In some cases, one or more pharmaceutical compositions are administered sequentially. In additional cases, one or more pharmaceutical compositions are administered at an interval period of time (e.g., the first administration of a first pharmaceutical composition is on day one followed by an interval of at least 1, 2, 3, 4, 5, or more days prior to the administration of at least a second pharmaceutical composition).

In some embodiments, two or more different pharmaceutical compositions are coadministered. In some instances, the two or more different pharmaceutical compositions are coadministered simultaneously. In some cases, the two or more different pharmaceutical compositions are coadministered sequentially without a gap of time between administrations. In other cases, the two or more different pharmaceutical compositions are coadministered sequentially with a gap of about 0.5 hour, 1 hour, 2 hour, 3 hour, 12 hours, 1 day, 2 days, or more between administrations.

In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the composition is given continuously; alternatively, the dose of the composition being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”). In some instances, the length of the drug holiday varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday is from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, are optionally reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained.

In some embodiments, the amount of a given agent that correspond to such an amount varies depending upon factors such as the particular compound, the severity of the disease, the identity (e.g., weight) of the subject or host in need of treatment, but nevertheless is routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, and the subject or host being treated. In some instances, the desired dose is conveniently presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

The foregoing ranges are merely suggestive, as the number of variables in regard to an individual treatment regime is large, and considerable excursions from these recommended values are not uncommon. Such dosages are altered depending on a number of variables, not limited to the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

In some embodiments, toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio between LD50 and ED50. Compounds exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage varies within this range depending upon the dosage form employed and the route of administration utilized.

Kits/Article of Manufacture

Disclosed herein, in certain embodiments, are kits and articles of manufacture for use with one or more of the compositions and methods described herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

For example, the container(s) include a molecule of Formula (I): A-X—B—Y—C, optionally conjugated to an endosomolytic moiety D as disclosed herein. Such kits optionally include an identifying description or label or instructions relating to its use in the methods described herein.

A kit typically includes labels listing contents and/or instructions for use and package inserts with instructions for use. A set of instructions will also typically be included.

In one embodiment, a label is on or associated with the container. In one embodiment, a label is on a container when letters, numbers, or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein. The pack, for example, contains metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is also accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. In one embodiment, compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

Certain Terminology

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 μL” means “about 5 μL” and also “5 μL.” Generally, the term “about” includes an amount that is expected to be within experimental error.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

As used herein, the terms “individual(s)”, “subject(s)” and “patient(s)” mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly or a hospice worker).

EXAMPLES

These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

Example 1. Sequences

Tables 1, 4, 7, 8, and 10 illustrate target sequences described herein. Tables 2, 3, 5, 6, 9, 11, and 12 illustrate polynucleic acid molecule sequences described herein.

TABLE 1
KRAS Target Sequences
	sequence
	position in		SEQ
Id #	NM_033360.2	target site in NM_033360.2	ID NO:
182	182-200	AAAUGACUGAAUAUAAACUUGUG	1
183	183-201	AAUGACUGAAUAUAAACUUGUGG	2
197	197-215	AACUUGUGGUAGUUGGAGCUGGU	3
224	224-242	UAGGCAAGAGUGCCUUGACGAUA	4
226	226-244	GGCAAGAGUGCCUUGACGAUACA	5
227	227-245	GCAAGAGUGCCUUGACGAUACAG	6
228	228-246	CAAGAGUGCCUUGACGAUACAGC	7
232	232-250	AGUGCCUUGACGAUACAGCUAAU	8
233	233-251	GUGCCUUGACGAUACAGCUAAUU	9
236	236-254	CCUUGACGAUACAGCUAAUUCAG	10
237	237-255	CUUGACGAUACAGCUAAUUCAGA	11
245	245-263	UACAGCUAAUUCAGAAUCAUUUU	12
266	266-284	UUGUGGACGAAUAUGAUCCAACA	13
269	269-287	UGGACGAAUAUGAUCCAACAAUA	14
270	270-288	GGACGAAUAUGAUCCAACAAUAG	15

TABLE 2
KRAS siRNA sequences
	sequence		SEQ		SEQ
	position in		ID	antisense strand sequence	ID
Id #	NM_033360.2	sense strand sequence (5′-3′)	NO:	(5′-3′)	NO:
182	182-200	AUGACUGAAUAUAAACU	16	CAAGUUUAUAUUCAG	17
		UGTT		UCAUTT
183	183-201	UGACUGAAUAUAAACUU	18	ACAAGUUUAUAUUCA	19
		GUTT		GUCATT
197	197-215	CUUGUGGUAGUUGGAGC	20	CAGCUCCAACUACCAC	21
		UGTT		AAGTT
224	224-242	GGCAAGAGUGCCUUGAC	22	UCGUCAAGGCACUCU	23
		GATT		UGCCTT
226	226-244	CAAGAGUGCCUUGACGA	24	UAUCGUCAAGGCACU	25
		UATT		CUUGTT
227	227-245	AAGAGUGCCUUGACGAU	26	GUAUCGUCAAGGCAC	27
		ACTT		UCUUTT
228	228-246	AGAGUGCCUUGACGAUA	28	UGUAUCGUCAAGGCA	29
		CATT		CUCUTT
232	232-250	UGCCUUGACGAUACAGC	30	UAGCUGUAUCGUCAA	31
		UATT		GGCATT
233	233-251	GCCUUGACGAUACAGCU	32	UUAGCUGUAUCGUCA	33
		AATT		AGGCTT
236	236-254	UUGACGAUACAGCUAAU	34	GAAUUAGCUGUAUCG	35
		UCTT		UCAATT
237	237-255	UGACGAUACAGCUAAUU	36	UGAAUUAGCUGUAUC	37
		CATT		GUCATT
245	245-263	CAGCUAAUUCAGAAUCA	38	AAUGAUUCUGAAUUA	39
		UUTT		GCUGTT
266	266-284	GUGGACGAAUAUGAUCC	40	UUGGAUCAUAUUCGU	41
		AATT		CCACTT
269	269-287	GACGAAUAUGAUCCAAC	42	UUGUUGGAUCAUAUU	43
		AATT		CGUCTT
270	270-288	ACGAAUAUGAUCCAACA	44	AUUGUUGGAUCAUAU	45
		AUTT		UCGUTT

TABLE 3
KRAS siRNA Sequences with Chemical Modification
		siRNA sequence with		siRNA sequence with
		chemical modification	SEQ	chemical modification	SEQ
	sequence position	sense strand sequence	ID	antisense strand sequence (5′-	ID
Id #	in NM_033360.2	(5′-3′)	NO:	3′)	NO:
182	182-200	auGfaCfuGfaAfuAfuAf	46	CfAfaGfuUfuAfuAfuUfcAfgU	47
		aAfcUfuGfdTsdT		fcAfudTsdT
183	183-201	ugAfcUfgAfaUfaUfaAf	48	AfCfaAfgUfuUfaUfaUfuCfaGf	49
		aCfuUfgUfdTsdT		uCfadTsdT
197	197-215	cuUfgUfgGfuAfgUfuGf	50	CfAfgCfuCfcAfaCfuAfcCfaCf	51
		gAfgCfuGfdTsdT		aAfgdTsdT
224	224-242	ggCfaAfgAfgUfgCfcUf	52	UfCfgUfcAfaGfgCfaCfuCfuUf	53
		uGfaCfgAfdTsdT		gCfcdTsdT
226	226-244	caAfgAfgUfgCfcUfuGf	54	UfAfuCfgUfcAfaGfgCfaCfuCf	55
		aCfgAfuAfdTsdT		uUfgdTsdT
227	227-245	aaGfaGfuGfcCfuUfgAf	56	GfUfaUfcGfuCfaAfgGfcAfcUf	57
		cGfaUfaCfdTsdT		cUfudTsdT
228	228-246	agAfgUfgCfcUfuGfaCf	58	UfGfuAfuCfgUfcAfaGfgCfaCf	59
		gAfuAfcAfdTsdT		uCfudTsdT
232	232-250	ugCfcUfuGfaCfgAfuAf	60	UfAfgCfuGfuAfuCfgUfcAfaGf	61
		cAfgCfuAfdTsdT		gCfadTsdT
233	233-251	gcCfuUfgAfcGfaUfaCf	62	UfUfaGfcUfgUfaUfcGfuCfaAf	63
		aGfcUfaAfdTsdT		gGfcdTsdT
236	236-254	uuGfaCfgAfuAfcAfgCf	64	GfAfaUfuAfgCfuGfuAfuCfgU	65
		uAfaUfuCfdTsdT		fcAfadTsdT
237	237-255	ugAfcGfaUfaCfaGfcUf	66	UfGfaAfuUfaGfcUfgUfaUfcGf	67
		aAfuUfcAfdTsdT		uCfadTsdT
245	245-263	caGfcUfaAfuUfcAfgAf	68	AfAfuGfaUfuCfuGfaAfuUfaGf	69
		aUfcAfuUfdTsdT		cUfgdTsdT
266	266-284	guGfgAfcGfaAfuAfuGf	70	UfUfgGfaUfcAfuAfuUfcGfuCf	71
		aUfcCfaAfdTsdT		cAfcdTsdT
269	269-287	gaCfgAfaUfaUfgAfuCf	72	UfUfgUfuGfgAfuCfaUfaUfuCf	73
		cAfaCfaAfdTsdT		gUfcdTsdT
270	270-288	acGfaAfuAfuGfaUfcCf	74	AfUfuGfuUfgGfaUfcAfuAfuU	75
		aAfcAfaUfdTsdT		fcGfudTsdT
siRNA Sequence with Chemical Modification Info
lower case (n) = 2′-O-Me;
Nf = 2′-F;
dT = deoxy-T residue;
s = phosphorothioate backbone modification;
iB = inverted abasic

TABLE 4
EGFR Target Sequences
hs	19mer pos. in	sequence of total 23mer	SEQ
Id #	NM_005228.3	target site in NM_005228.3	ID NO:
68	68-86	GGCGGCCGGAGUCCCGAGCUAGC	76
71	71-89	GGCCGGAGUCCCGAGCUAGCCCC	77
72	72-90	GCCGGAGUCCCGAGCUAGCCCCG	78
73	73-91	CCGGAGUCCCGAGCUAGCCCCGG	79
74	74-92	CGGAGUCCCGAGCUAGCCCCGGC	80
75	75-93	GGAGUCCCGAGCUAGCCCCGGCG	81
76	76-94	GAGUCCCGAGCUAGCCCCGGCGG	82
78	78-96	GUCCCGAGCUAGCCCCGGCGGCC	83
114	114-132	CCGGACGACAGGCCACCUCGUCG	84
115	115-133	CGGACGACAGGCCACCUCGUCGG	85
116	116-134	GGACGACAGGCCACCUCGUCGGC	86
117	117-135	GACGACAGGCCACCUCGUCGGCG	87
118	118-136	ACGACAGGCCACCUCGUCGGCGU	88
120	120-138	GACAGGCCACCUCGUCGGCGUCC	89
121	121-139	ACAGGCCACCUCGUCGGCGUCCG	90
122	122-140	CAGGCCACCUCGUCGGCGUCCGC	91
123	123-141	AGGCCACCUCGUCGGCGUCCGCC	92
124	124-142	GGCCACCUCGUCGGCGUCCGCCC	93
125	125-143	GCCACCUCGUCGGCGUCCGCCCG	94
126	126-144	CCACCUCGUCGGCGUCCGCCCGA	95
127	127-145	CACCUCGUCGGCGUCCGCCCGAG	96
128	128-146	ACCUCGUCGGCGUCCGCCCGAGU	97
129	129-147	CCUCGUCGGCGUCCGCCCGAGUC	98
130	130-148	CUCGUCGGCGUCCGCCCGAGUCC	99
131	131-149	UCGUCGGCGUCCGCCCGAGUCCC	100
132	132-150	CGUCGGCGUCCGCCCGAGUCCCC	101
135	135-153	CGGCGUCCGCCCGAGUCCCCGCC	102
136	136-154	GGCGUCCGCCCGAGUCCCCGCCU	103
141	141-159	CCGCCCGAGUCCCCGCCUCGCCG	104
164	164-182	CCAACGCCACAACCACCGCGCAC	105
165	165-183	CAACGCCACAACCACCGCGCACG	106
166	166-184	AACGCCACAACCACCGCGCACGG	107
168	168-186	CGCCACAACCACCGCGCACGGCC	108
169	169-187	GCCACAACCACCGCGCACGGCCC	109
170	170-188	CCACAACCACCGCGCACGGCCCC	110
247	247-265	CGAUGCGACCCUCCGGGACGGCC	111
248	248-266	GAUGCGACCCUCCGGGACGGCCG	112
249	249-267	AUGCGACCCUCCGGGACGGCCGG	113
251	251-269	GCGACCCUCCGGGACGGCCGGGG	114
252	252-270	CGACCCUCCGGGACGGCCGGGGC	115
254	254-272	ACCCUCCGGGACGGCCGGGGCAG	116
329	329-347	AAAGAAAGUUUGCCAAGGCACGA	117
330	330-348	AAGAAAGUUUGCCAAGGCACGAG	118
332	332-350	GAAAGUUUGCCAAGGCACGAGUA	119
333	333-351	AAAGUUUGCCAAGGCACGAGUAA	120
334	334-352	AAGUUUGCCAAGGCACGAGUAAC	121
335	335-353	AGUUUGCCAAGGCACGAGUAACA	122
336	336-354	GUUUGCCAAGGCACGAGUAACAA	123
337	337-355	UUUGCCAAGGCACGAGUAACAAG	124
338	338-356	UUGCCAAGGCACGAGUAACAAGC	125
361	361-379	UCACGCAGUUGGGCACUUUUGAA	126
362	362-380	CACGCAGUUGGGCACUUUUGAAG	127
363	363-381	ACGCAGUUGGGCACUUUUGAAGA	128
364	364-382	CGCAGUUGGGCACUUUUGAAGAU	129
365	365-383	GCAGUUGGGCACUUUUGAAGAUC	130
366	366-384	CAGUUGGGCACUUUUGAAGAUCA	131
367	367-385	AGUUGGGCACUUUUGAAGAUCAU	132
368	368-386	GUUGGGCACUUUUGAAGAUCAUU	133
369	369-387	UUGGGCACUUUUGAAGAUCAUUU	134
377	377-395	UUUUGAAGAUCAUUUUCUCAGCC	135
379	379-397	UUGAAGAUCAUUUUCUCAGCCUC	136
380	380-398	UGAAGAUCAUUUUCUCAGCCUCC	137
385	385-403	AUCAUUUUCUCAGCCUCCAGAGG	138
394	394-412	UCAGCCUCCAGAGGAUGUUCAAU	139
396	396-414	AGCCUCCAGAGGAUGUUCAAUAA	140
397	397-415	GCCUCCAGAGGAUGUUCAAUAAC	141
401	401-419	CCAGAGGAUGUUCAAUAACUGUG	142
403	403-421	AGAGGAUGUUCAAUAACUGUGAG	143
407	407-425	GAUGUUCAAUAACUGUGAGGUGG	144
409	409-427	UGUUCAAUAACUGUGAGGUGGUC	145
410	410-428	GUUCAAUAACUGUGAGGUGGUCC	146
411	411-429	UUCAAUAACUGUGAGGUGGUCCU	147
412	412-430	UCAAUAACUGUGAGGUGGUCCUU	148
413	413-431	CAAUAACUGUGAGGUGGUCCUUG	149
414	414-432	AAUAACUGUGAGGUGGUCCUUGG	150
416	416-434	UAACUGUGAGGUGGUCCUUGGGA	151
418	418-436	ACUGUGAGGUGGUCCUUGGGAAU	152
419	419-437	CUGUGAGGUGGUCCUUGGGAAUU	153
425	425-443	GGUGGUCCUUGGGAAUUUGGAAA	154
431	431-449	CCUUGGGAAUUUGGAAAUUACCU	155
432	432-450	CUUGGGAAUUUGGAAAUUACCUA	156
433	433-451	UUGGGAAUUUGGAAAUUACCUAU	157
434	434-452	UGGGAAUUUGGAAAUUACCUAUG	158
458	458-476	GCAGAGGAAUUAUGAUCUUUCCU	159
459	459-477	CAGAGGAAUUAUGAUCUUUCCUU	160
463	463-481	GGAAUUAUGAUCUUUCCUUCUUA	161
464	464-482	GAAUUAUGAUCUUUCCUUCUUAA	162
466	466-484	AUUAUGAUCUUUCCUUCUUAAAG	163
468	468-486	UAUGAUCUUUCCUUCUUAAAGAC	164
471	471-489	GAUCUUUCCUUCUUAAAGACCAU	165
476	476-494	UUCCUUCUUAAAGACCAUCCAGG	166
477	477-495	UCCUUCUUAAAGACCAUCCAGGA	167
479	479-497	CUUCUUAAAGACCAUCCAGGAGG	168
481	481-499	UCUUAAAGACCAUCCAGGAGGUG	169
482	482-500	CUUAAAGACCAUCCAGGAGGUGG	170
492	492-510	AUCCAGGAGGUGGCUGGUUAUGU	171
493	493-511	UCCAGGAGGUGGCUGGUUAUGUC	172
494	494-512	CCAGGAGGUGGCUGGUUAUGUCC	173
495	495-513	CAGGAGGUGGCUGGUUAUGUCCU	174
496	496-514	AGGAGGUGGCUGGUUAUGUCCUC	175
497	497-515	GGAGGUGGCUGGUUAUGUCCUCA	176
499	499-517	AGGUGGCUGGUUAUGUCCUCAUU	177
520	520-538	UUGCCCUCAACACAGUGGAGCGA	178
542	542-560	AAUUCCUUUGGAAAACCUGCAGA	179
543	543-561	AUUCCUUUGGAAAACCUGCAGAU	180
550	550-568	UGGAAAACCUGCAGAUCAUCAGA	181
551	551-569	GGAAAACCUGCAGAUCAUCAGAG	182
553	553-571	AAAACCUGCAGAUCAUCAGAGGA	183
556	556-574	ACCUGCAGAUCAUCAGAGGAAAU	184
586	586-604	ACGAAAAUUCCUAUGCCUUAGCA	185
587	587-605	CGAAAAUUCCUAUGCCUUAGCAG	186
589	589-607	AAAAUUCCUAUGCCUUAGCAGUC	187
592	592-610	AUUCCUAUGCCUUAGCAGUCUUA	188
593	593-611	UUCCUAUGCCUUAGCAGUCUUAU	189
594	594-612	UCCUAUGCCUUAGCAGUCUUAUC	190
596	596-614	CUAUGCCUUAGCAGUCUUAUCUA	191
597	597-615	UAUGCCUUAGCAGUCUUAUCUAA	192
598	598-616	AUGCCUUAGCAGUCUUAUCUAAC	193
599	599-617	UGCCUUAGCAGUCUUAUCUAACU	194
600	600-618	GCCUUAGCAGUCUUAUCUAACUA	195
601	601-619	CCUUAGCAGUCUUAUCUAACUAU	196
602	602-620	CUUAGCAGUCUUAUCUAACUAUG	197
603	603-621	UUAGCAGUCUUAUCUAACUAUGA	198
604	604-622	UAGCAGUCUUAUCUAACUAUGAU	199
605	605-623	AGCAGUCUUAUCUAACUAUGAUG	200
608	608-626	AGUCUUAUCUAACUAUGAUGCAA	201
609	609-627	GUCUUAUCUAACUAUGAUGCAAA	202
610	610-628	UCUUAUCUAACUAUGAUGCAAAU	203
611	611-629	CUUAUCUAACUAUGAUGCAAAUA	204
612	612-630	UUAUCUAACUAUGAUGCAAAUAA	205
613	613-631	UAUCUAACUAUGAUGCAAAUAAA	206
614	614-632	AUCUAACUAUGAUGCAAAUAAAA	207
616	616-634	CUAACUAUGAUGCAAAUAAAACC	208
622	622-640	AUGAUGCAAAUAAAACCGGACUG	209
623	623-641	UGAUGCAAAUAAAACCGGACUGA	210
624	624-642	GAUGCAAAUAAAACCGGACUGAA	211
626	626-644	UGCAAAUAAAACCGGACUGAAGG	212
627	627-645	GCAAAUAAAACCGGACUGAAGGA	213
628	628-646	CAAAUAAAACCGGACUGAAGGAG	214
630	630-648	AAUAAAACCGGACUGAAGGAGCU	215
631	631-649	AUAAAACCGGACUGAAGGAGCUG	216
632	632-650	UAAAACCGGACUGAAGGAGCUGC	217
633	633-651	AAAACCGGACUGAAGGAGCUGCC	218
644	644-662	GAAGGAGCUGCCCAUGAGAAAUU	219
665	665-683	UUUACAGGAAAUCCUGCAUGGCG	220
668	668-686	ACAGGAAAUCCUGCAUGGCGCCG	221
669	669-687	CAGGAAAUCCUGCAUGGCGCCGU	222
670	670-688	AGGAAAUCCUGCAUGGCGCCGUG	223
671	671-689	GGAAAUCCUGCAUGGCGCCGUGC	224
672	672-690	GAAAUCCUGCAUGGCGCCGUGCG	225
674	674-692	AAUCCUGCAUGGCGCCGUGCGGU	226
676	676-694	UCCUGCAUGGCGCCGUGCGGUUC	227
677	677-695	CCUGCAUGGCGCCGUGCGGUUCA	228
678	678-696	CUGCAUGGCGCCGUGCGGUUCAG	229
680	680-698	GCAUGGCGCCGUGCGGUUCAGCA	230
681	681-699	CAUGGCGCCGUGCGGUUCAGCAA	231
682	682-700	AUGGCGCCGUGCGGUUCAGCAAC	232
683	683-701	UGGCGCCGUGCGGUUCAGCAACA	233
684	684-702	GGCGCCGUGCGGUUCAGCAACAA	234
685	685-703	GCGCCGUGCGGUUCAGCAACAAC	235
686	686-704	CGCCGUGCGGUUCAGCAACAACC	236
688	688-706	CCGUGCGGUUCAGCAACAACCCU	237
690	690-708	GUGCGGUUCAGCAACAACCCUGC	238
692	692-710	GCGGUUCAGCAACAACCCUGCCC	239
698	698-716	CAGCAACAACCCUGCCCUGUGCA	240
700	700-718	GCAACAACCCUGCCCUGUGCAAC	241
719	719-737	CAACGUGGAGAGCAUCCAGUGGC	242
720	720-738	AACGUGGAGAGCAUCCAGUGGCG	243
721	721-739	ACGUGGAGAGCAUCCAGUGGCGG	244
724	724-742	UGGAGAGCAUCCAGUGGCGGGAC	245
725	725-743	GGAGAGCAUCCAGUGGCGGGACA	246
726	726-744	GAGAGCAUCCAGUGGCGGGACAU	247
733	733-751	UCCAGUGGCGGGACAUAGUCAGC	248
734	734-752	CCAGUGGCGGGACAUAGUCAGCA	249
736	736-754	AGUGGCGGGACAUAGUCAGCAGU	250
737	737-755	GUGGCGGGACAUAGUCAGCAGUG	251
763	763-781	UUCUCAGCAACAUGUCGAUGGAC	252
765	765-783	CUCAGCAACAUGUCGAUGGACUU	253
766	766-784	UCAGCAACAUGUCGAUGGACUUC	254
767	767-785	CAGCAACAUGUCGAUGGACUUCC	255
769	769-787	GCAACAUGUCGAUGGACUUCCAG	256
770	770-788	CAACAUGUCGAUGGACUUCCAGA	257
771	771-789	AACAUGUCGAUGGACUUCCAGAA	258
772	772-790	ACAUGUCGAUGGACUUCCAGAAC	259
775	775-793	UGUCGAUGGACUUCCAGAACCAC	260
789	789-807	CAGAACCACCUGGGCAGCUGCCA	261
798	798-816	CUGGGCAGCUGCCAAAAGUGUGA	262
800	800-818	GGGCAGCUGCCAAAAGUGUGAUC	263
805	805-823	GCUGCCAAAAGUGUGAUCCAAGC	264
806	806-824	CUGCCAAAAGUGUGAUCCAAGCU	265
807	807-825	UGCCAAAAGUGUGAUCCAAGCUG	266
810	810-828	CAAAAGUGUGAUCCAAGCUGUCC	267
814	814-832	AGUGUGAUCCAAGCUGUCCCAAU	268
815	815-833	GUGUGAUCCAAGCUGUCCCAAUG	269
817	817-835	GUGAUCCAAGCUGUCCCAAUGGG	270
818	818-836	UGAUCCAAGCUGUCCCAAUGGGA	271
819	819-837	GAUCCAAGCUGUCCCAAUGGGAG	272
820	820-838	AUCCAAGCUGUCCCAAUGGGAGC	273
821	821-839	UCCAAGCUGUCCCAAUGGGAGCU	274
823	823-841	CAAGCUGUCCCAAUGGGAGCUGC	275
826	826-844	GCUGUCCCAAUGGGAGCUGCUGG	276
847	847-865	GGGGUGCAGGAGAGGAGAACUGC	277
871	871-889	AGAAACUGACCAAAAUCAUCUGU	278
872	872-890	GAAACUGACCAAAAUCAUCUGUG	279
873	873-891	AAACUGACCAAAAUCAUCUGUGC	280
877	877-895	UGACCAAAAUCAUCUGUGCCCAG	281
878	878-896	GACCAAAAUCAUCUGUGCCCAGC	282
881	881-899	CAAAAUCAUCUGUGCCCAGCAGU	283
890	890-908	CUGUGCCCAGCAGUGCUCCGGGC	284
892	892-910	GUGCCCAGCAGUGCUCCGGGCGC	285
929	929-947	CCCCAGUGACUGCUGCCACAACC	286
930	930-948	CCCAGUGACUGCUGCCACAACCA	287
979	979-997	GGGAGAGCGACUGCCUGGUCUGC	288
980	980-998	GGAGAGCGACUGCCUGGUCUGCC	289
981	981-999	GAGAGCGACUGCCUGGUCUGCCG	290
982	982-1000	AGAGCGACUGCCUGGUCUGCCGC	291
983	983-1001	GAGCGACUGCCUGGUCUGCCGCA	292
984	984-1002	AGCGACUGCCUGGUCUGCCGCAA	293
989	989-1007	CUGCCUGGUCUGCCGCAAAUUCC	294
990	990-1008	UGCCUGGUCUGCCGCAAAUUCCG	295
991	991-1009	GCCUGGUCUGCCGCAAAUUCCGA	296
992	992-1010	CCUGGUCUGCCGCAAAUUCCGAG	297
994	994-1012	UGGUCUGCCGCAAAUUCCGAGAC	298
995	995-1013	GGUCUGCCGCAAAUUCCGAGACG	299
996	996-1014	GUCUGCCGCAAAUUCCGAGACGA	300
997	997-1015	UCUGCCGCAAAUUCCGAGACGAA	301
999	999-1017	UGCCGCAAAUUCCGAGACGAAGC	302
1004	1004-1022	CAAAUUCCGAGACGAAGCCACGU	303
1005	1005-1023	AAAUUCCGAGACGAAGCCACGUG	304
1006	1006-1024	AAUUCCGAGACGAAGCCACGUGC	305
1007	1007-1025	AUUCCGAGACGAAGCCACGUGCA	306
1008	1008-1026	UUCCGAGACGAAGCCACGUGCAA	307
1010	1010-1028	CCGAGACGAAGCCACGUGCAAGG	308
1013	1013-1031	AGACGAAGCCACGUGCAAGGACA	309
1014	1014-1032	GACGAAGCCACGUGCAAGGACAC	310
1015	1015-1033	ACGAAGCCACGUGCAAGGACACC	311
1016	1016-1034	CGAAGCCACGUGCAAGGACACCU	312
1040	1040-1058	CCCCCCACUCAUGCUCUACAACC	313
1042	1042-1060	CCCCACUCAUGCUCUACAACCCC	314
1044	1044-1062	CCACUCAUGCUCUACAACCCCAC	315
1047	1047-1065	CUCAUGCUCUACAACCCCACCAC	316
1071	1071-1089	UACCAGAUGGAUGUGAACCCCGA	317
1073	1073-1091	CCAGAUGGAUGUGAACCCCGAGG	318
1074	1074-1092	CAGAUGGAUGUGAACCCCGAGGG	319
1075	1075-1093	AGAUGGAUGUGAACCCCGAGGGC	320
1077	1077-1095	AUGGAUGUGAACCCCGAGGGCAA	321
1078	1078-1096	UGGAUGUGAACCCCGAGGGCAAA	322
1080	1080-1098	GAUGUGAACCCCGAGGGCAAAUA	323
1084	1084-1102	UGAACCCCGAGGGCAAAUACAGC	324
1085	1085-1103	GAACCCCGAGGGCAAAUACAGCU	325
1087	1087-1105	ACCCCGAGGGCAAAUACAGCUUU	326
1088	1088-1106	CCCCGAGGGCAAAUACAGCUUUG	327
1089	1089-1107	CCCGAGGGCAAAUACAGCUUUGG	328
1096	1096-1114	GCAAAUACAGCUUUGGUGCCACC	329
1097	1097-1115	CAAAUACAGCUUUGGUGCCACCU	330
1098	1098-1116	AAAUACAGCUUUGGUGCCACCUG	331
1104	1104-1122	AGCUUUGGUGCCACCUGCGUGAA	332
1106	1106-1124	CUUUGGUGCCACCUGCGUGAAGA	333
1112	1112-1130	UGCCACCUGCGUGAAGAAGUGUC	334
1116	1116-1134	ACCUGCGUGAAGAAGUGUCCCCG	335
1117	1117-1135	CCUGCGUGAAGAAGUGUCCCCGU	336
1118	1118-1136	CUGCGUGAAGAAGUGUCCCCGUA	337
1119	1119-1137	UGCGUGAAGAAGUGUCCCCGUAA	338
1120	1120-1138	GCGUGAAGAAGUGUCCCCGUAAU	339
1121	1121-1139	CGUGAAGAAGUGUCCCCGUAAUU	340
1122	1122-1140	GUGAAGAAGUGUCCCCGUAAUUA	341
1123	1123-1141	UGAAGAAGUGUCCCCGUAAUUAU	342
1124	1124-1142	GAAGAAGUGUCCCCGUAAUUAUG	343
1125	1125-1143	AAGAAGUGUCCCCGUAAUUAUGU	344
1126	1126-1144	AGAAGUGUCCCCGUAAUUAUGUG	345
1127	1127-1145	GAAGUGUCCCCGUAAUUAUGUGG	346
1128	1128-1146	AAGUGUCCCCGUAAUUAUGUGGU	347
1129	1129-1147	AGUGUCCCCGUAAUUAUGUGGUG	348
1130	1130-1148	GUGUCCCCGUAAUUAUGUGGUGA	349
1132	1132-1150	GUCCCCGUAAUUAUGUGGUGACA	350
1134	1134-1152	CCCCGUAAUUAUGUGGUGACAGA	351
1136	1136-1154	CCGUAAUUAUGUGGUGACAGAUC	352
1137	1137-1155	CGUAAUUAUGUGGUGACAGAUCA	353
1138	1138-1156	GUAAUUAUGUGGUGACAGAUCAC	354
1139	1139-1157	UAAUUAUGUGGUGACAGAUCACG	355
1140	1140-1158	AAUUAUGUGGUGACAGAUCACGG	356
1142	1142-1160	UUAUGUGGUGACAGAUCACGGCU	357
1145	1145-1163	UGUGGUGACAGAUCACGGCUCGU	358
1147	1147-1165	UGGUGACAGAUCACGGCUCGUGC	359
1148	1148-1166	GGUGACAGAUCACGGCUCGUGCG	360
1149	1149-1167	GUGACAGAUCACGGCUCGUGCGU	361
1150	1150-1168	UGACAGAUCACGGCUCGUGCGUC	362
1151	1151-1169	GACAGAUCACGGCUCGUGCGUCC	363
1152	1152-1170	ACAGAUCACGGCUCGUGCGUCCG	364
1153	1153-1171	CAGAUCACGGCUCGUGCGUCCGA	365
1154	1154-1172	AGAUCACGGCUCGUGCGUCCGAG	366
1155	1155-1173	GAUCACGGCUCGUGCGUCCGAGC	367
1156	1156-1174	AUCACGGCUCGUGCGUCCGAGCC	368
1157	1157-1175	UCACGGCUCGUGCGUCCGAGCCU	369
1160	1160-1178	CGGCUCGUGCGUCCGAGCCUGUG	370
1200	1200-1218	AUGGAGGAAGACGGCGUCCGCAA	371
1201	1201-1219	UGGAGGAAGACGGCGUCCGCAAG	372
1203	1203-1221	GAGGAAGACGGCGUCCGCAAGUG	373
1204	1204-1222	AGGAAGACGGCGUCCGCAAGUGU	374
1205	1205-1223	GGAAGACGGCGUCCGCAAGUGUA	375
1207	1207-1225	AAGACGGCGUCCGCAAGUGUAAG	376
1208	1208-1226	AGACGGCGUCCGCAAGUGUAAGA	377
1211	1211-1229	CGGCGUCCGCAAGUGUAAGAAGU	378
1212	1212-1230	GGCGUCCGCAAGUGUAAGAAGUG	379
1213	1213-1231	GCGUCCGCAAGUGUAAGAAGUGC	380
1214	1214-1232	CGUCCGCAAGUGUAAGAAGUGCG	381
1215	1215-1233	GUCCGCAAGUGUAAGAAGUGCGA	382
1216	1216-1234	UCCGCAAGUGUAAGAAGUGCGAA	383
1217	1217-1235	CCGCAAGUGUAAGAAGUGCGAAG	384
1219	1219-1237	GCAAGUGUAAGAAGUGCGAAGGG	385
1220	1220-1238	CAAGUGUAAGAAGUGCGAAGGGC	386
1221	1221-1239	AAGUGUAAGAAGUGCGAAGGGCC	387
1222	1222-1240	AGUGUAAGAAGUGCGAAGGGCCU	388
1223	1223-1241	GUGUAAGAAGUGCGAAGGGCCUU	389
1224	1224-1242	UGUAAGAAGUGCGAAGGGCCUUG	390
1225	1225-1243	GUAAGAAGUGCGAAGGGCCUUGC	391
1226	1226-1244	UAAGAAGUGCGAAGGGCCUUGCC	392
1229	1229-1247	GAAGUGCGAAGGGCCUUGCCGCA	393
1230	1230-1248	AAGUGCGAAGGGCCUUGCCGCAA	394
1231	1231-1249	AGUGCGAAGGGCCUUGCCGCAAA	395
1232	1232-1250	GUGCGAAGGGCCUUGCCGCAAAG	396
1233	1233-1251	UGCGAAGGGCCUUGCCGCAAAGU	397
1235	1235-1253	CGAAGGGCCUUGCCGCAAAGUGU	398
1236	1236-1254	GAAGGGCCUUGCCGCAAAGUGUG	399
1237	1237-1255	AAGGGCCUUGCCGCAAAGUGUGU	400
1238	1238-1256	AGGGCCUUGCCGCAAAGUGUGUA	401
1239	1239-1257	GGGCCUUGCCGCAAAGUGUGUAA	402
1241	1241-1259	GCCUUGCCGCAAAGUGUGUAACG	403
1261	1261-1279	ACGGAAUAGGUAUUGGUGAAUUU	404
1262	1262-1280	CGGAAUAGGUAUUGGUGAAUUUA	405
1263	1263-1281	GGAAUAGGUAUUGGUGAAUUUAA	406
1264	1264-1282	GAAUAGGUAUUGGUGAAUUUAAA	407
1266	1266-1284	AUAGGUAUUGGUGAAUUUAAAGA	408
1267	1267-1285	UAGGUAUUGGUGAAUUUAAAGAC	409
1289	1289-1307	CUCACUCUCCAUAAAUGCUACGA	410
1313	1313-1331	UAUUAAACACUUCAAAAACUGCA	411
1320	1320-1338	CACUUCAAAAACUGCACCUCCAU	412
1321	1321-1339	ACUUCAAAAACUGCACCUCCAUC	413
1322	1322-1340	CUUCAAAAACUGCACCUCCAUCA	414
1323	1323-1341	UUCAAAAACUGCACCUCCAUCAG	415
1324	1324-1342	UCAAAAACUGCACCUCCAUCAGU	416
1328	1328-1346	AAACUGCACCUCCAUCAGUGGCG	417
1332	1332-1350	UGCACCUCCAUCAGUGGCGAUCU	418
1333	1333-1351	GCACCUCCAUCAGUGGCGAUCUC	419
1335	1335-1353	ACCUCCAUCAGUGGCGAUCUCCA	420
1338	1338-1356	UCCAUCAGUGGCGAUCUCCACAU	421
1344	1344-1362	AGUGGCGAUCUCCACAUCCUGCC	422
1345	1345-1363	GUGGCGAUCUCCACAUCCUGCCG	423
1346	1346-1364	UGGCGAUCUCCACAUCCUGCCGG	424
1347	1347-1365	GGCGAUCUCCACAUCCUGCCGGU	425
1348	1348-1366	GCGAUCUCCACAUCCUGCCGGUG	426
1353	1353-1371	CUCCACAUCCUGCCGGUGGCAUU	427
1354	1354-1372	UCCACAUCCUGCCGGUGGCAUUU	428
1355	1355-1373	CCACAUCCUGCCGGUGGCAUUUA	429
1357	1357-1375	ACAUCCUGCCGGUGGCAUUUAGG	430
1360	1360-1378	UCCUGCCGGUGGCAUUUAGGGGU	431
1361	1361-1379	CCUGCCGGUGGCAUUUAGGGGUG	432
1362	1362-1380	CUGCCGGUGGCAUUUAGGGGUGA	433
1363	1363-1381	UGCCGGUGGCAUUUAGGGGUGAC	434
1366	1366-1384	CGGUGGCAUUUAGGGGUGACUCC	435
1369	1369-1387	UGGCAUUUAGGGGUGACUCCUUC	436
1370	1370-1388	GGCAUUUAGGGGUGACUCCUUCA	437
1371	1371-1389	GCAUUUAGGGGUGACUCCUUCAC	438
1372	1372-1390	CAUUUAGGGGUGACUCCUUCACA	439
1373	1373-1391	AUUUAGGGGUGACUCCUUCACAC	440
1374	1374-1392	UUUAGGGGUGACUCCUUCACACA	441
1404	1404-1422	CCUCUGGAUCCACAGGAACUGGA	442
1408	1408-1426	UGGAUCCACAGGAACUGGAUAUU	443
1409	1409-1427	GGAUCCACAGGAACUGGAUAUUC	444
1411	1411-1429	AUCCACAGGAACUGGAUAUUCUG	445
1412	1412-1430	UCCACAGGAACUGGAUAUUCUGA	446
1419	1419-1437	GAACUGGAUAUUCUGAAAACCGU	447
1426	1426-1444	AUAUUCUGAAAACCGUAAAGGAA	448
1427	1427-1445	UAUUCUGAAAACCGUAAAGGAAA	449
1430	1430-1448	UCUGAAAACCGUAAAGGAAAUCA	450
1431	1431-1449	CUGAAAACCGUAAAGGAAAUCAC	451

TABLE 5
EGFR siRNA Sequences
	Sequence		SEQ		SEQ
	position in	sense strand sequence	ID	antisense strand sequence	ID
hs Id #	NM_005228.3	(5′-3′)	NO:	(5′-3′)	NO:
68	68-86	CGGCCGGAGUCCCGAG	452	UAGCUCGGGACUCCGGC	453
		CUATT		CGTT
71	71-89	CCGGAGUCCCGAGCUA	454	GGCUAGCUCGGGACUCC	455
		GCCTT		GGTT
72	72-90	CGGAGUCCCGAGCUAG	456	GGGCUAGCUCGGGACUC	457
		CCCTT		CGTT
73	73-91	GGAGUCCCGAGCUAGC	458	GGGGCUAGCUCGGGACU	459
		CCCTT		CCTT
74	74-92	GAGUCCCGAGCUAGCC	460	CGGGGCUAGCUCGGGAC	461
		CCGTT		UCTT
75	75-93	AGUCCCGAGCUAGCCC	462	CCGGGGCUAGCUCGGGA	463
		CGGTT		CUTT
76	76-94	GUCCCGAGCUAGCCCC	464	GCCGGGGCUAGCUCGGG	465
		GGCTT		ACTT
78	78-96	CCCGAGCUAGCCCCGG	466	CCGCCGGGGCUAGCUCG	467
		CGGTT		GGTT
114	114-132	GGACGACAGGCCACCU	468	ACGAGGUGGCCUGUCGU	469
		CGUTT		CCTT
115	115-133	GACGACAGGCCACCUC	470	GACGAGGUGGCCUGUCG	471
		GUCTT		UCTT
116	116-134	ACGACAGGCCACCUCG	472	CGACGAGGUGGCCUGUC	473
		UCGTT		GUTT
117	117-135	CGACAGGCCACCUCGU	474	CCGACGAGGUGGCCUGU	475
		CGGTT		CGTT
118	118-136	GACAGGCCACCUCGUC	476	GCCGACGAGGUGGCCUG	477
		GGCTT		UCTT
120	120-138	CAGGCCACCUCGUCGG	478	ACGCCGACGAGGUGGCC	479
		CGUTT		UGTT
121	121-139	AGGCCACCUCGUCGGC	480	GACGCCGACGAGGUGGC	481
		GUCTT		CUTT
122	122-140	GGCCACCUCGUCGGCG	482	GGACGCCGACGAGGUGG	483
		UCCTT		CCTT
123	123-141	GCCACCUCGUCGGCGU	484	CGGACGCCGACGAGGUG	485
		CCGTT		GCTT
124	124-142	CCACCUCGUCGGCGUC	486	GCGGACGCCGACGAGGU	487
		CGCTT		GGTT
125	125-143	CACCUCGUCGGCGUCC	488	GGCGGACGCCGACGAGG	489
		GCCTT		UGTT
126	126-144	ACCUCGUCGGCGUCCG	490	GGGCGGACGCCGACGAG	491
		CCCTT		GUTT
127	127-145	CCUCGUCGGCGUCCGC	492	CGGGCGGACGCCGACGA	493
		CCGTT		GGTT
128	128-146	CUCGUCGGCGUCCGCC	494	UCGGGCGGACGCCGACG	495
		CGATT		AGTT
129	129-147	UCGUCGGCGUCCGCCC	496	CUCGGGCGGACGCCGAC	497
		GAGTT		GATT
130	130-148	CGUCGGCGUCCGCCCG	498	ACUCGGGCGGACGCCGA	499
		AGUTT		CGTT
131	131-149	GUCGGCGUCCGCCCGA	500	GACUCGGGCGGACGCCG	501
		GUCTT		ACTT
132	132-150	UCGGCGUCCGCCCGAG	502	GGACUCGGGCGGACGCC	503
		UCCTT		GATT
135	135-153	GCGUCCGCCCGAGUCC	504	CGGGGACUCGGGCGGAC	505
		CCGTT		GCTT
136	136-154	CGUCCGCCCGAGUCCC	506	GCGGGGACUCGGGCGGA	507
		CGCTT		CGTT
141	141-159	GCCCGAGUCCCCGCCU	508	GCGAGGCGGGGACUCGG	509
		CGCTT		GCTT
164	164-182	AACGCCACAACCACCG	510	GCGCGGUGGUUGUGGCG	511
		CGCTT		UUTT
165	165-183	ACGCCACAACCACCGC	512	UGCGCGGUGGUUGUGGC	513
		GCATT		GUTT
166	166-184	CGCCACAACCACCGCG	514	GUGCGCGGUGGUUGUGG	515
		CACTT		CGTT
168	168-186	CCACAACCACCGCGCA	516	CCGUGCGCGGUGGUUGU	517
		CGGTT		GGTT
169	169-187	CACAACCACCGCGCAC	518	GCCGUGCGCGGUGGUUG	519
		GGCTT		UGTT
170	170-188	ACAACCACCGCGCACG	520	GGCCGUGCGCGGUGGUU	521
		GCCTT		GUTT
247	247-265	AUGCGACCCUCCGGGA	522	CCGUCCCGGAGGGUCGC	523
		CGGTT		AUTT
248	248-266	UGCGACCCUCCGGGAC	524	GCCGUCCCGGAGGGUCG	525
		GGCTT		CATT
249	249-267	GCGACCCUCCGGGACG	526	GGCCGUCCCGGAGGGUC	527
		GCCTT		GCTT
251	251-269	GACCCUCCGGGACGGC	528	CCGGCCGUCCCGGAGGG	529
		CGGTT		UCTT
252	252-270	ACCCUCCGGGACGGCC	530	CCCGGCCGUCCCGGAGG	531
		GGGTT		GUTT
254	254-272	CCUCCGGGACGGCCGG	532	GCCCCGGCCGUCCCGGA	533
		GGCTT		GGTT
329	329-347	AGAAAGUUUGCCAAGG	534	GUGCCUUGGCAAACUUU	535
		CACTT		CUTT
330	330-348	GAAAGUUUGCCAAGGC	536	CGUGCCUUGGCAAACUU	537
		ACGTT		UCTT
332	332-350	AAGUUUGCCAAGGCAC	538	CUCGUGCCUUGGCAAAC	539
		GAGTT		UUTT
333	333-351	AGUUUGCCAAGGCACG	540	ACUCGUGCCUUGGCAAA	541
		AGUTT		CUTT
334	334-352	GUUUGCCAAGGCACGA	542	UACUCGUGCCUUGGCAA	543
		GUATT		ACTT
335	335-353	UUUGCCAAGGCACGAG	544	UUACUCGUGCCUUGGCA	545
		UAATT		AATT
336	336-354	UUGCCAAGGCACGAGU	546	GUUACUCGUGCCUUGGC	547
		AACTT		AATT
337	337-355	UGCCAAGGCACGAGUA	548	UGUUACUCGUGCCUUGG	549
		ACATT		CATT
338	338-356	GCCAAGGCACGAGUAA	550	UUGUUACUCGUGCCUUG	551
		CAATT		GCTT
361	361-379	ACGCAGUUGGGCACUU	552	CAAAAGUGCCCAACUGC	553
		UUGTT		GUTT
362	362-380	CGCAGUUGGGCACUUU	554	UCAAAAGUGCCCAACUG	555
		UGATT		CGTT
363	363-381	GCAGUUGGGCACUUUU	556	UUCAAAAGUGCCCAACU	557
		GAATT		GCTT
364	364-382	CAGUUGGGCACUUUUG	558	CUUCAAAAGUGCCCAAC	559
		AAGTT		UGTT
365	365-383	AGUUGGGCACUUUUGA	560	UCUUCAAAAGUGCCCAA	561
		AGATT		CUTT
366	366-384	GUUGGGCACUUUUGAA	562	AUCUUCAAAAGUGCCCA	563
		GAUTT		ACTT
367	367-385	UUGGGCACUUUUGAAG	564	GAUCUUCAAAAGUGCCC	565
		AUCTT		AATT
368	368-386	UGGGCACUUUUGAAGA	566	UGAUCUUCAAAAGUGCC	567
		UCATT		CATT
369	369-387	GGGCACUUUUGAAGAU	568	AUGAUCUUCAAAAGUGC	569
		CAUTT		CCTT
377	377-395	UUGAAGAUCAUUUUCU	570	CUGAGAAAAUGAUCUUC	571
		CAGTT		AATT
379	379-397	GAAGAUCAUUUUCUCA	572	GGCUGAGAAAAUGAUCU	573
		GCCTT		UCTT
380	380-398	AAGAUCAUUUUCUCAG	574	AGGCUGAGAAAAUGAUC	575
		CCUTT		UUTT
385	385-403	CAUUUUCUCAGCCUCC	576	UCUGGAGGCUGAGAAAA	577
		AGATT		UGTT
394	394-412	AGCCUCCAGAGGAUGU	578	UGAACAUCCUCUGGAGG	579
		UCATT		CUTT
396	396-414	CCUCCAGAGGAUGUUC	580	AUUGAACAUCCUCUGGA	581
		AAUTT		GGTT
397	397-415	CUCCAGAGGAUGUUCA	582	UAUUGAACAUCCUCUGG	583
		AUATT		AGTT
401	401-419	AGAGGAUGUUCAAUAA	584	CAGUUAUUGAACAUCCU	585
		CUGTT		CUTT
403	403-421	AGGAUGUUCAAUAACU	586	CACAGUUAUUGAACAUC	587
		GUGTT		CUTT
407	407-425	UGUUCAAUAACUGUGA	588	ACCUCACAGUUAUUGAA	589
		GGUTT		CATT
409	409-427	UUCAAUAACUGUGAGG	590	CCACCUCACAGUUAUUG	591
		UGGTT		AATT
410	410-428	UCAAUAACUGUGAGGU	592	ACCACCUCACAGUUAUU	593
		GGUTT		GATT
411	411-429	CAAUAACUGUGAGGUG	594	GACCACCUCACAGUUAU	595
		GUCTT		UGTT
412	412-430	AAUAACUGUGAGGUGG	596	GGACCACCUCACAGUUA	597
		UCCTT		UUTT
413	413-431	AUAACUGUGAGGUGGU	598	AGGACCACCUCACAGUU	599
		CCUTT		AUTT
414	414-432	UAACUGUGAGGUGGUC	600	AAGGACCACCUCACAGU	601
		CUUTT		UATT
416	416-434	ACUGUGAGGUGGUCCU	602	CCAAGGACCACCUCACA	603
		UGGTT		GUTT
418	418-436	UGUGAGGUGGUCCUUG	604	UCCCAAGGACCACCUCA	605
		GGATT		CATT
419	419-437	GUGAGGUGGUCCUUGG	606	UUCCCAAGGACCACCUC	607
		GAATT		ACTT
425	425-443	UGGUCCUUGGGAAUUU	608	UCCAAAUUCCCAAGGAC	609
		GGATT		CATT
431	431-449	UUGGGAAUUUGGAAAU	610	GUAAUUUCCAAAUUCCC	611
		UACTT		AATT
432	432-450	UGGGAAUUUGGAAAUU	612	GGUAAUUUCCAAAUUCC	613
		ACCTT		CATT
433	433-451	GGGAAUUUGGAAAUUA	614	AGGUAAUUUCCAAAUUC	615
		CCUTT		CCTT
434	434-452	GGAAUUUGGAAAUUAC	616	UAGGUAAUUUCCAAAUU	617
		CUATT		CCTT
458	458-476	AGAGGAAUUAUGAUCU	618	GAAAGAUCAUAAUUCCU	619
		UUCTT		CUTT
459	459-477	GAGGAAUUAUGAUCUU	620	GGAAAGAUCAUAAUUCC	621
		UCCTT		UCTT
463	463-481	AAUUAUGAUCUUUCCU	622	AGAAGGAAAGAUCAUAA	623
		UCUTT		UUTT
464	464-482	AUUAUGAUCUUUCCUU	624	AAGAAGGAAAGAUCAUA	625
		CUUTT		AUTT
466	466-484	UAUGAUCUUUCCUUCU	626	UUAAGAAGGAAAGAUCA	627
		UAATT		UATT
468	468-486	UGAUCUUUCCUUCUUA	628	CUUUAAGAAGGAAAGAU	629
		AAGTT		CATT
471	471-489	UCUUUCCUUCUUAAAG	630	GGUCUUUAAGAAGGAAA	631
		ACCTT		GATT
476	476-494	CCUUCUUAAAGACCAU	632	UGGAUGGUCUUUAAGAA	633
		CCATT		GGTT
477	477-495	CUUCUUAAAGACCAUC	634	CUGGAUGGUCUUUAAGA	635
		CAGTT		AGTT
479	479-497	UCUUAAAGACCAUCCA	636	UCCUGGAUGGUCUUUAA	637
		GGATT		GATT
481	481-499	UUAAAGACCAUCCAGG	638	CCUCCUGGAUGGUCUUU	639
		AGGTT		AATT
482	482-500	UAAAGACCAUCCAGGA	640	ACCUCCUGGAUGGUCUU	641
		GGUTT		UATT
492	492-510	CCAGGAGGUGGCUGGU	642	AUAACCAGCCACCUCCU	643
		UAUTT		GGTT
493	493-511	CAGGAGGUGGCUGGUU	644	CAUAACCAGCCACCUCC	645
		AUGTT		UGTT
494	494-512	AGGAGGUGGCUGGUUA	646	ACAUAACCAGCCACCUC	647
		UGUTT		CUTT
495	495-513	GGAGGUGGCUGGUUAU	648	GACAUAACCAGCCACCU	649
		GUCTT		CCTT
496	496-514	GAGGUGGCUGGUUAUG	650	GGACAUAACCAGCCACC	651
		UCCTT		UCTT
497	497-515	AGGUGGCUGGUUAUGU	652	AGGACAUAACCAGCCAC	653
		CCUTT		CUTT
499	499-517	GUGGCUGGUUAUGUCC	654	UGAGGACAUAACCAGCC	655
		UCATT		ACTT
520	520-538	GCCCUCAACACAGUGG	656	GCUCCACUGUGUUGAGG	657
		AGCTT		GCTT
542	542-560	UUCCUUUGGAAAACCU	658	UGCAGGUUUUCCAAAGG	659
		GCATT		AATT
543	543-561	UCCUUUGGAAAACCUG	660	CUGCAGGUUUUCCAAAG	661
		CAGTT		GATT
550	550-568	GAAAACCUGCAGAUCA	662	UGAUGAUCUGCAGGUUU	663
		UCATT		UCTT
551	551-569	AAAACCUGCAGAUCAU	664	CUGAUGAUCUGCAGGUU	665
		CAGTT		UUTT
553	553-571	AACCUGCAGAUCAUCA	666	CUCUGAUGAUCUGCAGG	667
		GAGTT		UUTT
556	556-574	CUGCAGAUCAUCAGAG	668	UUCCUCUGAUGAUCUGC	669
		GAATT		AGTT
586	586-604	GAAAAUUCCUAUGCCU	670	CUAAGGCAUAGGAAUUU	671
		UAGTT		UCTT
587	587-605	AAAAUUCCUAUGCCUU	672	GCUAAGGCAUAGGAAUU	673
		AGCTT		UUTT
589	589-607	AAUUCCUAUGCCUUAG	674	CUGCUAAGGCAUAGGAA	675
		CAGTT		UUTT
592	592-610	UCCUAUGCCUUAGCAG	676	AGACUGCUAAGGCAUAG	677
		UCUTT		GATT
593	593-611	CCUAUGCCUUAGCAGU	678	AAGACUGCUAAGGCAUA	679
		CUUTT		GGTT
594	594-612	CUAUGCCUUAGCAGUC	680	UAAGACUGCUAAGGCAU	681
		UUATT		AGTT
596	596-614	AUGCCUUAGCAGUCUU	682	GAUAAGACUGCUAAGGC	683
		AUCTT		AUTT
597	597-615	UGCCUUAGCAGUCUUA	684	AGAUAAGACUGCUAAGG	685
		UCUTT		CATT
598	598-616	GCCUUAGCAGUCUUAU	686	UAGAUAAGACUGCUAAG	687
		CUATT		GCTT
599	599-617	CCUUAGCAGUCUUAUC	688	UUAGAUAAGACUGCUAA	689
		UAATT		GGTT
600	600-618	CUUAGCAGUCUUAUCU	690	GUUAGAUAAGACUGCUA	691
		AACTT		AGTT
601	601-619	UUAGCAGUCUUAUCUA	692	AGUUAGAUAAGACUGCU	693
		ACUTT		AATT
602	602-620	UAGCAGUCUUAUCUAA	694	UAGUUAGAUAAGACUGC	695
		CUATT		UATT
603	603-621	AGCAGUCUUAUCUAAC	696	AUAGUUAGAUAAGACUG	697
		UAUTT		CUTT
604	604-622	GCAGUCUUAUCUAACU	698	CAUAGUUAGAUAAGACU	699
		AUGTT		GCTT
605	605-623	CAGUCUUAUCUAACUA	700	UCAUAGUUAGAUAAGAC	701
		UGATT		UGTT
608	608-626	UCUUAUCUAACUAUGA	702	GCAUCAUAGUUAGAUAA	703
		UGCTT		GATT
609	609-627	CUUAUCUAACUAUGAU	704	UGCAUCAUAGUUAGAUA	705
		GCATT		AGTT
610	610-628	UUAUCUAACUAUGAUG	706	UUGCAUCAUAGUUAGAU	707
		CAATT		AATT
611	611-629	UAUCUAACUAUGAUGC	708	UUUGCAUCAUAGUUAGA	709
		AAATT		UATT
612	612-630	AUCUAACUAUGAUGCA	710	AUUUGCAUCAUAGUUAG	711
		AAUTT		AUTT
613	613-631	UCUAACUAUGAUGCAA	712	UAUUUGCAUCAUAGUUA	713
		AUATT		GATT
614	614-632	CUAACUAUGAUGCAAA	714	UUAUUUGCAUCAUAGUU	715
		UAATT		AGTT
616	616-634	AACUAUGAUGCAAAUA	716	UUUUAUUUGCAUCAUAG	717
		AAATT		UUTT
622	622-640	GAUGCAAAUAAAACCG	718	GUCCGGUUUUAUUUGCA	719
		GACTT		UCTT
623	623-641	AUGCAAAUAAAACCGG	720	AGUCCGGUUUUAUUUGC	721
		ACUTT		AUTT
624	624-642	UGCAAAUAAAACCGGA	722	CAGUCCGGUUUUAUUUG	723
		CUGTT		CATT
626	626-644	CAAAUAAAACCGGACU	724	UUCAGUCCGGUUUUAUU	725
		GAATT		UGTT
627	627-645	AAAUAAAACCGGACUG	726	CUUCAGUCCGGUUUUAU	727
		AAGTT		UUTT
628	628-646	AAUAAAACCGGACUGA	728	CCUUCAGUCCGGUUUUA	729
		AGGTT		UUTT
630	630-648	UAAAACCGGACUGAAG	730	CUCCUUCAGUCCGGUUU	731
		GAGTT		UATT
631	631-649	AAAACCGGACUGAAGG	732	GCUCCUUCAGUCCGGUU	733
		AGCTT		UUTT
632	632-650	AAACCGGACUGAAGGA	734	AGCUCCUUCAGUCCGGU	735
		GCUTT		UUTT
633	633-651	AACCGGACUGAAGGAG	736	CAGCUCCUUCAGUCCGG	737
		CUGTT		UUTT
644	644-662	AGGAGCUGCCCAUGAG	738	UUUCUCAUGGGCAGCUC	739
		AAATT		CUTT
665	665-683	UACAGGAAAUCCUGCA	740	CCAUGCAGGAUUUCCUG	741
		UGGTT		UATT
668	668-686	AGGAAAUCCUGCAUGG	742	GCGCCAUGCAGGAUUUC	743
		CGCTT		CUTT
669	669-687	GGAAAUCCUGCAUGGC	744	GGCGCCAUGCAGGAUUU	745
		GCCTT		CCTT
670	670-688	GAAAUCCUGCAUGGCG	746	CGGCGCCAUGCAGGAUU	747
		CCGTT		UCTT
671	671-689	AAAUCCUGCAUGGCGC	748	ACGGCGCCAUGCAGGAU	749
		CGUTT		UUTT
672	672-690	AAUCCUGCAUGGCGCC	750	CACGGCGCCAUGCAGGA	751
		GUGTT		UUTT
674	674-692	UCCUGCAUGGCGCCGU	752	CGCACGGCGCCAUGCAG	753
		GCGTT		GATT
676	676-694	CUGCAUGGCGCCGUGC	754	ACCGCACGGCGCCAUGC	755
		GGUTT		AGTT
677	677-695	UGCAUGGCGCCGUGCG	756	AACCGCACGGCGCCAUG	757
		GUUTT		CATT
678	678-696	GCAUGGCGCCGUGCGG	758	GAACCGCACGGCGCCAU	759
		UUCTT		GCTT
680	680-698	AUGGCGCCGUGCGGUU	760	CUGAACCGCACGGCGCC	761
		CAGTT		AUTT
681	681-699	UGGCGCCGUGCGGUUC	762	GCUGAACCGCACGGCGC	763
		AGCTT		CATT
682	682-700	GGCGCCGUGCGGUUCA	764	UGCUGAACCGCACGGCG	765
		GCATT		CCTT
683	683-701	GCGCCGUGCGGUUCAG	766	UUGCUGAACCGCACGGC	767
		CAATT		GCTT
684	684-702	CGCCGUGCGGUUCAGC	768	GUUGCUGAACCGCACGG	769
		AACTT		CGTT
685	685-703	GCCGUGCGGUUCAGCA	770	UGUUGCUGAACCGCACG	771
		ACATT		GCTT
686	686-704	CCGUGCGGUUCAGCAA	772	UUGUUGCUGAACCGCAC	773
		CAATT		GGTT
688	688-706	GUGCGGUUCAGCAACA	774	GGUUGUUGCUGAACCGC	775
		ACCTT		ACTT
690	690-708	GCGGUUCAGCAACAAC	776	AGGGUUGUUGCUGAACC	777
		CCUTT		GCTT
692	692-710	GGUUCAGCAACAACCC	778	GCAGGGUUGUUGCUGAA	779
		UGCTT		CCTT
698	698-716	GCAACAACCCUGCCCU	780	CACAGGGCAGGGUUGUU	781
		GUGTT		GCTT
700	700-718	AACAACCCUGCCCUGU	782	UGCACAGGGCAGGGUUG	783
		GCATT		UUTT
719	719-737	ACGUGGAGAGCAUCCA	784	CACUGGAUGCUCUCCAC	785
		GUGTT		GUTT
720	720-738	CGUGGAGAGCAUCCAG	786	CCACUGGAUGCUCUCCA	787
		UGGTT		CGTT
721	721-739	GUGGAGAGCAUCCAGU	788	GCCACUGGAUGCUCUCC	789
		GGCTT		ACTT
724	724-742	GAGAGCAUCCAGUGGC	790	CCCGCCACUGGAUGCUC	791
		GGGTT		UCTT
725	725-743	AGAGCAUCCAGUGGCG	792	UCCCGCCACUGGAUGCU	793
		GGATT		CUTT
726	726-744	GAGCAUCCAGUGGCGG	794	GUCCCGCCACUGGAUGC	795
		GACTT		UCTT
733	733-751	CAGUGGCGGGACAUAG	796	UGACUAUGUCCCGCCAC	797
		UCATT		UGTT
734	734-752	AGUGGCGGGACAUAGU	798	CUGACUAUGUCCCGCCA	799
		CAGTT		CUTT
736	736-754	UGGCGGGACAUAGUCA	800	UGCUGACUAUGUCCCGC	801
		GCATT		CATT
737	737-755	GGCGGGACAUAGUCAG	802	CUGCUGACUAUGUCCCG	803
		CAGTT		CCTT
763	763-781	CUCAGCAACAUGUCGA	804	CCAUCGACAUGUUGCUG	805
		UGGTT		AGTT
765	765-783	CAGCAACAUGUCGAUG	806	GUCCAUCGACAUGUUGC	807
		GACTT		UGTT
766	766-784	AGCAACAUGUCGAUGG	808	AGUCCAUCGACAUGUUG	809
		ACUTT		CUTT
767	767-785	GCAACAUGUCGAUGGA	810	AAGUCCAUCGACAUGUU	811
		CUUTT		GCTT
769	769-787	AACAUGUCGAUGGACU	812	GGAAGUCCAUCGACAUG	813
		UCCTT		UUTT
770	770-788	ACAUGUCGAUGGACUU	814	UGGAAGUCCAUCGACAU	815
		CCATT		GUTT
771	771-789	CAUGUCGAUGGACUUC	816	CUGGAAGUCCAUCGACA	817
		CAGTT		UGTT
772	772-790	AUGUCGAUGGACUUCC	818	UCUGGAAGUCCAUCGAC	819
		AGATT		AUTT
775	775-793	UCGAUGGACUUCCAGA	820	GGUUCUGGAAGUCCAUC	821
		ACCTT		GATT
789	789-807	GAACCACCUGGGCAGC	822	GCAGCUGCCCAGGUGGU	823
		UGCTT		UCTT
798	798-816	GGGCAGCUGCCAAAAG	824	ACACUUUUGGCAGCUGC	825
		UGUTT		CCTT
800	800-818	GCAGCUGCCAAAAGUG	826	UCACACUUUUGGCAGCU	827
		UGATT		GCTT
805	805-823	UGCCAAAAGUGUGAUC	828	UUGGAUCACACUUUUGG	829
		CAATT		CATT
806	806-824	GCCAAAAGUGUGAUCC	830	CUUGGAUCACACUUUUG	831
		AAGTT		GCTT
807	807-825	CCAAAAGUGUGAUCCA	832	GCUUGGAUCACACUUUU	833
		AGCTT		GGTT
810	810-828	AAAGUGUGAUCCAAGC	834	ACAGCUUGGAUCACACU	835
		UGUTT		UUTT
814	814-832	UGUGAUCCAAGCUGUC	836	UGGGACAGCUUGGAUCA	837
		CCATT		CATT
815	815-833	GUGAUCCAAGCUGUCC	838	UUGGGACAGCUUGGAUC	839
		CAATT		ACTT
817	817-835	GAUCCAAGCUGUCCCA	840	CAUUGGGACAGCUUGGA	841
		AUGTT		UCTT
818	818-836	AUCCAAGCUGUCCCAA	842	CCAUUGGGACAGCUUGG	843
		UGGTT		AUTT
819	819-837	UCCAAGCUGUCCCAAU	844	CCCAUUGGGACAGCUUG	845
		GGGTT		GATT
820	820-838	CCAAGCUGUCCCAAUG	846	UCCCAUUGGGACAGCUU	847
		GGATT		GGTT
821	821-839	CAAGCUGUCCCAAUGG	848	CUCCCAUUGGGACAGCU	849
		GAGTT		UGTT
823	823-841	AGCUGUCCCAAUGGGA	850	AGCUCCCAUUGGGACAG	851
		GCUTT		CUTT
826	826-844	UGUCCCAAUGGGAGCU	852	AGCAGCUCCCAUUGGGA	853
		GCUTT		CATT
847	847-865	GGUGCAGGAGAGGAGA	854	AGUUCUCCUCUCCUGCA	855
		ACUTT		CCTT
871	871-889	AAACUGACCAAAAUCA	856	AGAUGAUUUUGGUCAGU	857
		UCUTT		UUTT
872	872-890	AACUGACCAAAAUCAU	858	CAGAUGAUUUUGGUCAG	859
		CUGTT		UUTT
873	873-891	ACUGACCAAAAUCAUC	860	ACAGAUGAUUUUGGUCA	861
		UGUTT		GUTT
877	877-895	ACCAAAAUCAUCUGUG	862	GGGCACAGAUGAUUUUG	863
		CCCTT		GUTT
878	878-896	CCAAAAUCAUCUGUGC	864	UGGGCACAGAUGAUUUU	865
		CCATT		GGTT
881	881-899	AAAUCAUCUGUGCCCA	866	UGCUGGGCACAGAUGAU	867
		GCATT		UUTT
890	890-908	GUGCCCAGCAGUGCUC	868	CCGGAGCACUGCUGGGC	869
		CGGTT		ACTT
892	892-910	GCCCAGCAGUGCUCCG	870	GCCCGGAGCACUGCUGG	871
		GGCTT		GCTT
929	929-947	CCAGUGACUGCUGCCA	872	UUGUGGCAGCAGUCACU	873
		CAATT		GGTT
930	930-948	CAGUGACUGCUGCCAC	874	GUUGUGGCAGCAGUCAC	875
		AACTT		UGTT
979	979-997	GAGAGCGACUGCCUGG	876	AGACCAGGCAGUCGCUC	877
		UCUTT		UCTT
980	980-998	AGAGCGACUGCCUGGU	878	CAGACCAGGCAGUCGCU	879
		CUGTT		CUTT
981	981-999	GAGCGACUGCCUGGUC	880	GCAGACCAGGCAGUCGC	881
		UGCTT		UCTT
982	982-1000	AGCGACUGCCUGGUCU	882	GGCAGACCAGGCAGUCG	883
		GCCTT		CUTT
983	983-1001	GCGACUGCCUGGUCUG	884	CGGCAGACCAGGCAGUC	885
		CCGTT		GCTT
984	984-1002	CGACUGCCUGGUCUGC	886	GCGGCAGACCAGGCAGU	887
		CGCTT		CGTT
989	989-1007	GCCUGGUCUGCCGCAA	888	AAUUUGCGGCAGACCAG	889
		AUUTT		GCTT
990	990-1008	CCUGGUCUGCCGCAAA	890	GAAUUUGCGGCAGACCA	891
		UUCTT		GGTT
991	991-1009	CUGGUCUGCCGCAAAU	892	GGAAUUUGCGGCAGACC	893
		UCCTT		AGTT
992	992-1010	UGGUCUGCCGCAAAUU	894	CGGAAUUUGCGGCAGAC	895
		CCGTT		CATT
994	994-1012	GUCUGCCGCAAAUUCC	896	CUCGGAAUUUGCGGCAG	897
		GAGTT		ACTT
995	995-1013	UCUGCCGCAAAUUCCG	898	UCUCGGAAUUUGCGGCA	899
		AGATT		GATT
996	996-1014	CUGCCGCAAAUUCCGA	900	GUCUCGGAAUUUGCGGC	901
		GACTT		AGTT
997	997-1015	UGCCGCAAAUUCCGAG	902	CGUCUCGGAAUUUGCGG	903
		ACGTT		CATT
999	999-1017	CCGCAAAUUCCGAGAC	904	UUCGUCUCGGAAUUUGC	905
		GAATT		GGTT
1004	1004-1022	AAUUCCGAGACGAAGC	906	GUGGCUUCGUCUCGGAA	907
		CACTT		UUTT
1005	1005-1023	AUUCCGAGACGAAGCC	908	CGUGGCUUCGUCUCGGA	909
		ACGTT		AUTT
1006	1006-1024	UUCCGAGACGAAGCCA	910	ACGUGGCUUCGUCUCGG	911
		CGUTT		AATT
1007	1007-1025	UCCGAGACGAAGCCAC	912	CACGUGGCUUCGUCUCG	913
		GUGTT		GATT
1008	1008-1026	CCGAGACGAAGCCACG	914	GCACGUGGCUUCGUCUC	915
		UGCTT		GGTT
1010	1010-1028	GAGACGAAGCCACGUG	916	UUGCACGUGGCUUCGUC	917
		CAATT		UCTT
1013	1013-1031	ACGAAGCCACGUGCAA	918	UCCUUGCACGUGGCUUC	919
		GGATT		GUTT
1014	1014-1032	CGAAGCCACGUGCAAG	920	GUCCUUGCACGUGGCUU	921
		GACTT		CGTT
1015	1015-1033	GAAGCCACGUGCAAGG	922	UGUCCUUGCACGUGGCU	923
		ACATT		UCTT
1016	1016-1034	AAGCCACGUGCAAGGA	924	GUGUCCUUGCACGUGGC	925
		CACTT		UUTT
1040	1040-1058	CCCCACUCAUGCUCUA	926	UUGUAGAGCAUGAGUGG	927
		CAATT		GGTT
1042	1042-1060	CCACUCAUGCUCUACA	928	GGUUGUAGAGCAUGAGU	929
		ACCTT		GGTT
1044	1044-1062	ACUCAUGCUCUACAAC	930	GGGGUUGUAGAGCAUGA	931
		CCCTT		GUTT
1047	1047-1065	CAUGCUCUACAACCCC	932	GGUGGGGUUGUAGAGCA	933
		ACCTT		UGTT
1071	1071-1089	CCAGAUGGAUGUGAAC	934	GGGGUUCACAUCCAUCU	935
		CCCTT		GGTT
1073	1073-1091	AGAUGGAUGUGAACCC	936	UCGGGGUUCACAUCCAU	937
		CGATT		CUTT
1074	1074-1092	GAUGGAUGUGAACCCC	938	CUCGGGGUUCACAUCCA	939
		GAGTT		UCTT
1075	1075-1093	AUGGAUGUGAACCCCG	940	CCUCGGGGUUCACAUCC	941
		AGGTT		AUTT
1077	1077-1095	GGAUGUGAACCCCGAG	942	GCCCUCGGGGUUCACAU	943
		GGCTT		CCTT
1078	1078-1096	GAUGUGAACCCCGAGG	944	UGCCCUCGGGGUUCACA	945
		GCATT		UCTT
1080	1080-1098	UGUGAACCCCGAGGGC	946	UUUGCCCUCGGGGUUCA	947
		AAATT		CATT
1084	1084-1102	AACCCCGAGGGCAAAU	948	UGUAUUUGCCCUCGGGG	949
		ACATT		UUTT
1085	1085-1103	ACCCCGAGGGCAAAUA	950	CUGUAUUUGCCCUCGGG	951
		CAGTT		GUTT
1087	1087-1105	CCCGAGGGCAAAUACA	952	AGCUGUAUUUGCCCUCG	953
		GCUTT		GGTT
1088	1088-1106	CCGAGGGCAAAUACAG	954	AAGCUGUAUUUGCCCUC	955
		CUUTT		GGTT
1089	1089-1107	CGAGGGCAAAUACAGC	956	AAAGCUGUAUUUGCCCU	957
		UUUTT		CGTT
1096	1096-1114	AAAUACAGCUUUGGUG	958	UGGCACCAAAGCUGUAU	959
		CCATT		UUTT
1097	1097-1115	AAUACAGCUUUGGUGC	960	GUGGCACCAAAGCUGUA	961
		CACTT		UUTT
1098	1098-1116	AUACAGCUUUGGUGCC	962	GGUGGCACCAAAGCUGU	963
		ACCTT		AUTT
1104	1104-1122	CUUUGGUGCCACCUGC	964	CACGCAGGUGGCACCAA	965
		GUGTT		AGTT
1106	1106-1124	UUGGUGCCACCUGCGU	966	UUCACGCAGGUGGCACC	967
		GAATT		AATT
1112	1112-1130	CCACCUGCGUGAAGAA	968	CACUUCUUCACGCAGGU	969
		GUGTT		GGTT
1116	1116-1134	CUGCGUGAAGAAGUGU	970	GGGACACUUCUUCACGC	971
		CCCTT		AGTT
1117	1117-1135	UGCGUGAAGAAGUGUC	972	GGGGACACUUCUUCACG	973
		CCCTT		CATT
1118	1118-1136	GCGUGAAGAAGUGUCC	974	CGGGGACACUUCUUCAC	975
		CCGTT		GCTT
1119	1119-1137	CGUGAAGAAGUGUCCC	976	ACGGGGACACUUCUUCA	977
		CGUTT		CGTT
1120	1120-1138	GUGAAGAAGUGUCCCC	978	UACGGGGACACUUCUUC	979
		GUATT		ACTT
1121	1121-1139	UGAAGAAGUGUCCCCG	980	UUACGGGGACACUUCUU	981
		UAATT		CATT
1122	1122-1140	GAAGAAGUGUCCCCGU	982	AUUACGGGGACACUUCU	983
		AAUTT		UCTT
1123	1123-1141	AAGAAGUGUCCCCGUA	984	AAUUACGGGGACACUUC	985
		AUUTT		UUTT
1124	1124-1142	AGAAGUGUCCCCGUAA	986	UAAUUACGGGGACACUU	987
		UUATT		CUTT
1125	1125-1143	GAAGUGUCCCCGUAAU	988	AUAAUUACGGGGACACU	989
		UAUTT		UCTT
1126	1126-1144	AAGUGUCCCCGUAAUU	990	CAUAAUUACGGGGACAC	991
		AUGTT		UUTT
1127	1127-1145	AGUGUCCCCGUAAUUA	992	ACAUAAUUACGGGGACA	993
		UGUTT		CUTT
1128	1128-1146	GUGUCCCCGUAAUUAU	994	CACAUAAUUACGGGGAC	995
		GUGTT		ACTT
1129	1129-1147	UGUCCCCGUAAUUAUG	996	CCACAUAAUUACGGGGA	997
		UGGTT		CATT
1130	1130-1148	GUCCCCGUAAUUAUGU	998	ACCACAUAAUUACGGGG	999
		GGUTT		ACTT
1132	1132-1150	CCCCGUAAUUAUGUGG	1000	UCACCACAUAAUUACGG	1001
		UGATT		GGTT
1134	1134-1152	CCGUAAUUAUGUGGUG	1002	UGUCACCACAUAAUUAC	1003
		ACATT		GGTT
1136	1136-1154	GUAAUUAUGUGGUGAC	1004	UCUGUCACCACAUAAUU	1005
		AGATT		ACTT
1137	1137-1155	UAAUUAUGUGGUGACA	1006	AUCUGUCACCACAUAAU	1007
		GAUTT		UATT
1138	1138-1156	AAUUAUGUGGUGACAG	1008	GAUCUGUCACCACAUAA	1009
		AUCTT		UUTT
1139	1139-1157	AUUAUGUGGUGACAGA	1010	UGAUCUGUCACCACAUA	1011
		UCATT		AUTT
1140	1140-1158	UUAUGUGGUGACAGAU	1012	GUGAUCUGUCACCACAU	1013
		CACTT		AATT
1142	1142-1160	AUGUGGUGACAGAUCA	1014	CCGUGAUCUGUCACCAC	1015
		CGGTT		AUTT
1145	1145-1163	UGGUGACAGAUCACGG	1016	GAGCCGUGAUCUGUCAC	1017
		CUCTT		CATT
1147	1147-1165	GUGACAGAUCACGGCU	1018	ACGAGCCGUGAUCUGUC	1019
		CGUTT		ACTT
1148	1148-1166	UGACAGAUCACGGCUC	1020	CACGAGCCGUGAUCUGU	1021
		GUGTT		CATT
1149	1149-1167	GACAGAUCACGGCUCG	1022	GCACGAGCCGUGAUCUG	1023
		UGCTT		UCTT
1150	1150-1168	ACAGAUCACGGCUCGU	1024	CGCACGAGCCGUGAUCU	1025
		GCGTT		GUTT
1151	1151-1169	CAGAUCACGGCUCGUG	1026	ACGCACGAGCCGUGAUC	1027
		CGUTT		UGTT
1152	1152-1170	AGAUCACGGCUCGUGC	1028	GACGCACGAGCCGUGAU	1029
		GUCTT		CUTT
1153	1153-1171	GAUCACGGCUCGUGCG	1030	GGACGCACGAGCCGUGA	1031
		UCCTT		UCTT
1154	1154-1172	AUCACGGCUCGUGCGU	1032	CGGACGCACGAGCCGUG	1033
		CCGTT		AUTT
1155	1155-1173	UCACGGCUCGUGCGUC	1034	UCGGACGCACGAGCCGU	1035
		CGATT		GATT
1156	1156-1174	CACGGCUCGUGCGUCC	1036	CUCGGACGCACGAGCCG	1037
		GAGTT		UGTT
1157	1157-1175	ACGGCUCGUGCGUCCG	1038	GCUCGGACGCACGAGCC	1039
		AGCTT		GUTT
1160	1160-1178	GCUCGUGCGUCCGAGC	1040	CAGGCUCGGACGCACGA	1041
		CUGTT		GCTT
1200	1200-1218	GGAGGAAGACGGCGUC	1042	GCGGACGCCGUCUUCCU	1043
		CGCTT		CCTT
1201	1201-1219	GAGGAAGACGGCGUCC	1044	UGCGGACGCCGUCUUCC	1045
		GCATT		UCTT
1203	1203-1221	GGAAGACGGCGUCCGC	1046	CUUGCGGACGCCGUCUU	1047
		AAGTT		CCTT
1204	1204-1222	GAAGACGGCGUCCGCA	1048	ACUUGCGGACGCCGUCU	1049
		AGUTT		UCTT
1205	1205-1223	AAGACGGCGUCCGCAA	1050	CACUUGCGGACGCCGUC	1051
		GUGTT		UUTT
1207	1207-1225	GACGGCGUCCGCAAGU	1052	UACACUUGCGGACGCCG	1053
		GUATT		UCTT
1208	1208-1226	ACGGCGUCCGCAAGUG	1054	UUACACUUGCGGACGCC	1055
		UAATT		GUTT
1211	1211-1229	GCGUCCGCAAGUGUAA	1056	UUCUUACACUUGCGGAC	1057
		GAATT		GCTT
1212	1212-1230	CGUCCGCAAGUGUAAG	1058	CUUCUUACACUUGCGGA	1059
		AAGTT		CGTT
1213	1213-1231	GUCCGCAAGUGUAAGA	1060	ACUUCUUACACUUGCGG	1061
		AGUTT		ACTT
1214	1214-1232	UCCGCAAGUGUAAGAA	1062	CACUUCUUACACUUGCG	1063
		GUGTT		GATT
1215	1215-1233	CCGCAAGUGUAAGAAG	1064	GCACUUCUUACACUUGC	1065
		UGCTT		GGTT
1216	1216-1234	CGCAAGUGUAAGAAGU	1066	CGCACUUCUUACACUUG	1067
		GCGTT		CGTT
1217	1217-1235	GCAAGUGUAAGAAGUG	1068	UCGCACUUCUUACACUU	1069
		CGATT		GCTT
1219	1219-1237	AAGUGUAAGAAGUGCG	1070	CUUCGCACUUCUUACAC	1071
		AAGTT		UUTT
1220	1220-1238	AGUGUAAGAAGUGCGA	1072	CCUUCGCACUUCUUACA	1073
		AGGTT		CUTT
1221	1221-1239	GUGUAAGAAGUGCGAA	1074	CCCUUCGCACUUCUUAC	1075
		GGGTT		ACTT
1222	1222-1240	UGUAAGAAGUGCGAAG	1076	GCCCUUCGCACUUCUUA	1077
		GGCTT		CATT
1223	1223-1241	GUAAGAAGUGCGAAGG	1078	GGCCCUUCGCACUUCUU	1079
		GCCTT		ACTT
1224	1224-1242	UAAGAAGUGCGAAGGG	1080	AGGCCCUUCGCACUUCU	1081
		CCUTT		UATT
1225	1225-1243	AAGAAGUGCGAAGGGC	1082	AAGGCCCUUCGCACUUC	1083
		CUUTT		UUTT
1226	1226-1244	AGAAGUGCGAAGGGCC	1084	CAAGGCCCUUCGCACUU	1085
		UUGTT		CUTT
1229	1229-1247	AGUGCGAAGGGCCUUG	1086	CGGCAAGGCCCUUCGCA	1087
		CCGTT		CUTT
1230	1230-1248	GUGCGAAGGGCCUUGC	1088	GCGGCAAGGCCCUUCGC	1089
		CGCTT		ACTT
1231	1231-1249	UGCGAAGGGCCUUGCC	1090	UGCGGCAAGGCCCUUCG	1091
		GCATT		CATT
1232	1232-1250	GCGAAGGGCCUUGCCG	1092	UUGCGGCAAGGCCCUUC	1093
		CAATT		GCTT
1233	1233-1251	CGAAGGGCCUUGCCGC	1094	UUUGCGGCAAGGCCCUU	1095
		AAATT		CGTT
1235	1235-1253	AAGGGCCUUGCCGCAA	1096	ACUUUGCGGCAAGGCCC	1097
		AGUTT		UUTT
1236	1236-1254	AGGGCCUUGCCGCAAA	1098	CACUUUGCGGCAAGGCC	1099
		GUGTT		CUTT
1237	1237-1255	GGGCCUUGCCGCAAAG	1100	ACACUUUGCGGCAAGGC	1101
		UGUTT		CCTT
1238	1238-1256	GGCCUUGCCGCAAAGU	1102	CACACUUUGCGGCAAGG	1103
		GUGTT		CCTT
1239	1239-1257	GCCUUGCCGCAAAGUG	1104	ACACACUUUGCGGCAAG	1105
		UGUTT		GCTT
1241	1241-1259	CUUGCCGCAAAGUGUG	1106	UUACACACUUUGCGGCA	1107
		UAATT		AGTT
1261	1261-1279	GGAAUAGGUAUUGGUG	1108	AUUCACCAAUACCUAUU	1109
		AAUTT		CCTT
1262	1262-1280	GAAUAGGUAUUGGUGA	1110	AAUUCACCAAUACCUAU	1111
		AUUTT		UCTT
1263	1263-1281	AAUAGGUAUUGGUGAA	1112	AAAUUCACCAAUACCUA	1113
		UUUTT		UUTT
1264	1264-1282	AUAGGUAUUGGUGAAU	1114	UAAAUUCACCAAUACCU	1115
		UUATT		AUTT
1266	1266-1284	AGGUAUUGGUGAAUUU	1116	UUUAAAUUCACCAAUAC	1117
		AAATT		CUTT
1267	1267-1285	GGUAUUGGUGAAUUUA	1118	CUUUAAAUUCACCAAUA	1119
		AAGTT		CCTT
1289	1289-1307	CACUCUCCAUAAAUGC	1120	GUAGCAUUUAUGGAGAG	1121
		UACTT		UGTT
1313	1313-1331	UUAAACACUUCAAAAA	1122	CAGUUUUUGAAGUGUUU	1123
		CUGTT		AATT
1320	1320-1338	CUUCAAAAACUGCACC	1124	GGAGGUGCAGUUUUUGA	1125
		UCCTT		AGTT
1321	1321-1339	UUCAAAAACUGCACCU	1126	UGGAGGUGCAGUUUUUG	1127
		CCATT		AATT
1322	1322-1340	UCAAAAACUGCACCUC	1128	AUGGAGGUGCAGUUUUU	1129
		CAUTT		GATT
1323	1323-1341	CAAAAACUGCACCUCC	1130	GAUGGAGGUGCAGUUUU	1131
		AUCTT		UGTT
1324	1324-1342	AAAAACUGCACCUCCA	1132	UGAUGGAGGUGCAGUUU	1133
		UCATT		UUTT
1328	1328-1346	ACUGCACCUCCAUCAG	1134	CCACUGAUGGAGGUGCA	1135
		UGGTT		GUTT
1332	1332-1350	CACCUCCAUCAGUGGC	1136	AUCGCCACUGAUGGAGG	1137
		GAUTT		UGTT
1333	1333-1351	ACCUCCAUCAGUGGCG	1138	GAUCGCCACUGAUGGAG	1139
		AUCTT		GUTT
1335	1335-1353	CUCCAUCAGUGGCGAU	1140	GAGAUCGCCACUGAUGG	1141
		CUCTT		AGTT
1338	1338-1356	CAUCAGUGGCGAUCUC	1142	GUGGAGAUCGCCACUGA	1143
		CACTT		UGTT
1344	1344-1362	UGGCGAUCUCCACAUC	1144	CAGGAUGUGGAGAUCGC	1145
		CUGTT		CATT
1345	1345-1363	GGCGAUCUCCACAUCC	1146	GCAGGAUGUGGAGAUCG	1147
		UGCTT		CCTT
1346	1346-1364	GCGAUCUCCACAUCCU	1148	GGCAGGAUGUGGAGAUC	1149
		GCCTT		GCTT
1347	1347-1365	CGAUCUCCACAUCCUG	1150	CGGCAGGAUGUGGAGAU	1151
		CCGTT		CGTT
1348	1348-1366	GAUCUCCACAUCCUGC	1152	CCGGCAGGAUGUGGAGA	1153
		CGGTT		UCTT
1353	1353-1371	CCACAUCCUGCCGGUG	1154	UGCCACCGGCAGGAUGU	1155
		GCATT		GGTT
1354	1354-1372	CACAUCCUGCCGGUGG	1156	AUGCCACCGGCAGGAUG	1157
		CAUTT		UGTT
1355	1355-1373	ACAUCCUGCCGGUGGC	1158	AAUGCCACCGGCAGGAU	1159
		AUUTT		GUTT
1357	1357-1375	AUCCUGCCGGUGGCAU	1160	UAAAUGCCACCGGCAGG	1161
		UUATT		AUTT
1360	1360-1378	CUGCCGGUGGCAUUUA	1162	CCCUAAAUGCCACCGGC	1163
		GGGTT		AGTT
1361	1361-1379	UGCCGGUGGCAUUUAG	1164	CCCCUAAAUGCCACCGG	1165
		GGGTT		CATT
1362	1362-1380	GCCGGUGGCAUUUAGG	1166	ACCCCUAAAUGCCACCG	1167
		GGUTT		GCTT
1363	1363-1381	CCGGUGGCAUUUAGGG	1168	CACCCCUAAAUGCCACC	1169
		GUGTT		GGTT
1366	1366-1384	GUGGCAUUUAGGGGUG	1170	AGUCACCCCUAAAUGCC	1171
		ACUTT		ACTT
1369	1369-1387	GCAUUUAGGGGUGACU	1172	AGGAGUCACCCCUAAAU	1173
		CCUTT		GCTT
1370	1370-1388	CAUUUAGGGGUGACUC	1174	AAGGAGUCACCCCUAAA	1175
		CUUTT		UGTT
1371	1371-1389	AUUUAGGGGUGACUCC	1176	GAAGGAGUCACCCCUAA	1177
		UUCTT		AUTT
1372	1372-1390	UUUAGGGGUGACUCCU	1178	UGAAGGAGUCACCCCUA	1179
		UCATT		AATT
1373	1373-1391	UUAGGGGUGACUCCUU	1180	GUGAAGGAGUCACCCCU	1181
		CACTT		AATT
1374	1374-1392	UAGGGGUGACUCCUUC	1182	UGUGAAGGAGUCACCCC	1183
		ACATT		UATT
1404	1404-1422	UCUGGAUCCACAGGAA	1184	CAGUUCCUGUGGAUCCA	1185
		CUGTT		GATT
1408	1408-1426	GAUCCACAGGAACUGG	1186	UAUCCAGUUCCUGUGGA	1187
		AUATT		UCTT
1409	1409-1427	AUCCACAGGAACUGGA	1188	AUAUCCAGUUCCUGUGG	1189
		UAUTT		AUTT
1411	1411-1429	CCACAGGAACUGGAUA	1190	GAAUAUCCAGUUCCUGU	1191
		UUCTT		GGTT
1412	1412-1430	CACAGGAACUGGAUAU	1192	AGAAUAUCCAGUUCCUG	1193
		UCUTT		UGTT
1419	1419-1437	ACUGGAUAUUCUGAAA	1194	GGUUUUCAGAAUAUCCA	1195
		ACCTT		GUTT
1426	1426-1444	AUUCUGAAAACCGUAA	1196	CCUUUACGGUUUUCAGA	1197
		AGGTT		AUTT
1427	1427-1445	UUCUGAAAACCGUAAA	1198	UCCUUUACGGUUUUCAG	1199
		GGATT		AATT
1430	1430-1448	UGAAAACCGUAAAGGA	1200	AUUUCCUUUACGGUUUU	1201
		AAUTT		CATT
1431	1431-1449	GAAAACCGUAAAGGAA	1202	GAUUUCCUUUACGGUUU	1203
		AUCTT		UCTT

TABLE 6
EGFR siRNA Sequences with Chemical Modifications
	Sequence		SEQ		SEQ
	position in	sense strand sequence	ID	antisense strand sequence	ID
hs Id #	NM_005228.3	(5′-3′)	NO:	(5′-3′)	NO:
68	68-86	cgGfcCfgGfaGfuCfcCfgAfg	1204	UfAfgCfuCfgGfgAfcUfcCfgGf	1205
		CfuAfdTsdT		cCfgdTsdT
71	71-89	ccGfgAfgUfcCfcGfaGfcUfa	1206	GfGfcUfaGfcUfcGfgGfaCfuCf	1207
		GfcCfdTsdT		cGfgdTsdT
72	72-90	cgGfaGfuCfcCfgAfgCfuAfg	1208	GfGfgCfuAfgCfuCfgGfgAfcUf	1209
		CfcCfdTsdT		cCfgdTsdT
73	73-91	ggAfgUfcCfcGfaGfcUfaGfc	1210	GfGfgGfcUfaGfcUfcGfgGfaCf	1211
		CfcCfdTsdT		uCfcdTsdT
74	74-92	gaGfuCfcCfgAfgCfuAfgCfc	1212	CfGfgGfgCfuAfgCfuCfgGfgAf	1213
		CfcGfdTsdT		cUfcdTsdT
75	75-93	agUfcCfcGfaGfcUfaGfcCfcC	1214	CfCfgGfgGfcUfaGfcUfcGfgGf	1215
		fgGfdTsdT		aCfudTsdT
76	76-94	guCfcCfgAfgCfuAfgCfcCfc	1216	GfCfcGfgGfgCfuAfgCfuCfgGf	1217
		GfgCfdTsdT		gAfcdTsdT
78	78-96	ccCfgAfgCfuAfgCfcCfcGfg	1218	CfCfgCfcGfgGfgCfuAfgCfuCf	1219
		CfgGfdTsdT		gGfgdTsdT
114	114-132	ggAfcGfaCfaGfgCfcAfcCfu	1220	AfCfgAfgGfuGfgCfcUfgUfcGf	1221
		CfgUfdTsdT		uCfcdTsdT
115	115-133	gaCfgAfcAfgGfcCfaCfcUfc	1222	GfAfcGfaGfgUfgGfcCfuGfuCf	1223
		GfuCfdTsdT		gUfcdTsdT
116	116-134	acGfaCfaGfgCfcAfcCfuCfg	1224	CfGfaCfgAfgGfuGfgCfcUfgUf	1225
		UfcGfdTsdT		cGfudTsdT
117	117-135	cgAfcAfgGfcCfaCfcUfcGfu	1226	CfCfgAfcGfaGfgUfgGfcCfuGf	1227
		CfgGfdTsdT		uCfgdTsdT
118	118-136	gaCfaGfgCfcAfcCfuCfgUfc	1228	GfCfcGfaCfgAfgGfuGfgCfcUf	1229
		GfgCfdTsdT		gUfcdTsdT
120	120-138	caGfgCfcAfcCfuCfgUfcGfg	1230	AfCfgCfcGfaCfgAfgGfuGfgCf	1231
		CfgUfdTsdT		cUfgdTsdT
121	121-139	agGfcCfaCfcUfcGfuCfgGfc	1232	GfAfcGfcCfgAfcGfaGfgUfgGf	1233
		GfuCfdTsdT		cCfudTsdT
122	122-140	ggCfcAfcCfuCfgUfcGfgCfg	1234	GfGfaCfgCfcGfaCfgAfgGfuGf	1235
		UfcCfdTsdT		gCfcdTsdT
123	123-141	gcCfaCfcUfcGfuCfgGfcGfu	1236	CfGfgAfcGfcCfgAfcGfaGfgUf	1237
		CfcGfdTsdT		gGfcdTsdT
124	124-142	ccAfcCfuCfgUfcGfgCfgUfc	1238	GfCfgGfaCfgCfcGfaCfgAfgGf	1239
		CfgCfdTsdT		uGfgdTsdT
125	125-143	caCfcUfcGfuCfgGfcGfuCfc	1240	GfGfcGfgAfcGfcCfgAfcGfaGf	1241
		GfcCfdTsdT		gUfgdTsdT
126	126-144	acCfuCfgUfcGfgCfgUfcCfg	1242	GfGfgCfgGfaCfgCfcGfaCfgAf	1243
		CfcCfdTsdT		gGfudTsdT
127	127-145	ccUfcGfuCfgGfcGfuCfcGfc	1244	CfGfgGfcGfgAfcGfcCfgAfcGf	1245
		CfcGfdTsdT		aGfgdTsdT
128	128-146	cuCfgUfcGfgCfgUfcCfgCfc	1246	UfCfgGfgCfgGfaCfgCfcGfaCf	1247
		CfgAfdTsdT		gAfgdTsdT
129	129-147	ucGfuCfgGfcGfuCfcGfcCfc	1248	CfUfcGfgGfcGfgAfcGfcCfgAf	1249
		GfaGfdTsdT		cGfadTsdT
130	130-148	cgUfcGfgCfgUfcCfgCfcCfg	1250	AfCfuCfgGfgCfgGfaCfgCfcGf	1251
		AfgUfdTsdT		aCfgdTsdT
131	131-149	guCfgGfcGfuCfcGfcCfcGfa	1252	GfAfcUfcGfgGfcGfgAfcGfcCf	1253
		GfuCfdTsdT		gAfcdTsdT
132	132-150	ucGfgCfgUfcCfgCfcCfgAfg	1254	GfGfaCfuCfgGfgCfgGfaCfgCf	1255
		UfcCfdTsdT		cGfadTsdT
135	135-153	gcGfuCfcGfcCfcGfaGfuCfc	1256	CfGfgGfgAfcUfcGfgGfcGfgAf	1257
		CfcGfdTsdT		cGfcdTsdT
136	136-154	cgUfcCfgCfcCfgAfgUfcCfc	1258	GfCfgGfgGfaCfuCfgGfgCfgGf	1259
		CfgCfdTsdT		aCfgdTsdT
141	141-159	gcCfcGfaGfuCfcCfcGfcCfuC	1260	GfCfgAfgGfcGfgGfgAfcUfcGf	1261
		fgCfdTsdT		gGfcdTsdT
164	164-182	aaCfgCfcAfcAfaCfcAfcCfgC	1262	GfCfgCfgGfuGfgUfuGfuGfgC	1263
		fgCfdTsdT		fgUfudTsdT
165	165-183	acGfcCfaCfaAfcCfaCfcGfcG	1264	UfGfcGfcGfgUfgGfuUfgUfgG	1265
		fcAfdTsdT		fcGfudTsdT
166	166-184	cgCfcAfcAfaCfcAfcCfgCfgC	1266	GfUfgCfgCfgGfuGfgUfuGfuG	1267
		faCfdTsdT		fgCfgdTsdT
168	168-186	ccAfcAfaCfcAfcCfgCfgCfaC	1268	CfCfgUfgCfgCfgGfuGfgUfuGf	1269
		fgGfdTsdT		uGfgdTsdT
169	169-187	caCfaAfcCfaCfcGfcGfcAfcG	1270	GfCfcGfuGfcGfcGfgUfgGfuUf	1271
		fgCfdTsdT		gUfgdTsdT
170	170-188	acAfaCfcAfcCfgCfgCfaCfgG	1272	GfGfcCfgUfgCfgCfgGfuGfgUf	1273
		fcCfdTsdT		uGfudTsdT
247	247-265	auGfcGfaCfcCfuCfcGfgGfa	1274	CfCfgUfcCfcGfgAfgGfgUfcGf	1275
		CfgGfdTsdT		cAfudTsdT
248	248-266	ugCfgAfcCfcUfcCfgGfgAfc	1276	GfCfcGfuCfcCfgGfaGfgGfuCf	1277
		GfgCfdTsdT		gCfadTsdT
249	249-267	gcGfaCfcCfuCfcGfgGfaCfg	1278	GfGfcCfgUfcCfcGfgAfgGfgUf	1279
		GfcCfdTsdT		cGfcdTsdT
251	251-269	gaCfcCfuCfcGfgGfaCfgGfc	1280	CfCfgGfcCfgUfcCfcGfgAfgGf	1281
		CfgGfdTsdT		gUfcdTsdT
252	252-270	acCfcUfcCfgGfgAfcGfgCfc	1282	CfCfcGfgCfcGfuCfcCfgGfaGf	1283
		GfgGfdTsdT		gGfudTsdT
254	254-272	ccUfcCfgGfgAfcGfgCfcGfg	1284	GfCfcCfcGfgCfcGfuCfcCfgGf	1285
		GfgCfdTsdT		aGfgdTsdT
329	329-347	agAfaAfgUfuUfgCfcAfaGfg	1286	GfUfgCfcUfuGfgCfaAfaCfuUf	1287
		CfaCfdTsdT		uCfudTsdT
330	330-348	gaAfaGfuUfuGfcCfaAfgGfc	1288	CfGfuGfcCfuUfgGfcAfaAfcUf	1289
		AfcGfdTsdT		uUfcdTsdT
332	332-350	aaGfuUfuGfcCfaAfgGfcAfc	1290	CfUfcGfuGfcCfuUfgGfcAfaAf	1291
		GfaGfdTsdT		cUfudTsdT
333	333-351	agUfuUfgCfcAfaGfgCfaCfg	1292	AfCfuCfgUfgCfcUfuGfgCfaAf	1293
		AfgUfdTsdT		aCfudTsdT
334	334-352	guUfuGfcCfaAfgGfcAfcGfa	1294	UfAfcUfcGfuGfcCfuUfgGfcAf	1295
		GfuAfdTsdT		aAfcdTsdT
335	335-353	uuUfgCfcAfaGfgCfaCfgAfg	1296	UfUfaCfuCfgUfgCfcUfuGfgCf	1297
		UfaAfdTsdT		aAfadTsdT
336	336-354	uuGfcCfaAfgGfcAfcGfaGfu	1298	GfUfuAfcUfcGfuGfcCfuUfgGf	1299
		AfaCfdTsdT		cAfadTsdT
337	337-355	ugCfcAfaGfgCfaCfgAfgUfa	1300	UfGfuUfaCfuCfgUfgCfcUfuGf	1301
		AfcAfdTsdT		gCfadTsdT
338	338-356	gcCfaAfgGfcAfcGfaGfuAfa	1302	UfUfgUfuAfcUfcGfuGfcCfuUf	1303
		CfaAfdTsdT		gGfcdTsdT
361	361-379	acGfcAfgUfuGfgGfcAfcUfu	1304	CfAfaAfaGfuGfcCfcAfaCfuGf	1305
		UfuGfdTsdT		cGfudTsdT
362	362-380	cgCfaGfuUfgGfgCfaCfuUfu	1306	UfCfaAfaAfgUfgCfcCfaAfcUf	1307
		UfgAfdTsdT		gCfgdTsdT
363	363-381	gcAfgUfuGfgGfcAfcUfuUfu	1308	UfUfcAfaAfaGfuGfcCfcAfaCf	1309
		GfaAfdTsdT		uGfcdTsdT
364	364-382	caGfuUfgGfgCfaCfuUfuUfg	1310	CfUfuCfaAfaAfgUfgCfcCfaAf	1311
		AfaGfdTsdT		cUfgdTsdT
365	365-383	agUfuGfgGfcAfcUfuUfuGfa	1312	UfCfuUfcAfaAfaGfuGfcCfcAf	1313
		AfgAfdTsdT		aCfudTsdT
366	366-384	guUfgGfgCfaCfuUfuUfgAfa	1314	AfUfcUfuCfaAfaAfgUfgCfcCf	1315
		GfaUfdTsdT		aAfcdTsdT
367	367-385	uuGfgGfcAfcUfuUfuGfaAfg	1316	GfAfuCfuUfcAfaAfaGfuGfcCf	1317
		AfuCfdTsdT		cAfadTsdT
368	368-386	ugGfgCfaCfuUfuUfgAfaGfa	1318	UfGfaUfcUfuCfaAfaAfgUfgCf	1319
		UfcAfdTsdT		cCfadTsdT
369	369-387	ggGfcAfcUfuUfuGfaAfgAfu	1320	AfUfgAfuCfuUfcAfaAfaGfuGf	1321
		CfaUfdTsdT		cCfcdTsdT
377	377-395	uuGfaAfgAfuCfaUfuUfuCfu	1322	CfUfgAfgAfaAfaUfgAfuCfuUf	1323
		CfaGfdTsdT		cAfadTsdT
379	379-397	gaAfgAfuCfaUfuUfuCfuCfa	1324	GfGfcUfgAfgAfaAfaUfgAfuCf	1325
		GfcCfdTsdT		uUfcdTsdT
380	380-398	aaGfaUfcAfuUfuUfcUfcAfg	1326	AfGfgCfuGfaGfaAfaAfuGfaUf	1327
		CfcUfdTsdT		cUfudTsdT
385	385-403	caUfuUfuCfuCfaGfcCfuCfc	1328	UfCfuGfgAfgGfcUfgAfgAfaA	1329
		AfgAfdTsdT		faUfgdTsdT
394	394-412	agCfcUfcCfaGfaGfgAfuGfu	1330	UfGfaAfcAfuCfcUfcUfgGfaGf	1331
		UfcAfdTsdT		gCfudTsdT
396	396-414	ccUfcCfaGfaGfgAfuGfuUfc	1332	AfUfuGfaAfcAfuCfcUfcUfgGf	1333
		AfaUfdTsdT		aGfgdTsdT
397	397-415	cuCfcAfgAfgGfaUfgUfuCfa	1334	UfAfuUfgAfaCfaUfcCfuCfuGf	1335
		AfuAfdTsdT		gAfgdTsdT
401	401-419	agAfgGfaUfgUfuCfaAfuAfa	1336	CfAfgUfuAfuUfgAfaCfaUfcCf	1337
		CfuGfdTsdT		uCfudTsdT
403	403-421	agGfaUfgUfuCfaAfuAfaCfu	1338	CfAfcAfgUfuAfuUfgAfaCfaUf	1339
		GfuGfdTsdT		cCfudTsdT
407	407-425	ugUfuCfaAfuAfaCfuGfuGfa	1340	AfCfcUfcAfcAfgUfuAfuUfgAf	1341
		GfgUfdTsdT		aCfadTsdT
409	409-427	uuCfaAfuAfaCfuGfuGfaGfg	1342	CfCfaCfcUfcAfcAfgUfuAfuUf	1343
		UfgGfdTsdT		gAfadTsdT
410	410-428	ucAfaUfaAfcUfgUfgAfgGfu	1344	AfCfcAfcCfuCfaCfaGfuUfaUf	1345
		GfgUfdTsdT		uGfadTsdT
411	411-429	caAfuAfaCfuGfuGfaGfgUfg	1346	GfAfcCfaCfcUfcAfcAfgUfuAf	1347
		GfuCfdTsdT		uUfgdTsdT
412	412-430	aaUfaAfcUfgUfgAfgGfuGfg	1348	GfGfaCfcAfcCfuCfaCfaGfuUf	1349
		UfcCfdTsdT		aUfudTsdT
413	413-431	auAfaCfuGfuGfaGfgUfgGfu	1350	AfGfgAfcCfaCfcUfcAfcAfgUf	1351
		CfcUfdTsdT		uAfudTsdT
414	414-432	uaAfcUfgUfgAfgGfuGfgUfc	1352	AfAfgGfaCfcAfcCfuCfaCfaGf	1353
		CfuUfdTsdT		uUfadTsdT
416	416-434	acUfgUfgAfgGfuGfgUfcCfu	1354	CfCfaAfgGfaCfcAfcCfuCfaCf	1355
		UfgGfdTsdT		aGfudTsdT
418	418-436	ugUfgAfgGfuGfgUfcCfuUfg	1356	UfCfcCfaAfgGfaCfcAfcCfuCf	1357
		GfgAfdTsdT		aCfadTsdT
419	419-437	guGfaGfgUfgGfuCfcUfuGfg	1358	UfUfcCfcAfaGfgAfcCfaCfcUf	1359
		GfaAfdTsdT		cAfcdTsdT
425	425-443	ugGfuCfcUfuGfgGfaAfuUfu	1360	UfCfcAfaAfuUfcCfcAfaGfgAf	1361
		GfgAfdTsdT		cCfadTsdT
431	431-449	uuGfgGfaAfuUfuGfgAfaAfu	1362	GfUfaAfuUfuCfcAfaAfuUfcCf	1363
		UfaCfdTsdT		cAfadTsdT
432	432-450	ugGfgAfaUfuUfgGfaAfaUfu	1364	GfGfuAfaUfuUfcCfaAfaUfuCf	1365
		AfcCfdTsdT		cCfadTsdT
433	433-451	ggGfaAfuUfuGfgAfaAfuUfa	1366	AfGfgUfaAfuUfuCfcAfaAfuUf	1367
		CfcUfdTsdT		cCfcdTsdT
434	434-452	ggAfaUfuUfgGfaAfaUfuAfc	1368	UfAfgGfuAfaUfuUfcCfaAfaUf	1369
		CfuAfdTsdT		uCfcdTsdT
458	458-476	agAfgGfaAfuUfaUfgAfuCfu	1370	GfAfaAfgAfuCfaUfaAfuUfcCf	1371
		UfuCfdTsdT		uCfudTsdT
459	459-477	gaGfgAfaUfuAfuGfaUfcUfu	1372	GfGfaAfaGfaUfcAfuAfaUfuCf	1373
		UfcCfdTsdT		cUfcdTsdT
463	463-481	aaUfuAfuGfaUfcUfuUfcCfu	1374	AfGfaAfgGfaAfaGfaUfcAfuAf	1375
		UfcUfdTsdT		aUfudTsdT
464	464-482	auUfaUfgAfuCfuUfuCfcUfu	1376	AfAfgAfaGfgAfaAfgAfuCfaUf	1377
		CfuUfdTsdT		aAfudTsdT
466	466-484	uaUfgAfuCfuUfuCfcUfuCfu	1378	UfUfaAfgAfaGfgAfaAfgAfuCf	1379
		UfaAfdTsdT		aUfadTsdT
468	468-486	ugAfuCfuUfuCfcUfuCfuUfa	1380	CfUfuUfaAfgAfaGfgAfaAfgAf	1381
		AfaGfdTsdT		uCfadTsdT
471	471-489	ucUfuUfcCfuUfcUfuAfaAfg	1382	GfGfuCfuUfuAfaGfaAfgGfaAf	1383
		AfcCfdTsdT		aGfadTsdT
476	476-494	ccUfuCfuUfaAfaGfaCfcAfu	1384	UfGfgAfuGfgUfcUfuUfaAfgA	1385
		CfcAfdTsdT		faGfgdTsdT
477	477-495	cuUfcUfuAfaAfgAfcCfaUfc	1386	CfUfgGfaUfgGfuCfuUfuAfaGf	1387
		CfaGfdTsdT		aAfgdTsdT
479	479-497	ucUfuAfaAfgAfcCfaUfcCfa	1388	UfCfcUfgGfaUfgGfuCfuUfuAf	1389
		GfgAfdTsdT		aGfadTsdT
481	481-499	uuAfaAfgAfcCfaUfcCfaGfg	1390	CfCfuCfcUfgGfaUfgGfuCfuUf	1391
		AfgGfdTsdT		uAfadTsdT
482	482-500	uaAfaGfaCfcAfuCfcAfgGfa	1392	AfCfcUfcCfuGfgAfuGfgUfcUf	1393
		GfgUfdTsdT		uUfadTsdT
492	492-510	ccAfgGfaGfgUfgGfcUfgGfu	1394	AfUfaAfcCfaGfcCfaCfcUfcCf	1395
		UfaUfdTsdT		uGfgdTsdT
493	493-511	caGfgAfgGfuGfgCfuGfgUfu	1396	CfAfuAfaCfcAfgCfcAfcCfuCf	1397
		AfuGfdTsdT		cUfgdTsdT
494	494-512	agGfaGfgUfgGfcUfgGfuUfa	1398	AfCfaUfaAfcCfaGfcCfaCfcUfc	1399
		UfgUfdTsdT		CfudTsdT
495	495-513	ggAfgGfuGfgCfuGfgUfuAfu	1400	GfAfcAfuAfaCfcAfgCfcAfcCf	1401
		GfuCfdTsdT		uCfcdTsdT
496	496-514	gaGfgUfgGfcUfgGfuUfaUfg	1402	GfGfaCfaUfaAfcCfaGfcCfaCfc	1403
		UfcCfdTsdT		UfcdTsdT
497	497-515	agGfuGfgCfuGfgUfuAfuGfu	1404	AfGfgAfcAfuAfaCfcAfgCfcAf	1405
		CfcUfdTsdT		cCfudTsdT
499	499-517	guGfgCfuGfgUfuAfuGfuCfc	1406	UfGfaGfgAfcAfuAfaCfcAfgCf	1407
		UfcAfdTsdT		cAfcdTsdT
520	520-538	gcCfcUfcAfaCfaCfaGfuGfg	1408	GfCfuCfcAfcUfgUfgUfuGfaGf	1409
		AfgCfdTsdT		gGfcdTsdT
542	542-560	uuCfcUfuUfgGfaAfaAfcCfu	1410	UfGfcAfgGfuUfuUfcCfaAfaGf	1411
		GfcAfdTsdT		gAfadTsdT
543	543-561	ucCfuUfuGfgAfaAfaCfcUfg	1412	CfUfgCfaGfgUfuUfuCfcAfaAf	1413
		CfaGfdTsdT		gGfadTsdT
550	550-568	gaAfaAfcCfuGfcAfgAfuCfa	1414	UfGfaUfgAfuCfuGfcAfgGfuU	1415
		UfcAfdTsdT		fuUfcdTsdT
551	551-569	aaAfaCfcUfgCfaGfaUfcAfu	1416	CfUfgAfuGfaUfcUfgCfaGfgUf	1417
		CfaGfdTsdT		uUfudTsdT
553	553-571	aaCfcUfgCfaGfaUfcAfuCfa	1418	CfUfcUfgAfuGfaUfcUfgCfaGf	1419
		GfaGfdTsdT		gUfudTsdT
556	556-574	cuGfcAfgAfuCfaUfcAfgAfg	1420	UfUfcCfuCfuGfaUfgAfuCfuGf	1421
		GfaAfdTsdT		cAfgdTsdT
586	586-604	gaAfaAfuUfcCfuAfuGfcCfu	1422	CfUfaAfgGfcAfuAfgGfaAfuUf	1423
		UfaGfdTsdT		uUfcdTsdT
587	587-605	aaAfaUfuCfcUfaUfgCfcUfu	1424	GfCfuAfaGfgCfaUfaGfgAfaUf	1425
		AfgCfdTsdT		uUfudTsdT
589	589-607	aaUfuCfcUfaUfgCfcUfuAfg	1426	CfUfgCfuAfaGfgCfaUfaGfgAf	1427
		CfaGfdTsdT		aUfudTsdT
592	592-610	ucCfuAfuGfcCfuUfaGfcAfg	1428	AfGfaCfuGfcUfaAfgGfcAfuAf	1429
		UfcUfdTsdT		gGfadTsdT
593	593-611	ccUfaUfgCfcUfuAfgCfaGfu	1430	AfAfgAfcUfgCfuAfaGfgCfaUf	1431
		CfuUfdTsdT		aGfgdTsdT
594	594-612	cuAfuGfcCfuUfaGfcAfgUfc	1432	UfAfaGfaCfuGfcUfaAfgGfcAf	1433
		UfuAfdTsdT		uAfgdTsdT
596	596-614	auGfcCfuUfaGfcAfgUfcUfu	1434	GfAfuAfaGfaCfuGfcUfaAfgGf	1435
		AfuCfdTsdT		cAfudTsdT
597	597-615	ugCfcUfuAfgCfaGfuCfuUfa	1436	AfGfaUfaAfgAfcUfgCfuAfaGf	1437
		UfcUfdTsdT		gCfadTsdT
598	598-616	gcCfuUfaGfcAfgUfcUfuAfu	1438	UfAfgAfuAfaGfaCfuGfcUfaAf	1439
		CfuAfdTsdT		gGfcdTsdT
599	599-617	ccUfuAfgCfaGfuCfuUfaUfc	1440	UfUfaGfaUfaAfgAfcUfgCfuAf	1441
		UfaAfdTsdT		aGfgdTsdT
600	600-618	cuUfaGfcAfgUfcUfuAfuCfu	1442	GfUfuAfgAfuAfaGfaCfuGfcUf	1443
		AfaCfdTsdT		aAfgdTsdT
601	601-619	uuAfgCfaGfuCfuUfaUfcUfa	1444	AfGfuUfaGfaUfaAfgAfcUfgCf	1445
		AfcUfdTsdT		uAfadTsdT
602	602-620	uaGfcAfgUfcUfuAfuCfuAfa	1446	UfAfgUfuAfgAfuAfaGfaCfuG	1447
		CfuAfdTsdT		fcUfadTsdT
603	603-621	agCfaGfuCfuUfaUfcUfaAfc	1448	AfUfaGfuUfaGfaUfaAfgAfcUf	1449
		UfaUfdTsdT		gCfudTsdT
604	604-622	gcAfgUfcUfuAfuCfuAfaCfu	1450	CfAfuAfgUfuAfgAfuAfaGfaCf	1451
		AfuGfdTsdT		uGfcdTsdT
605	605-623	caGfuCfuUfaUfcUfaAfcUfa	1452	UfCfaUfaGfuUfaGfaUfaAfgAf	1453
		UfgAfdTsdT		cUfgdTsdT
608	608-626	ucUfuAfuCfuAfaCfuAfuGfa	1454	GfCfaUfcAfuAfgUfuAfgAfuA	1455
		UfgCfdTsdT		faGfadTsdT
609	609-627	cuUfaUfcUfaAfcUfaUfgAfu	1456	UfGfcAfuCfaUfaGfuUfaGfaUf	1457
		GfcAfdTsdT		aAfgdTsdT
610	610-628	uuAfuCfuAfaCfuAfuGfaUfg	1458	UfUfgCfaUfcAfuAfgUfuAfgA	1459
		CfaAfdTsdT		fuAfadTsdT
611	611-629	uaUfcUfaAfcUfaUfgAfuGfc	1460	UfUfuGfcAfuCfaUfaGfuUfaGf	1461
		AfaAfdTsdT		aUfadTsdT
612	612-630	auCfuAfaCfuAfuGfaUfgCfa	1462	AfUfuUfgCfaUfcAfuAfgUfuA	1463
		AfaUfdTsdT		fgAfudTsdT
613	613-631	ucUfaAfcUfaUfgAfuGfcAfa	1464	UfAfuUfuGfcAfuCfaUfaGfuUf	1465
		AfuAfdTsdT		aGfadTsdT
614	614-632	cuAfaCfuAfuGfaUfgCfaAfa	1466	UfUfaUfuUfgCfaUfcAfuAfgUf	1467
		UfaAfdTsdT		uAfgdTsdT
616	616-634	aaCfuAfuGfaUfgCfaAfaUfa	1468	UfUfuUfaUfuUfgCfaUfcAfuAf	1469
		AfaAfdTsdT		gUfudTsdT
622	622-640	gaUfgCfaAfaUfaAfaAfcCfg	1470	GfUfcCfgGfuUfuUfaUfuUfgCf	1471
		GfaCfdTsdT		aUfcdTsdT
623	623-641	auGfcAfaAfuAfaAfaCfcGfg	1472	AfGfuCfcGfgUfuUfuAfuUfuG	1473
		AfcUfdTsdT		fcAfudTsdT
624	624-642	ugCfaAfaUfaAfaAfcCfgGfa	1474	CfAfgUfcCfgGfuUfuUfaUfuUf	1475
		CfuGfdTsdT		gCfadTsdT
626	626-644	caAfaUfaAfaAfcCfgGfaCfu	1476	UfUfcAfgUfcCfgGfuUfuUfaUf	1477
		GfaAfdTsdT		uUfgdTsdT
627	627-645	aaAfuAfaAfaCfcGfgAfcUfg	1478	CfUfuCfaGfuCfcGfgUfuUfuAf	1479
		AfaGfdTsdT		uUfudTsdT
628	628-646	aaUfaAfaAfcCfgGfaCfuGfa	1480	CfCfuUfcAfgUfcCfgGfuUfuUf	1481
		AfgGfdTsdT		aUfudTsdT
630	630-648	uaAfaAfcCfgGfaCfuGfaAfg	1482	CfUfcCfuUfcAfgUfcCfgGfuUf	1483
		GfaGfdTsdT		uUfadTsdT
631	631-649	aaAfaCfcGfgAfcUfgAfaGfg	1484	GfCfuCfcUfuCfaGfuCfcGfgUf	1485
		AfgCfdTsdT		uUfudTsdT
632	632-650	aaAfcCfgGfaCfuGfaAfgGfa	1486	AfGfcUfcCfuUfcAfgUfcCfgGf	1487
		GfcUfdTsdT		uUfudTsdT
633	633-651	aaCfcGfgAfcUfgAfaGfgAfg	1488	CfAfgCfuCfcUfuCfaGfuCfcGf	1489
		CfuGfdTsdT		gUfudTsdT
644	644-662	agGfaGfcUfgCfcCfaUfgAfg	1490	UfUfuCfuCfaUfgGfgCfaGfcUf	1491
		AfaAfdTsdT		cCfudTsdT
665	665-683	uaCfaGfgAfaAfuCfcUfgCfa	1492	CfCfaUfgCfaGfgAfuUfuCfcUf	1493
		UfgGfdTsdT		gUfadTsdT
668	668-686	agGfaAfaUfcCfuGfcAfuGfg	1494	GfCfgCfcAfuGfcAfgGfaUfuUf	1495
		CfgCfdTsdT		cCfudTsdT
669	669-687	ggAfaAfuCfcUfgCfaUfgGfc	1496	GfGfcGfcCfaUfgCfaGfgAfuUf	1497
		GfcCfdTsdT		uCfcdTsdT
670	670-688	gaAfaUfcCfuGfcAfuGfgCfg	1498	CfGfgCfgCfcAfuGfcAfgGfaUf	1499
		CfcGfdTsdT		uUfcdTsdT
671	671-689	aaAfuCfcUfgCfaUfgGfcGfc	1500	AfCfgGfcGfcCfaUfgCfaGfgAf	1501
		CfgUfdTsdT		uUfudTsdT
672	672-690	aaUfcCfuGfcAfuGfgCfgCfc	1502	CfAfcGfgCfgCfcAfuGfcAfgGf	1503
		GfuGfdTsdT		aUfudTsdT
674	674-692	ucCfuGfcAfuGfgCfgCfcGfu	1504	CfGfcAfcGfgCfgCfcAfuGfcAf	1505
		GfcGfdTsdT		gGfadTsdT
676	676-694	cuGfcAfuGfgCfgCfcGfuGfc	1506	AfCfcGfcAfcGfgCfgCfcAfuGf	1507
		GfgUfdTsdT		cAfgdTsdT
677	677-695	ugCfaUfgGfcGfcCfgUfgCfg	1508	AfAfcCfgCfaCfgGfcGfcCfaUf	1509
		GfuUfdTsdT		gCfadTsdT
678	678-696	gcAfuGfgCfgCfcGfuGfcGfg	1510	GfAfaCfcGfcAfcGfgCfgCfcAf	1511
		UfuCfdTsdT		uGfcdTsdT
680	680-698	auGfgCfgCfcGfuGfcGfgUfu	1512	CfUfgAfaCfcGfcAfcGfgCfgCf	1513
		CfaGfdTsdT		cAfudTsdT
681	681-699	ugGfcGfcCfgUfgCfgGfuUfc	1514	GfCfuGfaAfcCfgCfaCfgGfcGf	1515
		AfgCfdTsdT		cCfadTsdT
682	682-700	ggCfgCfcGfuGfcGfgUfuCfa	1516	UfGfcUfgAfaCfcGfcAfcGfgCf	1517
		GfcAfdTsdT		gCfcdTsdT
683	683-701	gcGfcCfgUfgCfgGfuUfcAfg	1518	UfUfgCfuGfaAfcCfgCfaCfgGf	1519
		CfaAfdTsdT		cGfcdTsdT
684	684-702	cgCfcGfuGfcGfgUfuCfaGfc	1520	GfUfuGfcUfgAfaCfcGfcAfcGf	1521
		AfaCfdTsdT		gCfgdTsdT
685	685-703	gcCfgUfgCfgGfuUfcAfgCfa	1522	UfGfuUfgCfuGfaAfcCfgCfaCf	1523
		AfcAfdTsdT		gGfcdTsdT
686	686-704	ccGfuGfcGfgUfuCfaGfcAfa	1524	UfUfgUfuGfcUfgAfaCfcGfcAf	1525
		CfaAfdTsdT		cGfgdTsdT
688	688-706	guGfcGfgUfuCfaGfcAfaCfa	1526	GfGfuUfgUfuGfcUfgAfaCfcGf	1527
		AfcCfdTsdT		cAfcdTsdT
690	690-708	gcGfgUfuCfaGfcAfaCfaAfc	1528	AfGfgGfuUfgUfuGfcUfgAfaC	1529
		CfcUfdTsdT		fcGfcdTsdT
692	692-710	ggUfuCfaGfcAfaCfaAfcCfc	1530	GfCfaGfgGfuUfgUfuGfcUfgA	1531
		UfgCfdTsdT		faCfcdTsdT
698	698-716	gcAfaCfaAfcCfcUfgCfcCfu	1532	CfAfcAfgGfgCfaGfgGfuUfgUf	1533
		GfuGfdTsdT		uGfcdTsdT
700	700-718	aaCfaAfcCfcUfgCfcCfuGfu	1534	UfGfcAfcAfgGfgCfaGfgGfuUf	1535
		GfcAfdTsdT		gUfudTsdT
719	719-737	acGfuGfgAfgAfgCfaUfcCfa	1536	CfAfcUfgGfaUfgCfuCfuCfcAf	1537
		GfuGfdTsdT		cGfudTsdT
720	720-738	cgUfgGfaGfaGfcAfuCfcAfg	1538	CfCfaCfuGfgAfuGfcUfcUfcCf	1539
		UfgGfdTsdT		aCfgdTsdT
721	721-739	guGfgAfgAfgCfaUfcCfaGfu	1540	GfCfcAfcUfgGfaUfgCfuCfuCf	1541
		GfgCfdTsdT		cAfcdTsdT
724	724-742	gaGfaGfcAfuCfcAfgUfgGfc	1542	CfCfcGfcCfaCfuGfgAfuGfcUf	1543
		GfgGfdTsdT		cUfcdTsdT
725	725-743	agAfgCfaUfcCfaGfuGfgCfg	1544	UfCfcCfgCfcAfcUfgGfaUfgCf	1545
		GfgAfdTsdT		uCfudTsdT
726	726-744	gaGfcAfuCfcAfgUfgGfcGfg	1546	GfUfcCfcGfcCfaCfuGfgAfuGf	1547
		GfaCfdTsdT		cUfcdTsdT
733	733-751	caGfuGfgCfgGfgAfcAfuAfg	1548	UfGfaCfuAfuGfuCfcCfgCfcAf	1549
		UfcAfdTsdT		cUfgdTsdT
734	734-752	agUfgGfcGfgGfaCfaUfaGfu	1550	CfUfgAfcUfaUfgUfcCfcGfcCf	1551
		CfaGfdTsdT		aCfudTsdT
736	736-754	ugGfcGfgGfaCfaUfaGfuCfa	1552	UfGfcUfgAfcUfaUfgUfcCfcGf	1553
		GfcAfdTsdT		cCfadTsdT
737	737-755	ggCfgGfgAfcAfuAfgUfcAfg	1554	CfUfgCfuGfaCfuAfuGfuCfcCf	1555
		CfaGfdTsdT		gCfcdTsdT
763	763-781	cuCfaGfcAfaCfaUfgUfcGfa	1556	CfCfaUfcGfaCfaUfgUfuGfcUf	1557
		UfgGfdTsdT		gAfgdTsdT
765	765-783	caGfcAfaCfaUfgUfcGfaUfg	1558	GfUfcCfaUfcGfaCfaUfgUfuGf	1559
		GfaCfdTsdT		cUfgdTsdT
766	766-784	agCfaAfcAfuGfuCfgAfuGfg	1560	AfGfuCfcAfuCfgAfcAfuGfuUf	1561
		AfcUfdTsdT		gCfudTsdT
767	767-785	gcAfaCfaUfgUfcGfaUfgGfa	1562	AfAfgUfcCfaUfcGfaCfaUfgUf	1563
		CfuUfdTsdT		uGfcdTsdT
769	769-787	aaCfaUfgUfcGfaUfgGfaCfu	1564	GfGfaAfgUfcCfaUfcGfaCfaUf	1565
		UfcCfdTsdT		gUfudTsdT
770	770-788	acAfuGfuCfgAfuGfgAfcUfu	1566	UfGfgAfaGfuCfcAfuCfgAfcAf	1567
		CfcAfdTsdT		uGfudTsdT
771	771-789	caUfgUfcGfaUfgGfaCfuUfc	1568	CfUfgGfaAfgUfcCfaUfcGfaCf	1569
		CfaGfdTsdT		aUfgdTsdT
772	772-790	auGfuCfgAfuGfgAfcUfuCfc	1570	UfCfuGfgAfaGfuCfcAfuCfgAf	1571
		AfgAfdTsdT		cAfudTsdT
775	775-793	ucGfaUfgGfaCfuUfcCfaGfa	1572	GfGfuUfcUfgGfaAfgUfcCfaUf	1573
		AfcCfdTsdT		cGfadTsdT
789	789-807	gaAfcCfaCfcUfgGfgCfaGfc	1574	GfCfaGfcUfgCfcCfaGfgUfgGf	1575
		UfgCfdTsdT		uUfcdTsdT
798	798-816	ggGfcAfgCfuGfcCfaAfaAfg	1576	AfCfaCfuUfuUfgGfcAfgCfuGf	1577
		UfgUfdTsdT		cCfcdTsdT
800	800-818	gcAfgCfuGfcCfaAfaAfgUfg	1578	UfCfaCfaCfuUfuUfgGfcAfgCf	1579
		UfgAfdTsdT		uGfcdTsdT
805	805-823	ugCfcAfaAfaGfuGfuGfaUfc	1580	UfUfgGfaUfcAfcAfcUfuUfuGf	1581
		CfaAfdTsdT		gCfadTsdT
806	806-824	gcCfaAfaAfgUfgUfgAfuCfc	1582	CfUfuGfgAfuCfaCfaCfuUfuUf	1583
		AfaGfdTsdT		gGfcdTsdT
807	807-825	ccAfaAfaGfuGfuGfaUfcCfa	1584	GfCfuUfgGfaUfcAfcAfcUfuUf	1585
		AfgCfdTsdT		uGfgdTsdT
810	810-828	aaAfgUfgUfgAfuCfcAfaGfc	1586	AfCfaGfcUfuGfgAfuCfaCfaCf	1587
		UfgUfdTsdT		uUfudTsdT
814	814-832	ugUfgAfuCfcAfaGfcUfgUfc	1588	UfGfgGfaCfaGfcUfuGfgAfuCf	1589
		CfcAfdTsdT		aCfadTsdT
815	815-833	guGfaUfcCfaAfgCfuGfuCfc	1590	UfUfgGfgAfcAfgCfuUfgGfaU	1591
		CfaAfdTsdT		fcAfcdTsdT
817	817-835	gaUfcCfaAfgCfuGfuCfcCfa	1592	CfAfuUfgGfgAfcAfgCfuUfgG	1593
		AfuGfdTsdT		faUfcdTsdT
818	818-836	auCfcAfaGfcUfgUfcCfcAfa	1594	CfCfaUfuGfgGfaCfaGfcUfuGf	1595
		UfgGfdTsdT		gAfudTsdT
819	819-837	ucCfaAfgCfuGfuCfcCfaAfu	1596	CfCfcAfuUfgGfgAfcAfgCfuUf	1597
		GfgGfdTsdT		gGfadTsdT
820	820-838	ccAfaGfcUfgUfcCfcAfaUfg	1598	UfCfcCfaUfuGfgGfaCfaGfcUf	1599
		GfgAfdTsdT		uGfgdTsdT
821	821-839	caAfgCfuGfuCfcCfaAfuGfg	1600	CfUfcCfcAfuUfgGfgAfcAfgCf	1601
		GfaGfdTsdT		uUfgdTsdT
823	823-841	agCfuGfuCfcCfaAfuGfgGfa	1602	AfGfcUfcCfcAfuUfgGfgAfcAf	1603
		GfcUfdTsdT		gCfudTsdT
826	826-844	ugUfcCfcAfaUfgGfgAfgCfu	1604	AfGfcAfgCfuCfcCfaUfuGfgGf	1605
		GfcUfdTsdT		aCfadTsdT
847	847-865	ggUfgCfaGfgAfgAfgGfaGfa	1606	AfGfuUfcUfcCfuCfuCfcUfgCf	1607
		AfcUfdTsdT		aCfcdTsdT
871	871-889	aaAfcUfgAfcCfaAfaAfuCfa	1608	AfGfaUfgAfuUfuUfgGfuCfaG	1609
		UfcUfdTsdT		fuUfudTsdT
872	872-890	aaCfuGfaCfcAfaAfaUfcAfu	1610	CfAfgAfuGfaUfuUfuGfgUfcA	1611
		CfuGfdTsdT		fgUfudTsdT
873	873-891	acUfgAfcCfaAfaAfuCfaUfc	1612	AfCfaGfaUfgAfuUfuUfgGfuCf	1613
		UfgUfdTsdT		aGfudTsdT
877	877-895	acCfaAfaAfuCfaUfcUfgUfg	1614	GfGfgCfaCfaGfaUfgAfuUfuUf	1615
		CfcCfdTsdT		gGfudTsdT
878	878-896	ccAfaAfaUfcAfuCfuGfuGfc	1616	UfGfgGfcAfcAfgAfuGfaUfuU	1617
		CfcAfdTsdT		fuGfgdTsdT
881	881-899	aaAfuCfaUfcUfgUfgCfcCfa	1618	UfGfcUfgGfgCfaCfaGfaUfgAf	1619
		GfcAfdTsdT		uUfudTsdT
890	890-908	guGfcCfcAfgCfaGfuGfcUfc	1620	CfCfgGfaGfcAfcUfgCfuGfgGf	1621
		CfgGfdTsdT		cAfcdTsdT
892	892-910	gcCfcAfgCfaGfuGfcUfcCfg	1622	GfCfcCfgGfaGfcAfcUfgCfuGf	1623
		GfgCfdTsdT		gGfcdTsdT
929	929-947	ccAfgUfgAfcUfgCfuGfcCfa	1624	UfUfgUfgGfcAfgCfaGfuCfaCf	1625
		CfaAfdTsdT		uGfgdTsdT
930	930-948	caGfuGfaCfuGfcUfgCfcAfc	1626	GfUfuGfuGfgCfaGfcAfgUfcAf	1627
		AfaCfdTsdT		cUfgdTsdT
979	979-997	gaGfaGfcGfaCfuGfcCfuGfg	1628	AfGfaCfcAfgGfcAfgUfcGfcUf	1629
		UfcUfdTsdT		cUfcdTsdT
980	980-998	agAfgCfgAfcUfgCfcUfgGfu	1630	CfAfgAfcCfaGfgCfaGfuCfgCf	1631
		CfuGfdTsdT		uCfudTsdT
981	981-999	gaGfcGfaCfuGfcCfuGfgUfc	1632	GfCfaGfaCfcAfgGfcAfgUfcGf	1633
		UfgCfdTsdT		cUfcdTsdT
982	982-1000	agCfgAfcUfgCfcUfgGfuCfu	1634	GfGfcAfgAfcCfaGfgCfaGfuCf	1635
		GfcCfdTsdT		gCfudTsdT
983	983-1001	gcGfaCfuGfcCfuGfgUfcUfg	1636	CfGfgCfaGfaCfcAfgGfcAfgUf	1637
		CfcGfdTsdT		cGfcdTsdT
984	984-1002	cgAfcUfgCfcUfgGfuCfuGfc	1638	GfCfgGfcAfgAfcCfaGfgCfaGf	1639
		CfgCfdTsdT		uCfgdTsdT
989	989-1007	gcCfuGfgUfcUfgCfcGfcAfa	1640	AfAfuUfuGfcGfgCfaGfaCfcAf	1641
		AfuUfdTsdT		gGfcdTsdT
990	990-1008	ccUfgGfuCfuGfcCfgCfaAfa	1642	GfAfaUfuUfgCfgGfcAfgAfcCf	1643
		UfuCfdTsdT		aGfgdTsdT
991	991-1009	cuGfgUfcUfgCfcGfcAfaAfu	1644	GfGfaAfuUfuGfcGfgCfaGfaCf	1645
		UfcCfdTsdT		cAfgdTsdT
992	992-1010	ugGfuCfuGfcCfgCfaAfaUfu	1646	CfGfgAfaUfuUfgCfgGfcAfgAf	1647
		CfcGfdTsdT		cCfadTsdT
994	994-1012	guCfuGfcCfgCfaAfaUfuCfc	1648	CfUfcGfgAfaUfuUfgCfgGfcAf	1649
		GfaGfdTsdT		gAfcdTsdT
995	995-1013	ucUfgCfcGfcAfaAfuUfcCfg	1650	UfCfuCfgGfaAfuUfuGfcGfgCf	1651
		AfgAfdTsdT		aGfadTsdT
996	996-1014	cuGfcCfgCfaAfaUfuCfcGfa	1652	GfUfcUfcGfgAfaUfuUfgCfgGf	1653
		GfaCfdTsdT		cAfgdTsdT
997	997-1015	ugCfcGfcAfaAfuUfcCfgAfg	1654	CfGfuCfuCfgGfaAfuUfuGfcGf	1655
		AfcGfdTsdT		gCfadTsdT
999	999-1017	ccGfcAfaAfuUfcCfgAfgAfc	1656	UfUfcGfuCfuCfgGfaAfuUfuGf	1657
		GfaAfdTsdT		cGfgdTsdT
1004	1004-1022	aaUfuCfcGfaGfaCfgAfaGfc	1658	GfUfgGfcUfuCfgUfcUfcGfgAf	1659
		CfaCfdTsdT		aUfudTsdT
1005	1005-1023	auUfcCfgAfgAfcGfaAfgCfc	1660	CfGfuGfgCfuUfcGfuCfuCfgGf	1661
		AfcGfdTsdT		aAfudTsdT
1006	1006-1024	uuCfcGfaGfaCfgAfaGfcCfa	1662	AfCfgUfgGfcUfuCfgUfcUfcGf	1663
		CfgUfdTsdT		gAfadTsdT
1007	1007-1025	ucCfgAfgAfcGfaAfgCfcAfc	1664	CfAfcGfuGfgCfuUfcGfuCfuCf	1665
		GfuGfdTsdT		gGfadTsdT
1008	1008-1026	ccGfaGfaCfgAfaGfcCfaCfg	1666	GfCfaCfgUfgGfcUfuCfgUfcUf	1667
		UfgCfdTsdT		cGfgdTsdT
1010	1010-1028	gaGfaCfgAfaGfcCfaCfgUfg	1668	UfUfgCfaCfgUfgGfcUfuCfgUf	1669
		CfaAfdTsdT		cUfcdTsdT
1013	1013-1031	acGfaAfgCfcAfcGfuGfcAfa	1670	UfCfcUfuGfcAfcGfuGfgCfuUf	1671
		GfgAfdTsdT		cGfudTsdT
1014	1014-1032	cgAfaGfcCfaCfgUfgCfaAfg	1672	GfUfcCfuUfgCfaCfgUfgGfcUf	1673
		GfaCfdTsdT		uCfgdTsdT
1015	1015-1033	gaAfgCfcAfcGfuGfcAfaGfg	1674	UfGfuCfcUfuGfcAfcGfuGfgCf	1675
		AfcAfdTsdT		uUfcdTsdT
1016	1016-1034	aaGfcCfaCfgUfgCfaAfgGfa	1676	GfUfgUfcCfuUfgCfaCfgUfgGf	1677
		CfaCfdTsdT		cUfudTsdT
1040	1040-1058	ccCfcAfcUfcAfuGfcUfcUfa	1678	UfUfgUfaGfaGfcAfuGfaGfuGf	1679
		CfaAfdTsdT		gGfgdTsdT
1042	1042-1060	ccAfcUfcAfuGfcUfcUfaCfa	1680	GfGfuUfgUfaGfaGfcAfuGfaGf	1681
		AfcCfdTsdT		uGfgdTsdT
1044	1044-1062	acUfcAfuGfcUfcUfaCfaAfc	1682	GfGfgGfuUfgUfaGfaGfcAfuG	1683
		CfcCfdTsdT		faGfudTsdT
1047	1047-1065	caUfgCfuCfuAfcAfaCfcCfc	1684	GfGfuGfgGfgUfuGfuAfgAfgC	1685
		AfcCfdTsdT		faUfgdTsdT
1071	1071-1089	ccAfgAfuGfgAfuGfuGfaAfc	1686	GfGfgGfuUfcAfcAfuCfcAfuCf	1687
		CfcCfdTsdT		uGfgdTsdT
1073	1073-1091	agAfuGfgAfuGfuGfaAfcCfc	1688	UfCfgGfgGfuUfcAfcAfuCfcAf	1689
		CfgAfdTsdT		uCfudTsdT
1074	1074-1092	gaUfgGfaUfgUfgAfaCfcCfc	1690	CfUfcGfgGfgUfuCfaCfaUfcCf	1691
		GfaGfdTsdT		aUfcdTsdT
1075	1075-1093	auGfgAfuGfuGfaAfcCfcCfg	1692	CfCfuCfgGfgGfuUfcAfcAfuCf	1693
		AfgGfdTsdT		cAfudTsdT
1077	1077-1095	ggAfuGfuGfaAfcCfcCfgAfg	1694	GfCfcCfuCfgGfgGfuUfcAfcAf	1695
		GfgCfdTsdT		uCfcdTsdT
1078	1078-1096	gaUfgUfgAfaCfcCfcGfaGfg	1696	UfGfcCfcUfcGfgGfgUfuCfaCf	1697
		GfcAfdTsdT		aUfcdTsdT
1080	1080-1098	ugUfgAfaCfcCfcGfaGfgGfc	1698	UfUfuGfcCfcUfcGfgGfgUfuCf	1699
		AfaAfdTsdT		aCfadTsdT
1084	1084-1102	aaCfcCfcGfaGfgGfcAfaAfu	1700	UfGfuAfuUfuGfcCfcUfcGfgGf	1701
		AfcAfdTsdT		gUfudTsdT
1085	1085-1103	acCfcCfgAfgGfgCfaAfaUfa	1702	CfUfgUfaUfuUfgCfcCfuCfgGf	1703
		CfaGfdTsdT		gGfudTsdT
1087	1087-1105	ccCfgAfgGfgCfaAfaUfaCfa	1704	AfGfcUfgUfaUfuUfgCfcCfuCf	1705
		GfcUfdTsdT		gGfgdTsdT
1088	1088-1106	ccGfaGfgGfcAfaAfuAfcAfg	1706	AfAfgCfuGfuAfuUfuGfcCfcUf	1707
		CfuUfdTsdT		cGfgdTsdT
1089	1089-1107	cgAfgGfgCfaAfaUfaCfaGfc	1708	AfAfaGfcUfgUfaUfuUfgCfcCf	1709
		UfuUfdTsdT		uCfgdTsdT
1096	1096-1114	aaAfuAfcAfgCfuUfuGfgUfg	1710	UfGfgCfaCfcAfaAfgCfuGfuAf	1711
		CfcAfdTsdT		uUfudTsdT
1097	1097-1115	aaUfaCfaGfcUfuUfgGfuGfc	1712	GfUfgGfcAfcCfaAfaGfcUfgUf	1713
		CfaCfdTsdT		aUfudTsdT
1098	1098-1116	auAfcAfgCfuUfuGfgUfgCfc	1714	GfGfuGfgCfaCfcAfaAfgCfuGf	1715
		AfcCfdTsdT		uAfudTsdT
1104	1104-1122	cuUfuGfgUfgCfcAfcCfuGfc	1716	CfAfcGfcAfgGfuGfgCfaCfcAf	1717
		GfuGfdTsdT		aAfgdTsdT
1106	1106-1124	uuGfgUfgCfcAfcCfuGfcGfu	1718	UfUfcAfcGfcAfgGfuGfgCfaCf	1719
		GfaAfdTsdT		cAfadTsdT
1112	1112-1130	ccAfcCfuGfcGfuGfaAfgAfa	1720	CfAfcUfuCfuUfcAfcGfcAfgGf	1721
		GfuGfdTsdT		uGfgdTsdT
1116	1116-1134	cuGfcGfuGfaAfgAfaGfuGfu	1722	GfGfgAfcAfcUfuCfuUfcAfcGf	1723
		CfcCfdTsdT		cAfgdTsdT
1117	1117-1135	ugCfgUfgAfaGfaAfgUfgUfc	1724	GfGfgGfaCfaCfuUfcUfuCfaCf	1725
		CfcCfdTsdT		gCfadTsdT
1118	1118-1136	gcGfuGfaAfgAfaGfuGfuCfc	1726	CfGfgGfgAfcAfcUfuCfuUfcAf	1727
		CfcGfdTsdT		cGfcdTsdT
1119	1119-1137	cgUfgAfaGfaAfgUfgUfcCfc	1728	AfCfgGfgGfaCfaCfuUfcUfuCf	1729
		CfgUfdTsdT		aCfgdTsdT
1120	1120-1138	guGfaAfgAfaGfuGfuCfcCfc	1730	UfAfcGfgGfgAfcAfcUfuCfuUf	1731
		GfuAfdTsdT		cAfcdTsdT
1121	1121-1139	ugAfaGfaAfgUfgUfcCfcCfg	1732	UfUfaCfgGfgGfaCfaCfuUfcUf	1733
		UfaAfdTsdT		uCfadTsdT
1122	1122-1140	gaAfgAfaGfuGfuCfcCfcGfu	1734	AfUfuAfcGfgGfgAfcAfcUfuCf	1735
		AfaUfdTsdT		uUfcdTsdT
1123	1123-1141	aaGfaAfgUfgUfcCfcCfgUfa	1736	AfAfuUfaCfgGfgGfaCfaCfuUf	1737
		AfuUfdTsdT		cUfudTsdT
1124	1124-1142	agAfaGfuGfuCfcCfcGfuAfa	1738	UfAfaUfuAfcGfgGfgAfcAfcUf	1739
		UfuAfdTsdT		uCfudTsdT
1125	1125-1143	gaAfgUfgUfcCfcCfgUfaAfu	1740	AfUfaAfuUfaCfgGfgGfaCfaCf	1741
		UfaUfdTsdT		uUfcdTsdT
1126	1126-1144	aaGfuGfuCfcCfcGfuAfaUfu	1742	CfAfuAfaUfuAfcGfgGfgAfcAf	1743
		AfuGfdTsdT		cUfudTsdT
1127	1127-1145	agUfgUfcCfcCfgUfaAfuUfa	1744	AfCfaUfaAfuUfaCfgGfgGfaCf	1745
		UfgUfdTsdT		aCfudTsdT
1128	1128-1146	guGfuCfcCfcGfuAfaUfuAfu	1746	CfAfcAfuAfaUfuAfcGfgGfgAf	1747
		GfuGfdTsdT		cAfcdTsdT
1129	1129-1147	ugUfcCfcCfgUfaAfuUfaUfg	1748	CfCfaCfaUfaAfuUfaCfgGfgGf	1749
		UfgGfdTsdT		aCfadTsdT
1130	1130-1148	guCfcCfcGfuAfaUfuAfuGfu	1750	AfCfcAfcAfuAfaUfuAfcGfgGf	1751
		GfgUfdTsdT		gAfcdTsdT
1132	1132-1150	ccCfcGfuAfaUfuAfuGfuGfg	1752	UfCfaCfcAfcAfuAfaUfuAfcGf	1753
		UfgAfdTsdT		gGfgdTsdT
1134	1134-1152	ccGfuAfaUfuAfuGfuGfgUfg	1754	UfGfuCfaCfcAfcAfuAfaUfuAf	1755
		AfcAfdTsdT		cGfgdTsdT
1136	1136-1154	guAfaUfuAfuGfuGfgUfgAfc	1756	UfCfuGfuCfaCfcAfcAfuAfaUf	1757
		AfgAfdTsdT		uAfcdTsdT
1137	1137-1155	uaAfuUfaUfgUfgGfuGfaCfa	1758	AfUfcUfgUfcAfcCfaCfaUfaAf	1759
		GfaUfdTsdT		uUfadTsdT
1138	1138-1156	aaUfuAfuGfuGfgUfgAfcAfg	1760	GfAfuCfuGfuCfaCfcAfcAfuAf	1761
		AfuCfdTsdT		aUfudTsdT
1139	1139-1157	auUfaUfgUfgGfuGfaCfaGfa	1762	UfGfaUfcUfgUfcAfcCfaCfaUf	1763
		UfcAfdTsdT		aAfudTsdT
1140	1140-1158	uuAfuGfuGfgUfgAfcAfgAfu	1764	GfUfgAfuCfuGfuCfaCfcAfcAf	1765
		CfaCfdTsdT		uAfadTsdT
1142	1142-1160	auGfuGfgUfgAfcAfgAfuCfa	1766	CfCfgUfgAfuCfuGfuCfaCfcAf	1767
		CfgGfdTsdT		cAfudTsdT
1145	1145-1163	ugGfuGfaCfaGfaUfcAfcGfg	1768	GfAfgCfcGfuGfaUfcUfgUfcAf	1769
		CfuCfdTsdT		cCfadTsdT
1147	1147-1165	guGfaCfaGfaUfcAfcGfgCfu	1770	AfCfgAfgCfcGfuGfaUfcUfgUf	1771
		CfgUfdTsdT		cAfcdTsdT
1148	1148-1166	ugAfcAfgAfuCfaCfgGfcUfc	1772	CfAfcGfaGfcCfgUfgAfuCfuGf	1773
		GfuGfdTsdT		uCfadTsdT
1149	1149-1167	gaCfaGfaUfcAfcGfgCfuCfg	1774	GfCfaCfgAfgCfcGfuGfaUfcUf	1775
		UfgCfdTsdT		gUfcdTsdT
1150	1150-1168	acAfgAfuCfaCfgGfcUfcGfu	1776	CfGfcAfcGfaGfcCfgUfgAfuCf	1777
		GfcGfdTsdT		uGfudTsdT
1151	1151-1169	caGfaUfcAfcGfgCfuCfgUfg	1778	AfCfgCfaCfgAfgCfcGfuGfaUf	1779
		CfgUfdTsdT		cUfgdTsdT
1152	1152-1170	agAfuCfaCfgGfcUfcGfuGfc	1780	GfAfcGfcAfcGfaGfcCfgUfgAf	1781
		GfuCfdTsdT		uCfudTsdT
1153	1153-1171	gaUfcAfcGfgCfuCfgUfgCfg	1782	GfGfaCfgCfaCfgAfgCfcGfuGf	1783
		UfcCfdTsdT		aUfcdTsdT
1154	1154-1172	auCfaCfgGfcUfcGfuGfcGfu	1784	CfGfgAfcGfcAfcGfaGfcCfgUf	1785
		CfcGfdTsdT		gAfudTsdT
1155	1155-1173	ucAfcGfgCfuCfgUfgCfgUfc	1786	UfCfgGfaCfgCfaCfgAfgCfcGf	1787
		CfgAfdTsdT		uGfadTsdT
1156	1156-1174	caCfgGfcUfcGfuGfcGfuCfc	1788	CfUfcGfgAfcGfcAfcGfaGfcCf	1789
		GfaGfdTsdT		gUfgdTsdT
1157	1157-1175	acGfgCfuCfgUfgCfgUfcCfg	1790	GfCfuCfgGfaCfgCfaCfgAfgCf	1791
		AfgCfdTsdT		cGfudTsdT
1160	1160-1178	gcUfcGfuGfcGfuCfcGfaGfc	1792	CfAfgGfcUfcGfgAfcGfcAfcGf	1793
		CfuGfdTsdT		aGfcdTsdT
1200	1200-1218	ggAfgGfaAfgAfcGfgCfgUfc	1794	GfCfgGfaCfgCfcGfuCfuUfcCf	1795
		CfgCfdTsdT		uCfcdTsdT
1201	1201-1219	gaGfgAfaGfaCfgGfcGfuCfc	1796	UfGfcGfgAfcGfcCfgUfcUfuCf	1797
		GfcAfdTsdT		cUfcdTsdT
1203	1203-1221	ggAfaGfaCfgGfcGfuCfcGfc	1798	CfUfuGfcGfgAfcGfcCfgUfcUf	1799
		AfaGfdTsdT		uCfcdTsdT
1204	1204-1222	gaAfgAfcGfgCfgUfcCfgCfa	1800	AfCfuUfgCfgGfaCfgCfcGfuCf	1801
		AfgUfdTsdT		uUfcdTsdT
1205	1205-1223	aaGfaCfgGfcGfuCfcGfcAfa	1802	CfAfcUfuGfcGfgAfcGfcCfgUf	1803
		GfuGfdTsdT		cUfudTsdT
1207	1207-1225	gaCfgGfcGfuCfcGfcAfaGfu	1804	UfAfcAfcUfuGfcGfgAfcGfcCf	1805
		GfuAfdTsdT		gUfcdTsdT
1208	1208-1226	acGfgCfgUfcCfgCfaAfgUfg	1806	UfUfaCfaCfuUfgCfgGfaCfgCf	1807
		UfaAfdTsdT		cGfudTsdT
1211	1211-1229	gcGfuCfcGfcAfaGfuGfuAfa	1808	UfUfcUfuAfcAfcUfuGfcGfgAf	1809
		GfaAfdTsdT		cGfcdTsdT
1212	1212-1230	cgUfcCfgCfaAfgUfgUfaAfg	1810	CfUfuCfuUfaCfaCfuUfgCfgGf	1811
		AfaGfdTsdT		aCfgdTsdT
1213	1213-1231	guCfcGfcAfaGfuGfuAfaGfa	1812	AfCfuUfcUfuAfcAfcUfuGfcGf	1813
		AfgUfdTsdT		gAfcdTsdT
1214	1214-1232	ucCfgCfaAfgUfgUfaAfgAfa	1814	CfAfcUfuCfuUfaCfaCfuUfgCf	1815
		GfuGfdTsdT		gGfadTsdT
1215	1215-1233	ccGfcAfaGfuGfuAfaGfaAfg	1816	GfCfaCfuUfcUfuAfcAfcUfuGf	1817
		UfgCfdTsdT		cGfgdTsdT
1216	1216-1234	cgCfaAfgUfgUfaAfgAfaGfu	1818	CfGfcAfcUfuCfuUfaCfaCfuUf	1819
		GfcGfdTsdT		gCfgdTsdT
1217	1217-1235	gcAfaGfuGfuAfaGfaAfgUfg	1820	UfCfgCfaCfuUfcUfuAfcAfcUf	1821
		CfgAfdTsdT		uGfcdTsdT
1219	1219-1237	aaGfuGfuAfaGfaAfgUfgCfg	1822	CfUfuCfgCfaCfuUfcUfuAfcAf	1823
		AfaGfdTsdT		cUfudTsdT
1220	1220-1238	agUfgUfaAfgAfaGfuGfcGfa	1824	CfCfuUfcGfcAfcUfuCfuUfaCf	1825
		AfgGfdTsdT		aCfudTsdT
1221	1221-1239	guGfuAfaGfaAfgUfgCfgAfa	1826	CfCfcUfuCfgCfaCfuUfcUfuAf	1827
		GfgGfdTsdT		cAfcdTsdT
1222	1222-1240	ugUfaAfgAfaGfuGfcGfaAfg	1828	GfCfcCfuUfcGfcAfcUfuCfuUf	1829
		GfgCfdTsdT		aCfadTsdT
1223	1223-1241	guAfaGfaAfgUfgCfgAfaGfg	1830	GfGfcCfcUfuCfgCfaCfuUfcUf	1831
		GfcCfdTsdT		uAfcdTsdT
1224	1224-1242	uaAfgAfaGfuGfcGfaAfgGfg	1832	AfGfgCfcCfuUfcGfcAfcUfuCf	1833
		CfcUfdTsdT		uUfadTsdT
1225	1225-1243	aaGfaAfgUfgCfgAfaGfgGfc	1834	AfAfgGfcCfcUfuCfgCfaCfuUf	1835
		CfuUfdTsdT		cUfudTsdT
1226	1226-1244	agAfaGfuGfcGfaAfgGfgCfc	1836	CfAfaGfgCfcCfuUfcGfcAfcUf	1837
		UfuGfdTsdT		uCfudTsdT
1229	1229-1247	agUfgCfgAfaGfgGfcCfuUfg	1838	CfGfgCfaAfgGfcCfcUfuCfgCf	1839
		CfcGfdTsdT		aCfudTsdT
1230	1230-1248	guGfcGfaAfgGfgCfcUfuGfc	1840	GfCfgGfcAfaGfgCfcCfuUfcGf	1841
		CfgCfdTsdT		cAfcdTsdT
1231	1231-1249	ugCfgAfaGfgGfcCfuUfgCfc	1842	UfGfcGfgCfaAfgGfcCfcUfuCf	1843
		GfcAfdTsdT		gCfadTsdT
1232	1232-1250	gcGfaAfgGfgCfcUfuGfcCfg	1844	UfUfgCfgGfcAfaGfgCfcCfuUf	1845
		CfaAfdTsdT		cGfcdTsdT
1233	1233-1251	cgAfaGfgGfcCfuUfgCfcGfc	1846	UfUfuGfcGfgCfaAfgGfcCfcUf	1847
		AfaAfdTsdT		uCfgdTsdT
1235	1235-1253	aaGfgGfcCfuUfgCfcGfcAfa	1848	AfCfuUfuGfcGfgCfaAfgGfcCf	1849
		AfgUfdTsdT		cUfudTsdT
1236	1236-1254	agGfgCfcUfuGfcCfgCfaAfa	1850	CfAfcUfuUfgCfgGfcAfaGfgCf	1851
		GfuGfdTsdT		cCfudTsdT
1237	1237-1255	ggGfcCfuUfgCfcGfcAfaAfg	1852	AfCfaCfuUfuGfcGfgCfaAfgGf	1853
		UfgUfdTsdT		cCfcdTsdT
1238	1238-1256	ggCfcUfuGfcCfgCfaAfaGfu	1854	CfAfcAfcUfuUfgCfgGfcAfaGf	1855
		GfuGfdTsdT		gCfcdTsdT
1239	1239-1257	gcCfuUfgCfcGfcAfaAfgUfg	1856	AfCfaCfaCfuUfuGfcGfgCfaAf	1857
		UfgUfdTsdT		gGfcdTsdT
1241	1241-1259	cuUfgCfcGfcAfaAfgUfgUfg	1858	UfUfaCfaCfaCfuUfuGfcGfgCf	1859
		UfaAfdTsdT		aAfgdTsdT
1261	1261-1279	ggAfaUfaGfgUfaUfuGfgUfg	1860	AfUfuCfaCfcAfaUfaCfcUfaUf	1861
		AfaUfdTsdT		uCfcdTsdT
1262	1262-1280	gaAfuAfgGfuAfuUfgGfuGfa	1862	AfAfuUfcAfcCfaAfuAfcCfuAf	1863
		AfuUfdTsdT		uUfcdTsdT
1263	1263-1281	aaUfaGfgUfaUfuGfgUfgAfa	1864	AfAfaUfuCfaCfcAfaUfaCfcUf	1865
		UfuUfdTsdT		aUfudTsdT
1264	1264-1282	auAfgGfuAfuUfgGfuGfaAfu	1866	UfAfaAfuUfcAfcCfaAfuAfcCf	1867
		UfuAfdTsdT		uAfudTsdT
1266	1266-1284	agGfuAfuUfgGfuGfaAfuUfu	1868	UfUfuAfaAfuUfcAfcCfaAfuAf	1869
		AfaAfdTsdT		cCfudTsdT
1267	1267-1285	ggUfaUfuGfgUfgAfaUfuUfa	1870	CfUfuUfaAfaUfuCfaCfcAfaUf	1871
		AfaGfdTsdT		aCfcdTsdT
1289	1289-1307	caCfuCfuCfcAfuAfaAfuGfc	1872	GfUfaGfcAfuUfuAfuGfgAfgA	1873
		UfaCfdTsdT		fgUfgdTsdT
1313	1313-1331	uuAfaAfcAfcUfuCfaAfaAfa	1874	CfAfgUfuUfuUfgAfaGfuGfuU	1875
		CfuGfdTsdT		fuAfadTsdT
1320	1320-1338	cuUfcAfaAfaAfcUfgCfaCfc	1876	GfGfaGfgUfgCfaGfuUfuUfuG	1877
		UfcCfdTsdT		faAfgdTsdT
1321	1321-1339	uuCfaAfaAfaCfuGfcAfcCfu	1878	UfGfgAfgGfuGfcAfgUfuUfuU	1879
		CfcAfdTsdT		fgAfadTsdT
1322	1322-1340	ucAfaAfaAfcUfgCfaCfcUfc	1880	AfUfgGfaGfgUfgCfaGfuUfuU	1881
		CfaUfdTsdT		fuGfadTsdT
1323	1323-1341	caAfaAfaCfuGfcAfcCfuCfc	1882	GfAfuGfgAfgGfuGfcAfgUfuU	1883
		AfuCfdTsdT		fuUfgdTsdT
1324	1324-1342	aaAfaAfcUfgCfaCfcUfcCfaU	1884	UfGfaUfgGfaGfgUfgCfaGfuUf	1885
		fcAfdTsdT		uUfudTsdT
1328	1328-1346	acUfgCfaCfcUfcCfaUfcAfg	1886	CfCfaCfuGfaUfgGfaGfgUfgCf	1887
		UfgGfdTsdT		aGfudTsdT
1332	1332-1350	caCfcUfcCfaUfcAfgUfgGfc	1888	AfUfcGfcCfaCfuGfaUfgGfaGf	1889
		GfaUfdTsdT		gUfgdTsdT
1333	1333-1351	acCfuCfcAfuCfaGfuGfgCfg	1890	GfAfuCfgCfcAfcUfgAfuGfgAf	1891
		AfuCfdTsdT		gGfudTsdT
1335	1335-1353	cuCfcAfuCfaGfuGfgCfgAfu	1892	GfAfgAfuCfgCfcAfcUfgAfuGf	1893
		CfuCfdTsdT		gAfgdTsdT
1338	1338-1356	caUfcAfgUfgGfcGfaUfcUfc	1894	GfUfgGfaGfaUfcGfcCfaCfuGf	1895
		CfaCfdTsdT		aUfgdTsdT
1344	1344-1362	ugGfcGfaUfcUfcCfaCfaUfc	1896	CfAfgGfaUfgUfgGfaGfaUfcGf	1897
		CfuGfdTsdT		cCfadTsdT
1345	1345-1363	ggCfgAfuCfuCfcAfcAfuCfc	1898	GfCfaGfgAfuGfuGfgAfgAfuC	1899
		UfgCfdTsdT		fgCfcdTsdT
1346	1346-1364	gcGfaUfcUfcCfaCfaUfcCfu	1900	GfGfcAfgGfaUfgUfgGfaGfaUf	1901
		GfcCfdTsdT		cGfcdTsdT
1347	1347-1365	cgAfuCfuCfcAfcAfuCfcUfg	1902	CfGfgCfaGfgAfuGfuGfgAfgA	1903
		CfcGfdTsdT		fuCfgdTsdT
1348	1348-1366	gaUfcUfcCfaCfaUfcCfuGfcC	1904	CfCfgGfcAfgGfaUfgUfgGfaGf	1905
		fgGfdTsdT		aUfcdTsdT
1353	1353-1371	ccAfcAfuCfcUfgCfcGfgUfg	1906	UfGfcCfaCfcGfgCfaGfgAfuGf	1907
		GfcAfdTsdT		uGfgdTsdT
1354	1354-1372	caCfaUfcCfuGfcCfgGfuGfg	1908	AfUfgCfcAfcCfgGfcAfgGfaUf	1909
		CfaUfdTsdT		gUfgdTsdT
1355	1355-1373	acAfuCfcUfgCfcGfgUfgGfc	1910	AfAfuGfcCfaCfcGfgCfaGfgAf	1911
		AfuUfdTsdT		uGfudTsdT
1357	1357-1375	auCfcUfgCfcGfgUfgGfcAfu	1912	UfAfaAfuGfcCfaCfcGfgCfaGf	1913
		UfuAfdTsdT		gAfudTsdT
1360	1360-1378	cuGfcCfgGfuGfgCfaUfuUfa	1914	CfCfcUfaAfaUfgCfcAfcCfgGf	1915
		GfgGfdTsdT		cAfgdTsdT
1361	1361-1379	ugCfcGfgUfgGfcAfuUfuAfg	1916	CfCfcCfuAfaAfuGfcCfaCfcGf	1917
		GfgGfdTsdT		gCfadTsdT
1362	1362-1380	gcCfgGfuGfgCfaUfuUfaGfg	1918	AfCfcCfcUfaAfaUfgCfcAfcCf	1919
		GfgUfdTsdT		gGfcdTsdT
1363	1363-1381	ccGfgUfgGfcAfuUfuAfgGfg	1920	CfAfcCfcCfuAfaAfuGfcCfaCf	1921
		GfuGfdTsdT		cGfgdTsdT
1366	1366-1384	guGfgCfaUfuUfaGfgGfgUfg	1922	AfGfuCfaCfcCfcUfaAfaUfgCf	1923
		AfcUfdTsdT		cAfcdTsdT
1369	1369-1387	gcAfuUfuAfgGfgGfuGfaCfu	1924	AfGfgAfgUfcAfcCfcCfuAfaAf	1925
		CfcUfdTsdT		uGfcdTsdT
1370	1370-1388	caUfuUfaGfgGfgUfgAfcUfc	1926	AfAfgGfaGfuCfaCfcCfcUfaAf	1927
		CfuUfdTsdT		aUfgdTsdT
1371	1371-1389	auUfuAfgGfgGfuGfaCfuCfc	1928	GfAfaGfgAfgUfcAfcCfcCfuAf	1929
		UfuCfdTsdT		aAfudTsdT
1372	1372-1390	uuUfaGfgGfgUfgAfcUfcCfu	1930	UfGfaAfgGfaGfuCfaCfcCfcUf	1931
		UfcAfdTsdT		aAfadTsdT
1373	1373-1391	uuAfgGfgGfuGfaCfuCfcUfu	1932	GfUfgAfaGfgAfgUfcAfcCfcCf	1933
		CfaCfdTsdT		uAfadTsdT
1374	1374-1392	uaGfgGfgUfgAfcUfcCfuUfc	1934	UfGfuGfaAfgGfaGfuCfaCfcCf	1935
		AfcAfdTsdT		cUfadTsdT
1404	1404-1422	ucUfgGfaUfcCfaCfaGfgAfa	1936	CfAfgUfuCfcUfgUfgGfaUfcCf	1937
		CfuGfdTsdT		aGfadTsdT
1408	1408-1426	gaUfcCfaCfaGfgAfaCfuGfg	1938	UfAfuCfcAfgUfuCfcUfgUfgGf	1939
		AfuAfdTsdT		aUfcdTsdT
1409	1409-1427	auCfcAfcAfgGfaAfcUfgGfa	1940	AfUfaUfcCfaGfuUfcCfuGfuGf	1941
		UfaUfdTsdT		gAfudTsdT
1411	1411-1429	ccAfcAfgGfaAfcUfgGfaUfa	1942	GfAfaUfaUfcCfaGfuUfcCfuGf	1943
		UfuCfdTsdT		uGfgdTsdT
1412	1412-1430	caCfaGfgAfaCfuGfgAfuAfu	1944	AfGfaAfuAfuCfcAfgUfuCfcUf	1945
		UfcUfdTsdT		gUfgdTsdT
1419	1419-1437	acUfgGfaUfaUfuCfuGfaAfa	1946	GfGfuUfuUfcAfgAfaUfaUfcCf	1947
		AfcCfdTsdT		aGfudTsdT
1426	1426-1444	auUfcUfgAfaAfaCfcGfuAfa	1948	CfCfuUfuAfcGfgUfuUfuCfaGf	1949
		AfgGfdTsdT		aAfudTsdT
1427	1427-1445	uuCfuGfaAfaAfcCfgUfaAfa	1950	UfCfcUfuUfaCfgGfuUfuUfcAf	1951
		GfgAfdTsdT		gAfadTsdT
1430	1430-1448	ugAfaAfaCfcGfuAfaAfgGfa	1952	AfUfuUfcCfuUfuAfcGfgUfuU	1953
		AfaUfdTsdT		fuCfadTsdT
1431	1431-1449	gaAfaAfcCfgUfaAfaGfgAfa	1954	GfAfuUfuCfcUfuUfaCfgGfuUf	1955
		AfuCfdTsdT		uUfcdTsdT
siRNA Sequence with Chemical Modification Info
lower case (n) = 2′-O-Me;
Nf = 2′-F;
dT = deoxy-T residue;
s = phosphorothioate backbone modification;
iB = inverted abasic

TABLE 7
AR Target Sequences
			SEQ ID
ID	Code	Target Sequence	NO:	NM_000044.3	Exon	Species
XD-	17	CAAAGGUUCUCUGCUAGAC	1956	1987-2005	1	h
01817K1		GACA
XD-	27	UCUGGGUGUCACUAUGGAG	1957	2819-2837	2	h
01827K1		CUCU
XD-	28	CUGGGUGUCACUAUGGAGC	1958	2820-2838	2	h
01828K1		UCUC
XD-	29	GGGUGUCACUAUGGAGCUC	1959	2822-2840	2	h
01829K1		UCAC
XD-01821K1	21	UACUACAACUUUCCACUGGC	1960	2207-2225	1	h
		UCU
XD-01825K1	25	AAGCUUCUGGGUGUCACUA	1961	2814-2832	2	h, m
		UGGA
XD-01826K1	26	CUUCUGGGUGUCACUAUGG	1962	2817-2835	2	h
		AGCU

TABLE 8
β-catenin Target Sequences
	Generic
R #	name	Gene	Target sequences
R-	1797mfm	CTNNB1	CUGUUGGAUUGAU	SEQ ID	UUUCGAAUCAAUCCA	SEQ ID
1146			UCGAAAUU	NO.	ACAGUU	NO:
				1963		1964
R-	1870mfm	CTNNB1	ACGACUAGUUCAGU	SEQ ID	AAGCAACUGAACUAG	SEQ ID
1147			UGCUUUU	NO.	UCGUUU	NO:
				1965		1966

TABLE 9
β-catenin and β-catenin associated siRNA Sequences
			Sense Strand Sequence	SEQ	Antisense Strand	SEQ
	Generic		(5′-3′)	ID	Sequence (5′-3′)	ID
R #	name	Gene	Passenger Strand (PS)2	NO:	Guide Strand (GS)3	NO:
R-	1797mfm	CTNNB1	iBcsuGfuUfgGfaUfuGfa	1967	usUfsusCfgAfaUfcAfaUfcCfa	1968
1146			UfuCfgAfaAfusuiB		AfcAfgusu
R-	1870mfm	CTNNB1	iBascGfaCfuAfgUfuCfa	1969	asAfsgsCfaAfcUfgAfaCfuA	1970
1147			GfuUfgCfuUfusuiB		fgUfcGfuusu
R-	PA1746	1746	GCUCAAAGCAAUUU	1971	UGUAGAAAUUGCUUU	1972
1150			CUACAdTsdT		GAGCdTsdT
R-	PA2328	2328	GGAUGAAACACAAA	1973	UACCUUUUGUGUUUCA	1974
1151			AGGUAdTsdT		UCCdTsdT
R-	PA2522	2522	UGUCAGAGUUACUG	1975	UGAAACAGUAACUCUG	1976
1152			UUUCAdTsdT		ACAdTsdT
R-	PA3484	3484	AGCAAGAACAGAAA	1977	UUUUAUUUCUGUUCU	1978
1153			UAAAAdTsdT		UGCUdTsdT
R-	PA5018	5018	CUAGUUCAUUUCAA	1979	UAAUUUUGAAAUGAA	1980
1154			AAUUAdTsdT		CUAGdTsdT
R-	PB183	183	CAAGUUCACAAUUA	1981	UUGGGUAAUUGUGAA	1982
1155			CCCAAdTsdT		CUUGdTsdT
R-	PB272	272	GCUUGAAGAUGAAA	1983	UCGUGUUUCAUCUUCA	1984
1156			CACGAdTsdT		AGCdTsdT
R-	PB862	862	AGAUCAAGAAAAUG	1985	UCAUACAUUUUCUUGA	1986
1157			UAUGAdTsdT		UCUdTsdT
R-	PB948	948	CCAAAGAAAACACG	1987	UAAUUCGUGUUUUCU	1988
1158			AAUUAdTsdT		UUGGdTsdT
R-	PB1520	1520	CUUCGAUAAGAUUA	1989	UUCAAUAAUCUUAUCG	1990
1159			UUGAAdTsdT		AAGdTsdT
R-	Myc953U	953	AGGAACUAUGACCU	1991	AGUCGAGGUCAUAGU	1992
1160			CGACUdTsdT		UCCUdTsdT
R-	Myc622U	622	ACGACGAGACCUUC	1993	UUGAUGAAGGUCUCG	1994
1161			AUCAAdTsdT		UCGUdTsdT
R-	Myc1370U	1370	AAGAUGAGGAAGAA	1995	UCGAUUUCUUCCUCAU	1996
1162			AUCGAdTsdT		CUUdTsdT
R-	Myc1364U	1364	AGGAAGAAAUCGAU	1997	ACAACAUCGAUUUCUU	1998
1163			GUUGUdTsdT		CCUdTsdT
R-	Myc1711U	1711	AGCUUUUUUGCCCU	1999	CACGCAGGGCAAAAAA	2000
1164			GCGUGdTsdT		GCUdTsdT
R-	Myc1769U	1769	AGGUAGUUAUCCUU	2001	UUUUUAAGGAUAACU	2002
1165			AAAAAdTsdT		ACCUdTsdT
siRNA Sequence with Chemical Modification Info
lower case (n) = 2′-O-Me;
Nf = 2′-F;
dT = deoxy-T residue;
s = phosphorothioate backbone modification;
iB = inverted abasic

TABLE 10
PIK3CA* and PIK3CB* Target Sequences
Gene	Gene			SEQ ID
Symbol	ID	Name	Target Sequences (97-mer)	NO:
PIK3CA	5290	PIK3CA_1746	TGCTGTTGACAGTGAGCGCCAGCTCAAAGCAATTT	2003
			CTACATAGTGAAGCCACAGATGTATGTAGAAATTG
			CTTTGAGCTGTTGCCTACTGCCTCGGA
PIK3CA	5290	PIK3CA_2328	TGCTGTTGACAGTGAGCGAAAGGATGAAACACAAA	2004
			AGGTATAGTGAAGCCACAGATGTATACCTTTTGTGT
			TTCATCCTTCTGCCTACTGCCTCGGA
PIK3CA	5290	PIK3CA_2522	TGCTGTTGACAGTGAGCGCCATGTCAGAGTTACTG	2005
			TTTCATAGTGAAGCCACAGATGTATGAAACAGTAA
			CTCTGACATGATGCCTACTGCCTCGGA
PIK3CA	5290	PIK3CA_3555	TGCTGTTGACAGTGAGCGCAACTAGTTCATTTCAA	2006
			AATTATAGTGAAGCCACAGATGTATAATTTTGAAA
			TGAACTAGTTTTGCCTACTGCCTCGGA
PIK3CA	5290	PIK3CA_3484	TGCTGTTGACAGTGAGCGCACAGCAAGAACAGAAA	2007
			TAAAATAGTGAAGCCACAGATGTATTTTATTTCTGT
			TCTTGCTGTATGCCTACTGCCTCGGA
PIK3CB	5291	PIK3CB_862	TGCTGTTGACAGTGAGCGACAAGATCAAGAAAATG	2008
			TATGATAGTGAAGCCACAGATGTATCATACATTTTC
			TTGATCTTGCTGCCTACTGCCTCGGA
PIK3CB	5291	PIK3CB_183	TGCTGTTGACAGTGAGCGCAGCAAGTTCACAATTA	2009
			CCCAATAGTGAAGCCACAGATGTATTGGGTAATTG
			TGAACTTGCTTTGCCTACTGCCTCGGA
PIK3CB	5291	PIK3CB_1520	TGCTGTTGACAGTGAGCGCCCCTTCGATAAGATTAT	2010
			TGAATAGTGAAGCCACAGATGTATTCAATAATCTT
			ATCGAAGGGATGCCTACTGCCTCGGA
PIK3CB	5291	PIK3CB_272	TGCTGTTGACAGTGAGCGAGAGCTTGAAGATGAAA	2011
			CACGATAGTGAAGCCACAGATGTATCGTGTTTCAT
			CTTCAAGCTCCTGCCTACTGCCTCGGA
PIK3CB	5291	PIK3CB_948	TGCTGTTGACAGTGAGCGACACCAAAGAAAACACG	2012
			AATTATAGTGAAGCCACAGATGTATAATTCGTGTTT
			TCTTTGGTGGTGCCTACTGCCTCGGA
*Species is Homo sapiens.

TABLE 11
PIK3CA and PIK3CB siRNA Sequences
				SEQ		SEQ
Gene	Gene			ID		ID
Symbol	ID	Name	siRNA Guide	NO:	siRNA passenger	NO:
PIK3CA	5290	PIK3CA_1746	UGUAGAAAUUGCUU	2013	AGCUCAAAGCAAUUU	2014
			UGAGCUGU		CUACAUA
PIK3CA	5290	PIK3CA_2328	UACCUUUUGUGUUU	2015	AGGAUGAAACACAAA	2016
			CAUCCUUC		AGGUAUA
PIK3CA	5290	PIK3CA_2522	UGAAACAGUAACUC	2017	AUGUCAGAGUUACUG	2018
			UGACAUGA		UUUCAUA
PIK3CA	5290	PIK3CA_3555	UAAUUUUGAAAUGA	2019	ACUAGUUCAUUUCAA	2020
			ACUAGUUU		AAUUAUA
PIK3CA	5290	PIK3CA_3484	UUUUAUUUCUGUUC	2021	CAGCAAGAACAGAAA	2022
			UUGCUGUA		UAAAAUA
PIK3CB	5291	PIK3CB_862	UCAUACAUUUUCUU	2023	AAGAUCAAGAAAAUG	2024
			GAUCUUGC		UAUGAUA
PIK3CB	5291	PIK3CB_183	UUGGGUAAUUGUGA	2025	GCAAGUUCACAAUUA	2026
			ACUUGCUU		CCCAAUA
PIK3CB	5291	PIK3CB_1520	UUCAAUAAUCUUAU	2027	CCUUCGAUAAGAUUA	2028
			CGAAGGGA		UUGAAUA
PIK3CB	5291	PIK3CB_272	UCGUGUUUCAUCUU	2029	AGCUUGAAGAUGAAA	2030
			CAAGCUCC		CACGAUA
PIK3CB	5291	PIK3CB_948	UAAUUCGUGUUUUC	2031	ACCAAAGAAAACACG	2032
			UUUGGUGG		AAUUAUA

TABLE 12
Additional polynucleic acid molecule sequences
	Base		SEQ
	start		ID		SEQ ID
	position	Guide strand	NO:	Passenger strand	NO:
EGFR	333	ACUCGUGCCUUGGCAA	2082	AGUUUGCCAAGGCACGAG	2083
R1246		ACUUU		UUU
EGFR	333	ACUCGUGCCUUGGCAA	2084	AGUUUGCCAAGGCACGAG	2085
R1195		ACUUU		UUU
EGFR	333	ACUCGUGCCUUGGCAA	2086	AGUUUGCCAAGGCACGAG	2087
R1449		ACUUU		UUU
KRAS	237	UGAAUUAGCUGUAUCG	2088	TGACGAUACAGCUAAUUC	2089
R1450		UCAUU		AUU
KRAS	237	UGAAUUAGCUGUAUCG	2090	UGACGAUACAGCUAAUUC	2091
R1443		UCAUU		AUU
KRAS	237	UGAAUUAGCUGUAUCG	2092	UGACGAUACAGCUAAUUC	2093
R1194		UCAUU		AUU
CTNNB1	1248	UAAGUAUAGGUCCUCA	2094	UAAUGAGGACCUAUACU	2095
R1442		UUAUU		UAUU
CTNNB1	1797	TUUCGAAUCAAUCCAA	2096	CUGUUGGAUUGAUUCGA	2097
R1404		CAGUU		AAUU
CTNNB1	1797	UUUCGAAUCAAUCCAA	2098	CUGUUGGAUUGAUUCGA	2099
R1441		CAGUU		AAUU
CTNNB1	1797	UUUCGAAUCAAUCCAA	2100	CUGUUGGAUUGAUUCGA	2101
R1523		CAGUU		AAUU
HPRT	425	AUAAAAUCUACAGUCA	2102	CUAUGACUGUAGAUUUU	2103
R1492		UAGUU		AUUU
HPRT	425	UUAAAAUCUACAGUCA	2104	CUAUGACUGUAGAUUUU	2105
R1526		UAGUU		AAUU
HPRT	425	UUAAAAUCUACAGUCA	2106	CUAUGACUGUAGAUUUU	2107
R1527		UAGUU		AAUU
AR	2822	GAGAGCUCCAUAGUGA	2108	GUGUCACUAUGGAGCUCU	2109
R1245		CACUU		CUU

Example 2. General Experimental Protocol

Stem-Loop qPCR Assay for Quantification of siRNA

Plasma samples were directly diluted in TE buffer. 50 mg tissue pieces were homogenized in 1 mL of Trizol using a FastPrep-24 tissue homogenizer (MP Biomedicals) and then diluted in TE buffer. Standard curves were generated by spiking siRNA into plasma or homogenized tissue from untreated animals and then serially diluting with TE buffer. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit (Applied Biosystems) with 25 nM of a sequence-specific stem-loop RT primer. The cDNA from the RT step was utilized for real-time PCR using TaqMan Fast Advanced Master Mix (Applied Biosystems) with 1.5 μM of forward primer, 0.75 μM of reverse primer, and 0.2 μM of probe. The sequences of KRAS and EGFR siRNA antisense strands and all primers and probes used to measure them are shown in Table 13. Quantitative PCR reactions were performed using standard cycling conditions in a ViiA 7 Real-Time PCR System (Life Technologies). The Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 13
Sequences for all siRNA antisense strands, primers, and probes
used in the stem-loop qPCR assay.
Target	Name	Sequence (5′-3′)	SEQ ID NO:
KRAS	Anti sense	UGAAUUAGCUGUAUCGUCAUU	2033
KRAS	RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGA	2034
		TACGACAATGACG
KRAS	Forward	GGCGGCTGAATTAGCTGTATCGT	2035
KRAS	Reverse	AGTGCAGGGTCCGAG	2036
KRAS	Probe	(6FAM)-TGGATACGACAATGAC-(NFQ-MGB)	2037
EGFR	Antisense	ACUCGUGCCUUGGCAAACUUU	2038
EGFR	RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGA	2039
		TACGACAAAGTTTG
EGFR	Forward	GGCGGCACTCGTGCCTTGGCA	2040
EGFR	Reverse	AGTGCAGGGTCCGAG	2041
EGFR	Probe	(6FAM)-TGGATACGACAAAGTT-(NFQ-MGB)	2042

Comparative qPCR Assay for Determination of mRNA Knockdown

Tissue samples were homogenized in Trizol as described above. Total RNA was isolated using RNeasy RNA isolation 96-well plates (Qiagen), then 500 ng RNA was reverse transcribed with a High Capacity RNA to cDNA kit (ThermoFisher). KRAS, EGFR, CTNNB1 and PPIB mRNA was quantified by TaqMan qPCR analysis performed with a ViiA 7 Real-Time PCR System. The TaqMan primers and probes for KRAS were designed and validated by Avidity and are shown in Table 14. The TaqMan primers and probes for EGFR and CTNNB1 were purchased from Applied Biosystems as pre-validated gene expression assays. PPIB (housekeeping gene) was used as an internal RNA loading control, with all TaqMan primers and probes for PPIB purchased from Applied Biosystems as pre-validated gene expression assays. Results are calculated by the comparative Ct method, where the difference between the target gene (KRAS, CTNNB1, or EGFR) Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt).

TABLE 14
Sequences of primers and probes for KRAS mRNA detection
using comparative qPCR assay.
Target	Species	Name	Sequence (5′-3′)	SEQ ID NO:
KRAS	Mouse	Forward	CGCCTTGACGATACAGCTAAT	2043
KRAS	Mouse	Reverse	TGTTTCCTGTAGGAGTCCTCTAT	2044
KRAS	Mouse	Probe	(6FAM)-	2045 and 2114
			TCACTTTGT(Zen)GGATGAGTATGACCCTACG-
			(IABkFQ)
KRAS	Human	Forward	GTGCCTTGACGATACAGCTAAT	2046
KRAS	Human	Reverse	CCAAGAGACAGGTTTCTCCATC	2047
KRAS	Human	Probe	(6FAM)-	2048 and 2115
			CCAACAATA(Zen)GAGGATTCCTACAGGAAGCA-
			(IABkFQ)

Animals

All animal studies were conducted following protocols in accordance with the Institutional Animal Care and Use Committee (IACUC) at Explora BioLabs, which adhere to the regulations outlined in the USDA Animal Welfare Act as well as the “Guide for the Care and Use of Laboratory Animals” (National Research Council publication, 8th Ed., revised in 2011). All mice were obtained from either Charles River Laboratories or Harlan Laboratories.

H358, HCC827, and Hep-3B2 1-7 Subcutaneous Flank Tumor Model

For the H358 subcutaneous flank tumor model, tumor cells were inoculated and tumors were established according to the following methods. Female NCr nu/nu mice were identified by ear-tag the day before cell injection. Mice were weighed prior to inoculation. H358 cells were cultured with 10% FBS/RPMI medium and harvested with 0.05% Trypsin and Cell Stripper (MediaTech). 5 million H358 cells in 0.05 ml Hank's Balanced Salt Solution (HBSS) with Matrigel (1:1) were injected subcutaneously (SC) into the upper right flank of each mouse. Tumor growth was monitored by tumor volume measurement using a digital caliper starting on day 7 after inoculation, and followed 2 times per week until average tumor volume reaches >100 & ≤300 mm³. Once tumors were staged to the desired volume (average from 100 to 300 mm³), animals were randomized and mice with very large or small tumors were culled. Mice were divided into the required groups and randomized by tumor volume. Mice were then treated as described in the individual experiments.

For the Hep3B orthotropic liver tumor model, tumor cells were inoculated and tumors were established according to the following methods. Female NCr nu/nu mice were identified by ear-tag the day before, mice will be anesthetized with isoflurane. The mice were then placed in a supine position on a water circulating heating pad to maintain body temperature. A small transverse incision below the sternum will be made to expose the liver. Cancer cells were slowly injected into the upper left lobe of the liver using a 28-gauge needle. The cells were injected at a 30-degree angle into the liver, so that a transparent bleb of cells can be seen through the liver capsule. Hep 3B2.1 7 cells were prepared by suspending in cold PBS (0.1-5×10⁶ cells) and mixing with diluted matrigel (30× in PBS). 30-50 ul of the cell/matrigel was inoculated. After injection, a small piece of sterile gauze was placed on the injection site, and light pressure was applied for 1 min to prevent bleeding. The abdomen was then closed with a 6-0 silk suture. After tumor cell implantation, animals were kept in a warm cage, observed for 1-2 h, and subsequently returned to the animal room after full recovery from the anesthesia. 7-10 days after tumor implantation animals were randomized, divided into the required groups and then treated as described in the individual experiments.

LNCap Subcutaneous Flank Tumor Model

LNCaP cells (ATCC®CRL-174™) were grown in RPMI+10% FBS supplemented with non-essential amino acids and sodium pyruvate to a confluency of about 80%. Cells were mixed 1:1 with matrigel and 5-7*106 cells injected subcutaneously into male SCID mice (6-8 weeks). Tumors usually developed within 3-5 weeks to a size of 100-350 mm³. Animals bearing tumors within this range were randomized and treated with ASCs by injections into the tail vein. For PD studies animals were sacrificed 96 hours after injection and organ fragments harvested, weighted, and frozen in liquid nitrogen. For RNA isolation, organ samples were homogenized in Trizol and RNA prepared using a Qiagen RNeasy 96 Plus kit following the instructions by the manufacturer. RNA concentrations were determined spectroscopically. RNAs were converted into cDNAs by reverse transcription and expression of specific targets quantified by qPCR using the ΔΔCT method and validated Taqman assays (Thermofisher). Samples were standardize to the expression levels of PPIB.

Cholesterol siRNA Conjugate Synthesis

All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. Structure of cholesterol conjugated to the passenger strand is illustrated in FIG. 2. Table 15 shows KRAS, EGFR, and CTNNB1 siRNA sequences.

TABLE 15
			MW	SEQ ID
siRNA	Strand	Sequence (5′-3′)	observed	NO:
KRAS	Passenger	Chol-iBusgAfcGfaUfaCfaGfcUfaAfuUfcAfusuiB	7813.6	2049
KRAS	Guide	UfsGfsasAfuUfaGfcUfgUfaUfcGfuCfausu	6874.6	2050
EGFR	Passenger	Chol-iBasgUfuUfgCfcAfaGfgCfaCfgAfgUfusuiB	7884.6	2051
EGFR	Guide	asCfsusCfgUfgCfcUfuGfgCfaAfaCfuusu	6860.6	2052
CTNNB1	Passenger	Chol-iBcsuGfuUfgGfaUfuGfaUfuCfgAfaAfusuiB	7847.5	2053
CTNNB1	Guide	usUfsusCfgAfaUfcAfaUfcCfaAfcAfgusu	6852.6	2054

The siRNA chemical modifications include:

• • upper case (N)=2′-OH (ribo);
  • lower case (n)=2′-O-Me (methyl);
  • dN=2′-H (deoxy);
  • Nf=2′-F (fluoro);
  • s=phosphorothioate backbone modification;
  • iB=inverted abasic

Peptide Synthesis

Peptides were synthesized on solid phase using standard Fmoc chemistry. Both peptides have cysteine at the N-terminus and the cleaved peptides were purified by HPLC and confirmed by mass spectroscopy. INF7 peptide is as illustrated in FIG. 3 (SEQ ID NO: 2055). Melittin peptide is as illustrated in FIG. 4 (SEQ ID NO: 2060).

Anti-EGFR Antibody

Anti-EGFR antibody is a fully human IgG1κ monoclonal antibody directed against the human epidermal growth factor receptor (EGFR). It is produced in the Chinese Hamster Ovary cell line DJT33, which has been derived from the CHO cell line CHO-K1SV by transfection with a GS vector carrying the antibody genes derived from a human anti-EGFR antibody producing hybridoma cell line (2F8). Standard mammalian cell culture and purification technologies are employed in the manufacturing of anti-EGFR antibody.

The theoretical molecular weight (MW) of anti-EGFR antibody without glycans is 146.6 kDa. The experimental MW of the major glycosylated isoform of the antibody is 149 kDa as determined by mass spectrometry. Using SDS-PAGE under reducing conditions the MW of the light chain was found to be approximately 25 kDa and the MW of the heavy chain to be approximately 50 kDa. The heavy chains are connected to each other by two inter-chain disulfide bonds, and one light chain is attached to each heavy chain by a single inter-chain disulfide bond. The light chain has two intra-chain disulfide bonds and the heavy chain has four intra-chain disulfide bonds. The antibody is N-linked glycosylated at Asn305 of the heavy chain with glycans composed of N-acetyl-glucosamine, mannose, fucose and galactose. The predominant glycans present are fucosylated bi-antennary structures containing zero or one terminal galactose residue.

The charged isoform pattern of the IgG1κ antibody has been investigated using imaged capillary IEF, agarose IEF and analytical cation exchange HPLC. Multiple charged isoforms are found, with the main isoform having an isoelectric point of approximately 8.7.

The major mechanism of action of anti-EGFR antibody is a concentration dependent inhibition of EGF-induced EGFR phosphorylation in A431 cancer cells. Additionally, induction of antibody-dependent cell-mediated cytotoxicity (ADCC) at low antibody concentrations has been observed in pre-clinical cellular in vitro studies.

Example 3: Synthesis, Purification and Analysis of Antibody-PEG-EGFR and Antibody-EGFR Conjugates—Conjugation Scheme 1

Step 1: Antibody Conjugation with Maleimide-PEG-NHS Followed by SH-EGFR

Anti-EGFR antibody (EGFR-Ab) was exchanged with 1× Phosphate buffer (pH 7.4) and made up to 5 mg/ml concentration. To this solution, 2 equivalents of SMCC linker or maleimide-PEGxkDa-NHS (x=1, 5, 10, 20) was added and rotated for 4 hours at room temperature. Unreacted maleimide-PEG was removed by spin filtration using 50 kDa MWCO Amicon spin filters and PBS pH 7.4. The antibody-PEG-Mal conjugate was collected and transferred into a reaction vessel. SH-C6-EGFR (2 equivalents) was added at RT to the antibody-PEG-maleimide in PBS and rotated overnight, see FIG. 88. The reaction mixture was analyzed by analytical SAX column chromatography and conjugate along with unreacted antibody and siRNA was seen.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1. Fractions containing the antibody-PEG-EGFR conjugate were pooled, concentrated and buffer exchanged with PBS, pH 7.4. Antibody siRNA conjugates with SMCC linker, PEG1kDa, PEG5kDa and PEG10kDa were separated based on the siRNA loading. Conjugates with PEG20kDa gave poor separation.

Step-3: Analysis of the Purified Conjugate

The isolated conjugate was characterized by either mass spec or SDS-PAGE. The purity of the conjugate was assessed by analytical HPLC using either anion exchange chromatography method-2 or anion exchange chromatography method-3. Examples of all the conjugates made using these methods are described in Table 16.

TABLE 16
List of AXCYB conjugates
	HPLC retention time (minutes) with Anion
	exchange chromatography method-2
Conjugate	DAR = 1	DAR = 2	DAR => 2
EGFR-Ab-EGFR	9.0	9.9	10.4
EGFR-Ab-PEG1kDa-EGFR	9.2	10.0	10.6
EGFR-Ab-PEG5kDa-EGFR	8.7	9.3	ND
EGFR-Ab-PEG10kDa-EGFR	8.6	8.8 to 10; mix of DAR 2-3
EGFR-Ab-PEG20kDa-EGFR	8.6; Mixture of DAR of 1-3
Holo-anti-B cell Ab-	9.2		9.5
PEG20kDa-EGFR

Anion Exchange Chromatography Method-1

• 1. Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5 mm ID×15 cm, 13 um
• 2. Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 mM TRIS, 1.5 M NaCl, pH 8.0; Flow Rate: 6.0 ml/min
• 3. Gradient:

	a.	% A	% B	Column Volume
	b.	100	0	1.00
	c.	60	40	18.00
	d.	40	60	2.00
	e.	40	60	5.00
	f.	0	100	2.00
	g.	100	0	2.00

Anion Exchange Chromatography Method-2

• 1. Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm
• 2. Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80% 10 mM TRIS pH 8, 20% ethanol, 1.5 M NaCl; Flow Rate: 1.0 ml/min
• 3. Gradient:

	a.	Time	% A	% B
	b.	0.0	90	10
	c.	3.00	90	10
	d.	11.00	40	60
	e.	13.00	40	60
	f.	15.00	90	10
	g.	20.00	90	10

Anion Exchange Chromatography Method-3

• 1. Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm
• 2. Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80% 10 mM TRIS pH 8, 20% ethanol, 1.5 M NaCl
• 3. Flow Rate: 0.75 ml/min
• 4. Gradient:

	a.	Time	% A	% B
	b.	0.0	90	10
	c.	3.00	90	10
	d.	11.00	40	60
	e.	23.00	40	60
	f.	25.00	90	10
	g.	30.00	90	10

The analytical data for EGFR antibody-PEG20kDa-EGFR are illustrated in FIG. 5 and FIG. 6. FIG. 5 shows the analytical HPLC of EGFR antibody-PEG20kDa-EGFR. FIG. 6 shows a SDS-PAGE analysis of EGFR antibody-PEG20kDa-EGFR conjugate. The analytical chromatogram of EGFR antibody-PEG10kDa-EGFR is illustrated in FIG. 7. The analytical data for EGFR antibody-PEG5kDa-EGFR are illustrated in FIG. 8 and FIG. 9. FIG. 8 shows the analytical chromatogram of EGFR antibody-PEG5kDa-EGFR. FIG. 9 shows SDS PAGE analysis of EGFR antibody-PEG10kDa-EGFR and EGFR antibody-PEG5kDa-EGFR conjugates. The analytical data for EGFR antibody-PEG1kDa-EGFR conjugates with different siRNA loading is illustrated in FIG. 10.

Example 4: Synthesis, Purification and Analysis of Antibody-siRNA-PEG Conjugates—Conjugation Scheme-2

Step 1: Antibody Conjugation with SMCC Linker Followed by SH-KRAS-PEG5kDa

Anti-EGFR antibody was exchanged with 1× Phosphate buffer (pH 7.4) and made up to 5 mg/ml concentration. To this solution, 2 equivalents of SMCC linker (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) was added and rotated for 4 hours at room temperature, see FIG. 89. Unreacted SMCC linker was removed by spin filtration using 50 kDa MWCO Amicon spin filters and PBS buffer pH 7.4. The retentate was collected and 2 equivalents of SH-C6-KRAS-PEG5kDa was added at RT and rotated overnight. The reaction mixture was analyzed by analytical SAX column chromatography and the conjugate along with unreacted antibody and siRNA was observed.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1. Fractions containing the antibody-KRAS-PEG conjugate were pooled, concentrated and buffer exchanged with PBS, pH 7.4.

Step-3: Analysis of the Purified Conjugate

The isolated conjugate was characterized by either mass spec or SDS-PAGE. The purity of the conjugate was assessed by analytical HPLC using anion exchange chromatography method-3 (described in example 1). Examples of the conjugates made using the methods described in Examples 4 and 5 are illustrated in Table 17.

TABLE 17
List of A-X-B-Y-C conjugates
	HPLC retention time (minutes) with Anion
	exchange chromatography method-3
Conjugate	DAR = 1	DAR = 2	DAR => 2
EGFR-Ab-KRAS-PEG5kDa	9.2
EGFR-Ab-S-S-KRAS-	9.0
PEG5kDa
Holo-anti-B cell Ab-KRAS-	9.2	9.7	10.1
PEG5kDa
Panitumumab-KRAS-	9.2	9.7	10.2
PEG5kDa

The HPLC chromatogram of EGFR Antibody-KRAS-PEG5kDa is illustrated in FIG. 11. The HPLC chromatogram of Panitumumab-KRAS-PEG5kDa is as shown in FIG. 12.

Example 5: Synthesis, Purification and Analysis of Antibody-S—S-siRNA-PEG Conjugates—Conjugation Scheme-3

Step 1: Antibody Conjugation with SPDP Linker Followed by SH-siRNA-PEG5kDa

Anti-EGFR antibody was exchanged with 1× Phosphate buffer (pH 7.4) and made up to 5 mg/ml concentration. To this solution, 2 equivalents of SPDP linker (succinimidyl 3-(2-pyridyldithio)propionate) was added and rotated for 4 hours at room temperature. Unreacted SPDP linker was removed by spin filtration using 50 kDa MWCO Amicon spin filters and pH 7.4 PBS buffer. The retentate was collected and 2 equivalents of SH-C6-siRNA-PEG5kDa was added at room temperature and rotated overnight, see FIG. 90. The reaction mixture was analyzed by analytical SAX column chromatography and conjugate along with unreacted antibody and siRNA was seen.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1. Fractions containing the antibody-PEG-siRNA conjugate were pooled, concentrated and buffer exchanged with PBS, pH 7.4.

Step-3: Analysis of the Purified Conjugate

The isolated conjugate was characterized by either mass spec or SDS-PAGE. The purity of the conjugate was assessed by analytical HPLC using anion exchange chromatography method-2. The HPLC chromatogram of EGFR Antibody-S—S-siRNA-PEG5kDa (DAR=1) is as shown in FIG. 13.

Example 6: Synthesis, Purification and Analysis of Antibody-SMCC-Endosomal Escape Peptide Conjugates—Conjugation Scheme-4

Step 1: Antibody Conjugation with SMCC Linker or Maleimide-PEG-NHS Followed by SH-Cys-Peptide-CONH₂

Anti-EGFR antibody was exchanged with 1× Phosphate buffer (pH 7.4) and made up to 10 mg/ml concentration. To this solution, 3 equivalents of SMCC linker (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) or maleimide-PEG1kDa-NHS was added and rotated for 1.5 hours at room temperature. Unreacted SMCC linker or PEG linker was removed by spin filtration using 50 kDa MWCO Amicon spin filters and PBS buffer pH 7.4 (25 mM MES pH=6.1 for Melittin conjugates). The retentate was collected and 3 equivalents of SH-Cys-Peptide-CONH₂ was added at RT and rotated overnight. See FIG. 91. The reaction mixture was then purified by either HIC chromatography or cation exchange chromatography to isolate the anti-EGFR antibody-Peptide or anti-EGFR antibody-PEG1k-Peptide.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC using either hydrophobic interaction chromatography (HIC) method-1 or cation exchange chromatography method-1. Fractions containing the antibody-peptide conjugates were pooled, concentrated and buffer exchanged with PBS, pH 7.4 (10 mM Acetate pH=6.0 for Melittin conjugates).

Step-3: Analysis of the Purified Conjugate

The isolated conjugate was characterized by either mass spec or SDS-PAGE. Purity and peptide loading was assessed by analytical HPLC using either HIC method-2 or cation exchange chromatography method-2. Examples of all the conjugates made using the method of Example 6 are described in Tables 18 and 19.

TABLE 18
List of AXYD conjugates
	HPLC retention time (minutes)
	with HIC method-2
Conjugate	DAR = 1	DAR = 2	DAR => 2
EGFR-Ab-INF7	7.7	9.3	11.2
EGFR-Ab-PEG24-INF7	8.4	12.2	15.2

TABLE 19
List of AXYD conjugates
	HPLC retention time (minutes) with cation
	exchange chromatography method-2
Conjugate	DAR = 1	DAR => 1	DAR => 2
EGFR-Ab-Melittin	40.9	54.8
EGFR-Ab-PEG1kDa-	48.	53.4	55.8
melittin

Cation Exchange Chromatography Method-1

• • 1. Column: GE Healthcare HiPrep SP HP 16/10
  • 2. Solvent A: 50 mM MES pH=6.0; Solvent B: 50 mM MES+0.5M NaCl pH=6.0; Flow Rate: 2.0 ml/min
  • 3. Gradient:

	a.	% A	% B	Column Volume
	b.	100	0	0.1
	c.	100	0	Flush loop 12 ml
	d.	100	0	2.5
	e.	0	100	15
	f.	0	100	5
	g.	100	0	0.5
	h.	100	0	5

Cation Exchange Chromatography Method-2

• • 1. Column: Thermo Scientific, MAbPac™ SCX-10, Bio LC™, 4×250 mm (product #074625)
  • 2. Solvent A: 20 mM MES pH=5.5; Solvent B: 20 mM MES+0.3 M NaCl pH=5.5; Flow Rate: 0.5 ml/min
  • 3. Gradient:

	a.	Time	% A	% B
	b.	0.0	100	0
	c.	5	100	0
	d.	50	0	100
	e.	80	0	100
	f.	85	100	0
	g.	90	100	0

Hydrophobic Interaction Chromatography Method-1 (HIC Method-1)

• • 1. Column: GE Healthcare Butyl Sepharose High Performance (17-5432-02) 200 ml
  • 2. Solvent A: 50 mM Sodium Phosphate+0.8M ammonium sulfate (pH=7.0); Solvent B: 80% 50 mM Sodium Phosphate (pH=7.0), 20% IPA; Flow Rate: 3.0 ml/min
  • 3. Gradient:

	a.	% A	% B	Column Volume
	b.	100	0	0.1
	c.	0	100	3
	d.	0	100	1.35
	e.	100	0	0.1
	f.	100	0	0.5

Hydrophobic Interaction Chromatography Method-2 (HIC Method-2)

• • 1. Column: Tosoh Bioscience TSKgel Butyl-NPR 4.6 mm ID×10 cm 2.5 μm
  • 2. Solvent A: 100 mM Sodium phosphate+1.8 M ammonium sulfate (pH=7.0); Solvent B: 80% 100 mM sodium phosphate (pH=7.0), 20% IPA; Flow Rate: 0.5 ml/min
  • 3. Gradient:

	a.	Time	% A	% B
	b.	0	100	0
	c.	3	50	50
	d.	21	0	100
	e.	23	0	100
	f.	25	100	0

FIG. 14 illustrates the HPLC chromatogram of EGFR antibody-PEG24-Melittin (loading=˜1). FIG. 15 illustrates the HPLC chromatogram of EGFR antibody-Melittin (n=˜1). FIG. 16 shows the mass spectrum of EGFR antibody-Melittin (n=1). FIG. 17 shows the HIC chromatogram of EGFR antibody-PEG1kDa-INF7 (Peptide loading=˜1). FIG. 18 shows the HPLC chromatogram of EGFR antibody-INF7 (Peptide Loading=˜1).

Example 7: Synthesis, Purification and Analysis of EEP-Antibody-siRNA-PEG Conjugates—Conjugation Scheme-5

Step 1: Conjugation of PEG24 Linker Followed by SH-Cys-Peptide-CONH₂ to EGFR-Ab-siRNA-PEG

EGFR-Ab-siRNA-PEG conjugate with a siRNA loading of 1 was conjugated with 4 equivalents of PEG1k linker (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) in PBS, pH 7.4 buffer and rotated for 1.5 hours at room temperature. See FIG. 92. Unreacted PEG1k linker was removed by spin filtration using 50 kDa MWCO Amicon spin filters and PBS buffer pH 7.4. The retentate was collected and 4 equivalents of SH-Cys-Peptide-CONH₂ was added at RT and rotated overnight.

Step 2: Purification

The reaction mixture was then purified by repeated spin filtration using PBS buffer pH7.4 and 50 kDa Amicon spin filters until the unreacted peptide was removed as monitored by HPLC. The product contains a mixture of conjugates with 0, 1, 2, 3 or more peptides conjugated to the antibody backbone.

Step-3: Analysis of the Purified Conjugate

The isolated conjugate was characterized by either mass spec or SDS-PAGE. The purity and the peptide loading of the conjugate was assessed by analytical HPLC using either HIC method-2 or cation exchange chromatography method-2. Examples of the conjugates made using the method described in Example 7 are shown in Table 20.

TABLE 20
List of (A-X-B-Y-Cn)-L-D conjugates
	HPLC retention time (minutes) with cation
	exchange chromatography method-2
Conjugate	DAR = 0	DAR = 1	DAR = 2	DAR = 3
(EGFR-Ab-siRNA-	24	38	27	9
PEG5kDa)-PEG1k-
INF7
(EGFR-Ab-siRNA-	24	11.79 (broad peak)
PEG5kDa)-PEG1k-
melittin

FIG. 19 shows INF7-PEG1kDa-(EGFR antibody-KRAS-PEG5kDa). FIG. 20 shows Melittin-PEG1kDa-(EGFR antibody-KRAS-PEG5kDa).

Example 8: In Vivo Pharmacokinetics Study of a EGFR Antibody-siRNA-PEG Conjugate (PK-055)

Groups (n=3) of female NCr nu/nu mice bearing subcutaneous flank H358 tumors 100-150 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control groups (n=4) of the same mice received one i.v. injection of PBS as a vehicle control. Treatment groups that received EGFR antibody-siRNA-PEG conjugates were dosed at 0.5 mg/kg (based on the weight of siRNA) and groups that received cholesterol-siRNA conjugates were dosed at 15 mg/kg. All groups (treatments and controls) were administered a dose volume of 5 mL/kg. Non-terminal blood samples were collected at 2, 15, or 60 minutes post-dose via puncture of the retro-orbital plexus and centrifuged to generate plasma for PK analysis. Mice were sacrificed by CO₂ asphyxiation at 24, 96, or 168 h post-dose. Table 21 describes the study design in more detail and provides a cross-reference to the conjugate synthesis and characterization. Terminal blood samples were collected via cardiac puncture and processed to generate plasma for PK analysis. 50 mg pieces of tumor, liver, kidney, and lung were collected and snap-frozen in liquid nitrogen. mRNA knockdown analysis and siRNA quantitation were performed as described in Examples 2-7.

TABLE 21
Study design for a EGFR antibody-siRNA-PEG Conjugate (PK-055) with a cross-reference to
the synthesis and characterization of the conjugates tested.
				siRNA:EGFR-
			siRNA	Ab	melittin:siRNA			Survival	Terminal	Harvest	Cross-reference
	Test		Dose	Ratio	Ratio		Dose	Bleed	Bleed	Time	to synthesis and
Group	Article	N	(mg/kg)	(mol/mol)	(mol/mol)	ROA	Schedule	(min)	(h)	(h)	characterization
4	EGFR-	3	0.5	1.4	—	IV	t = 0	2	24	24	Example 3
5	Ab-	3	0.5	1.4	—	IV	t = 0	15	96	96
6	PEG10k-	3	0.5	1.4	—	IV	t = 0	60	168	168
	EGFR
7	EGFR-	3	0.5	1.25	—	IV	t = 0	2	24	24	Example 3
8	Ab-	3	0.5	1.25	—	IV	t = 0	15	96	96
9	PEG5k-	3	0.5	1.25	—	IV	t = 0	60	168	168
	EGFR
10	EGFR-	3	0.5	1.25	—	IV	t = 0	2	24	24	Example 3
11	Ab-	3	0.5	1.25	—	IV	t = 0	15	96	96
12	PEG1k-	3	0.5	1.25	—	IV	t = 0	60	168	168
	EGFR
13	EGFR-	3	0.5	1.3	—	IV	t = 0	2	24	24	Example 3
14	Ab-	3	0.5	1.3	—	IV	t = 0	15	96	96
15	EGFR	3	0.5	1.3	—	IV	t = 0	60	168	168
16	EGFR-	3	0.5	2.6	—	IV	t = 0	2	24	24	Example 4
17	Ab-	3	0.5	2.6	—	IV	t = 0	15	96	96
18	KRAS-	3	0.5	2.6	—	IV	t = 0	60	168	168
	PEG5k
	(n = 2
	siRNAs
	per
	EGFR-
	Ab)
19	EGFR-	3	0.5	1.0	—	IV	t = 0	2	24	24	Example 4
20	Ab-	3	0.5	1.0	—	IV	t = 0	15	96	96
21	KRAS-	3	0.5	1.0	—	IV	t = 0	60	168	168
	PEG5k
	(n = 1
	siRNA
	per
	EGFR-
	Ab)
22	EGFR-	3	0.5	1.0	1:1	IV	t = 0	2	24	24	Example 4 and 6
23	Ab-	3	0.5	1.0	1:1	IV	t = 0	15	96	96
24	KRAS-	3	0.5	1.0	1:1	IV	t = 0	60	168	168
	PEG5k
	(n = 1) +
	EGFR-
	Ab-
	melittin
25	Chol-	3	15	—	—	IV	t = 0	2	24	24	General
26	EGFR-	3	15	—	—	IV	t = 0	15	96	96	experimental
27	333mfm	3	15	—	—	IV	t = 0	60	168	168	(Example 2)
28	Chol-	3	15	—	—	IV	t = 0	2	24	24	General
29	KRAS-	3	15	—	—	IV	t = 0	15	96	96	experimental
30	237ffm	3	15	—	—	IV	t = 0	60	168	168	(Example 2)
31	Vehicle	4	—	—	—	IV	t = 0	—	—	24
32		4	—	—	—	IV	t = 0	—	—	96
33		4	—	—	—	IV	t = 0	—	—	168

PEG linkers of various molecular weights and a small molecule linker were used to attach EGFR siRNA to an EGFR antibody (EGFR-Ab) and the PK was assessed to determine the effect of the linker molecular weight on the behavior of the mAb-siRNA conjugate in plasma. As illustrated in FIG. 21, the molecular weight of the PEG linker does not have a large impact on the plasma PK, except for the 10 kDa PEG leads to a faster siRNA clearance (i.e. lower plasma concentrations at later times). The orientation of the siRNA and PEG relative to the EGFR-Ab was also explored. As illustrated in FIG. 22, having the siRNA in between the EGFR-Ab and the PEG5k (EGFR antibody-KRAS-PEG5k) results in significantly higher plasma concentrations than the alternative conjugate where PEG5k is in between the EGFR-Ab and the siRNA (EGFR antibody-PEG5k-EGFR). In some instances, the use of two different siRNAs on these conjugates does not impact the plasma kinetics.

The drug loading on the EGFR-Ab was also investigated, with n=1 and n=2 siRNAs per EGFR-Ab. As illustrated in FIG. 23, having only one siRNA per EGFR-Ab resulted in much higher plasma concentrations, whereas the higher loading of n=2 siRNA per EGFR-Ab resulted in faster clearance from plasma. The impact of adding an endosomal escape peptide (melittin) was assessed. EGFR antibody-KRAS-PEG5k and EGFR antibody-melittin were mixed together in solution and co-injected. As illustrated in FIG. 24, the presence of EGFR antibody-melittin increases the clearance from plasma of EGFR antibody-KRAS-PEG5k at later times.

The plasma PK of cholesterol-siRNA conjugates was next compared to the mAb-siRNA conjugates after intravenous administration via tail vein injection. As illustrated in FIG. 25, the chol-siRNA conjugates are cleared much faster from plasma than the mAb-siRNA conjugates. As illustrated from the PK profile, having either EGFR or KRAS siRNA on the conjugate did not affect the plasma kinetics.

In addition to the plasma PK analysis, siRNA concentrations were determined in tissues at various times post-dose to determine the tissue PK. Tissue concentrations were measured pmol/g and then converted to pmol/mL by assuming the density of tissue equals 1 g/mL. In FIG. 26, a concentration of 1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue. As illustrated in FIG. 26A, a single i.v. dose of 0.5 mg/kg of EGFR antibody-siRNA resulted in approximately 100 nM concentrations of siRNA in tumor at 24 h post-dose for virtually all of the conjugates. In the case of these EGFR antibody-linker-siRNA conjugates, the molecular weight of the linker between the EGFR-Ab and the EGFR siRNA does not seem to alter the PK of these conjugates in the s.c. flank H358 tumors. As illustrated in FIG. 26B, the concentration of siRNA in liver following a single i.v. dose of 0.5 mg/kg of EGFR antibody-siRNA is approximately 100 nM at 24 h post-dose, similar to that seen in tumor. Only the small molecule linker at 24 h post-dose produces a siRNA concentration in liver approximately half of what is seen with longer PEG linkers. siRNA concentrations decrease over time in both tumor and liver tissue with these EGFR antibody-linker-siRNA conjugates.

The orientation of the siRNA and PEG relative to the EGFR-Ab was also explored relative to the tissue PK profiles. As illustrated in FIG. 27, both the EGFR antibody-KRAS-PEG5k and the EGFR antibody-PEG5k-EGFR conjugates deliver approximately 100 nM siRNA into both tumor and liver following a single i.v. dose of 0.5 mg/kg. However, while the EGFR antibody-KRAS-PEG5k maintains the siRNA concentration in tumor at approximately 100 nM until 168 h post-dose, the other 3 curves decline in concentration over time. Next, the tissue PK as a function of drug loading was assessed. As illustrated from FIG. 28, n=1 siRNA per EGFR-Ab delivered higher amounts of siRNA into tumor compared to liver. However, increasing the siRNA loading to n=2 siRNA per EGFR-Ab increased the amount of siRNA delivered to liver and decreased the amount of siRNA delivered to tumor. Additionally, EGFR antibody-melittin was mixed with some formulations in order to introduce endosomal escape functionality. As illustrated from FIG. 29, mixing and co-administering EGFR antibody-melittin with EGFR antibody-siRNA did not have a large impact on the tissue PK. The addition of melittin decreased uptake of siRNA in tumor and increased the uptake of siRNA in liver.

The tissue PK profiles of cholesterol-siRNA conjugates (using both EGFR and KRAS siRNA) in liver and in s.c. flank H358 tumors was also assessed. As illustrated from FIG. 30, both chol-siRNA conjugates delivered approximately 5 μM concentrations of siRNA into liver 24 h following a single i.v. dose of 15 mg/kg. In liver, the chol-KRAS appears to clear slightly faster than the chol-EGFR on the 1-week time scale. The two different chol-siRNA conjugates further show different PK profiles in tumor. Both cholesterol conjugates deliver less siRNA into tumor compared to liver, but the chol-EGFR delivers more siRNA into tumor when compared to the chol-KRAS conjugate. Both chol-siRNA conjugates are cleared from tumor over time and with a similar slope.

A PD analysis followed the PK analysis. As illustrated in FIG. 31A, the chol-KRAS conjugate produced only marginal (˜25%) mRNA knockdown of the KRAS target gene in tumor following a single i.v. dose of 15 mg/kg. However, as illustrated in FIG. 31B, the same 15 mg/kg dose of chol-KRAS was able to produce >50% mRNA knockdown in the mouse liver. The chol-EGFR conjugate was able to produce >50% mRNA knockdown in tumor, as illustrated in FIG. 32. In some instances, the higher knockdown with chol-EGFR in tumor compared to chol-KRAS is due to the higher siRNA concentrations observed in tumor with chol-EGFR compared to chol-KRAS (FIG. 30). Finally, as illustrated in FIGS. 33 and 34, most of the EGFR antibody-siRNA conjugates resulted in approximately 25-50% EGFR or KRAS mRNA knockdown in tumors after a single IV dose, but at a much lower dose (0.5 mg/kg) compared to the chol-siRNA conjugates.

Example 9: Synthesis, Purification and Analysis of Additional Antibody-siRNA Conjugates—Scheme-6: Antibody-Lys-siRNA-PEG Conjugates Via Antibody Lysine Conjugation of SMCC Linker

Step 1: Antibody Conjugation with SMCC Linker Followed by SH-siRNA

Antibody was buffer exchanged with 1× Phosphate buffer (pH 7.4) and made up to 10 mg/ml concentration. To this solution, 2 equivalents of SMCC linker dissolved in DMSO was added and rotated for 4 hours at room temperature. Unreacted SMCC linker was removed by spin filtration using 50 kDa MWCO Amicon spin filters and PBS pH 7.4. The antibody-maleimide conjugate was collected into a reaction vessel and SH-C6-siRNA or SH-C6-siRNA-C6-NHCO-PEG-XkDa (2 equivalents) (X=0.5 kDa to 10 kDa) was added at RT in pH 7.4 PBS with 5 mM EDTA and rotated overnight. See FIG. 93. Analysis of the reaction mixture by analytical SAX column chromatography method-2 showed antibody siRNA conjugate along with unreacted antibody and siRNA.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1. Fractions containing DAR1 and DAR>2 antibody-siRNA-PEG conjugates were separated, concentrated and buffer exchanged with pH 7.4 PBS.

Step-3: Analysis of the Purified Conjugate

The isolated conjugates were characterized by SAX chromatography, SEC chromatography and SDS-PAGE analysis. The purity of the conjugate was assessed by analytical HPLC using either anion exchange chromatography method-2. All DAR1 conjugate generally eluted at 9.0±0.4 minutes while the DAR2 and DAR3 conjugates generally eluted at 9.7±0.2 minutes. Typical DAR1 conjugate is greater than 90% pure after purification while typical DAR>2 lysine conjugates contains 70-80% DAR2 and 20-30% DAR3.

Scheme-7: Antibody-Cys-siRNA-PEG Conjugates Via Antibody Cysteine Conjugation

Step 1: Antibody Interchain Disulfide Reduction with TCEP

Antibody was buffer exchanged with borax buffer (pH 8) and made up to 10 mg/ml concentration. To this solution, 2 equivalents of TCEP in water was added and rotated for 2 hours at RT. The resultant reaction mixture was buffer exchanged with pH 7.4 PBS containing 5 mM EDTA and added to a solution of SMCC-C6-siRNA or SMCC-C6-siRNA-C6-NHCO-PEG-XkDa (2 equivalents) (X=0.5 kDa to 10 kDa) in pH 7.4 PBS containing 5 mM EDTA at RT and rotated overnight. Analysis of the reaction mixture by analytical SAX column chromatography showed antibody siRNA conjugate along with unreacted antibody and siRNA. See FIG. 94.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1. Fractions containing DAR1 and DAR>2 antibody-PEG-siRNA conjugates were separated, concentrated and buffer exchanged with pH 7.4 PBS.

Step-3: Analysis of the Purified Conjugate

The isolated conjugates were characterized by SEC, SAX chromatography and SDS-PAGE. The purity of the conjugate was assessed by analytical HPLC using either anion exchange chromatography method-2 or anion exchange chromatography method-3. Isolated DAR1 conjugates are typically eluted at 9.0+0.3 min on analytical SAX method-2 and are greater than 90% pure. The typical DAR>2 cysteine conjugate contains more than 85% DAR2 and less than 15% DAR3.

Scheme-8: Antibody siRNA Conjugates Via Antibody Inter-Chain Cysteine Conjugation

Step 1: Antibody Interchain Disulfide Reduction with TCEP

Antibody was buffer exchanged with borax buffer (pH 8) and made up to 10 mg/ml concentration. To this solution, 2 equivalents of TCEP in water was added and rotated for 2 hours at RT. The resultant reaction mixture was buffer exchanged with pH 7.4 PBS containing 5 mM EDTA and added to a solution of CBTF-C6-siRNA-C6-NHCO-PEG-5 kDa (2 equivalents) in pH 7.4 PBS containing 5 mM EDTA at RT and rotated overnight. Analysis of the reaction mixture by analytical SAX column chromatography showed antibody siRNA conjugate along with unreacted antibody and siRNA. See FIG. 95.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1. Fractions containing DAR1 and DAR>2 antibody-siRNA conjugates were separated, concentrated and buffer exchanged with pH 7.4 PBS. Typical DAR>2 cysteine conjugate contains greater than 85% DAR2 and less than 15% DAR3 or higher.

Step-3: Analysis of the Purified Conjugate

The isolated conjugates were characterized by either mass spec or SDS-PAGE. The purity of the conjugate was assessed by analytical HPLC using either anion exchange chromatography method-2 or anion exchange chromatography method-3.

Scheme-9: Antibody siRNA Conjugates Via Antibody Inter-Chain Cysteine Conjugation

Step 1: Antibody Reduction with TCEP

Antibody was buffer exchanged with borax buffer (pH 8) and made up to 5 mg/ml concentration. To this solution, 2 equivalents of TCEP in water was added and rotated for 2 hours at RT. The resultant reaction mixture was exchanged with pH 7.4 PBS containing 5 mM EDTA and added to a solution of MBS-C6-siRNA-C6-NHCO-PEG-5 kDa (2 equivalents) in pH 7.4 PBS containing 5 mM EDTA at RT and rotated overnight. Analysis of the reaction mixture by analytical SAX column chromatography showed antibody siRNA conjugate along with unreacted antibody and siRNA. See FIG. 96.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1. Fractions containing DAR1 and DAR>2 antibody-siRNA conjugates were separated, concentrated and buffer exchanged with pH 7.4 PBS. Typical DAR>2 cysteine conjugate contains greater than 85% DAR2 and less than 15% DAR3 or higher.

Step-3: Analysis of the Purified Conjugate

The isolated conjugates were characterized by either mass spec or SDS-PAGE. The purity of the conjugate was assessed by analytical HPLC using either anion exchange chromatography method-2 or anion exchange chromatography method-3.

Scheme-10: Antibody siRNA Conjugates Via Antibody Inter-Chain Cysteine Conjugation

Step 1: Antibody Reduction with TCEP

Antibody was buffer exchanged with borax buffer (pH 8) and made up to 5 mg/ml concentration. To this solution, 2 equivalents of TCEP in water was added and rotated for 2 hours at RT. The resultant reaction mixture was exchanged with pH 7.4 PBS containing 5 mM EDTA and added to a solution of MBS-C6-siRNA-C6-NHCO-PEG-5 kDa (2 equivalents) in pH 7.4 PBS containing 5 mM EDTA at RT and rotated overnight. Analysis of the reaction mixture by analytical SAX column chromatography showed antibody siRNA conjugate along with unreacted antibody and siRNA. See FIG. 97.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1. Fractions containing DAR1 and DAR>2 antibody-siRNA conjugates were separated, concentrated and buffer exchanged with pH 7.4 PBS. Typical DAR>2 cysteine conjugate contains greater than 85% DAR2 and less than 15% DAR3 or higher.

Step-3: Analysis of the Purified Conjugate

The isolated conjugates were characterized by either mass spec or SDS-PAGE. The purity of the conjugate was assessed by analytical HPLC using either anion exchange chromatography method-2 or anion exchange chromatography method-3.

Scheme-11: Synthesis of Antibody-Lysine-S—S-siRNA-PEG Conjugates

Step 1: Antibody Conjugation with SPDP Linker Followed by SH-siRNA-PEG5kDa

Antibody was buffer exchanged with pH 7.4 1×PBS and made up to 10 mg/ml concentration. To this solution, 2 equivalents of SPDP linker [succinimidyl 3-(2-pyridyldithio)propionate] or its methylated version was added and rotated for 4 hours at room temperature. Unreacted SPDP linker was removed by spin filtration using 50 kDa MWCO Amicon spin filters and pH 7.4 PBS buffer. The retentate was collected and 2 equivalents of SH-C6-siRNA-PEG5kDa in pH 7.4 PBS was added at room temperature and rotated overnight. The reaction mixture was analyzed by analytical SAX column chromatography and the conjugate along with unreacted antibody and siRNA was seen. See FIG. 98.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1. Fractions containing DAR1 and DAR>2 antibody-siRNA conjugates were separated, concentrated and buffer exchanged with pH 7.4 PBS. Typical DAR>2 lysine conjugate contains 70 to 80% DAR2 and 20 to 30% DAR3 or higher.

Step-3: Analysis of the Purified Conjugate

The isolated conjugate was characterized by either mass spec or SDS-PAGE. The purity of the conjugate was assessed by analytical HPLC using anion exchange chromatography method-2.

Scheme-12: Synthesis of Antibody-Cysteine-S—S-siRNA-PEG Conjugates

Step 1: Antibody Reduction and Conjugation with Pyridyldithio-siRNA-PEG5kDa

Antibody was buffer exchanged with pH 8.0 borax buffer and made up to 10 mg/ml concentration. To this solution, 1.5 equivalents of TCEP was added and the reaction mixture was rotated for 1 hour at room temperature. Unreacted TCEP was removed by spin filtration using 50 kDa MWCO Amicon spin filters and buffer exchanged with pH 7.4 PBS buffer. The retentate was collected and 2 equivalents of pyridyldithio-C6-siRNA-PEG5kDa in pH 7.4 PBS was added at room temperature and rotated overnight. The reaction mixture was analyzed by analytical SAX column chromatography and conjugate along with unreacted antibody and siRNA was seen. See FIG. 99.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1. Fractions containing DAR1 and DAR>2 antibody-siRNA conjugates were separated, concentrated and buffer exchanged with pH 7.4 PBS.

Step-3: Analysis of the Purified Conjugate

The isolated conjugate was characterized by either mass spec or SDS-PAGE. The purity of the conjugate was assessed by analytical HPLC using anion exchange chromatography method-2. Typical DAR>2 cysteine conjugate contains 90% DAR2 and 10% DAR3 or higher.

Scheme-13: Synthesis of Antibody-Cysteine-ECL-siRNA-PEG Conjugates

Step 1: Antibody Reduction and Conjugation with Maleimide-ECL-siRNA-PEG5kDa

Antibody was buffer exchanged with pH 8.0 borax buffer and made up to 10 mg/ml concentration. To this solution, 1.5 equivalents of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) reagent was added and rotated for 1 hour at room temperature. Unreacted TCEP was removed by spin filtration using 50 kDa MWCO Amicon spin filters and pH 7.4 PBS buffer with 5 mM EDTA. The retentate was collected and 1.5 equivalents of maleimide-ECL-C6-siRNA-PEG5kDa in pH 7.4 PBS was added at room temperature and rotated overnight. The reaction mixture was analyzed by analytical SAX column chromatography and conjugate along with unreacted antibody and siRNA was seen. See FIG. 100.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1. Fractions containing DAR1 and DAR>2 antibody-siRNA conjugates were separated, concentrated and buffer exchanged with pH 7.4 PBS.

Step-3: Analysis of the Purified Conjugate

The isolated conjugate was characterized by either mass spec or SDS-PAGE. The purity of the conjugate was assessed by analytical HPLC using anion exchange chromatography method-2. Typical DAR>2 lysine conjugate contains 70 to 80% DAR2 and 20 to 30% DAR3 or higher.

Scheme-14: Antibody Lysine Conjugation with TCO/Tetrazine Linker

Step 1: Antibody Conjugation with NHS-PEG4-TCO Followed by Methyltetrazine-PEG4-siRNA-PEG5kDa

Antibody was buffer exchanged with pH 7.4 PBS and made up to 5 mg/ml concentration. To this solution, 2 equivalents of NHS-PEG4-TCO linker was added and rotated for 4 hours at room temperature. Unreacted linker was removed by spin filtration using 50 kDa MWCO Amicon spin filters and pH 7.4 PBS. The retentate was collected and 2 equivalents of methyltetrazine-PEG4-siRNA-PEG5kDa in pH 7.4 PBS was added at room temperature. The reaction mixture was analyzed by analytical SAX column chromatography and the antibody-siRNA conjugate was seen along with the unreacted antibody and siRNA. See FIG. 101.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1. Fractions containing DAR1 and DAR>2 antibody-siRNA conjugates were separated, concentrated and buffer exchanged with pH 7.4 PBS. Typical DAR>2 lysine conjugate contains 70-80% DAR2 and 20-30% DAR3 or higher.

Step-3: Analysis of the Purified Conjugate

The characterization and purity of the isolated conjugate was characterized by either mass spec or SDS-PAGE. The purity of the conjugate was assessed by analytical HPLC using anion exchange chromatography method-2.

Scheme-15: Site Specific Conjugation at Antibody Glycans

Step 1: Antibody Glycan Modification and Gal-N3 Addition

Antibody was buffer exchanged with pH 6.0, 50 mM sodium phosphate buffer and treated with EndoS2 at 37° C. for 16 hrs. The reaction mixture was buffer exchanged into TBS buffer (20 mM Tris, 0.9% NaCl, pH 7.4) and UDP-GalNAz was added followed by MnCl₂, and Gal-T(Y289L) in 50 mM Tris, 5 mM EDTA (pH 8). The final solution contained concentrations of 0.4 mg/mL antibody, 10 mM MnCl₂, 1 mM UDP-GalNAz, and 0.2 mg/mL Gal-T(Y289L) and was incubated overnight at 30° C. See FIG. 102.

Step 2: DIBO-PEG-TCO Conjugation to Azide Modified Antibody

The reaction mixture from step-1 was buffer exchanged with PBS and 2 equivalents of DIBO-PEG4-TCO linker was added and rotated for 6 hours at room temperature. Unreacted linker was removed by spin filtration using 50 kDa MWCO Amicon spin filters and pH 7.4 PBS. The retentate was collected and used as is in step-3.

Step 3: Methyl Tetrazine-siRNA Conjugation to TCO Labeled Antibody

2 equivalents of methyltetrazine-PEG4-siRNA-PEG5kDa in pH 7.4 PBS was added to the retentate from step-2 and rotated at room temperature for 1 hour. The reaction mixture was analyzed by analytical SAX column chromatography and the antibody-siRNA conjugate was seen along with the unreacted antibody and siRNA.

Step 4: Purification

The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1. Fractions containing DAR1 and DAR>2 antibody-siRNA conjugates were separated, concentrated and buffer exchanged with pH 7.4 PBS. Typical DAR>2 lysine conjugate contains 70-80% DAR2 and 20-30% DAR3 or higher.

Step-5: Analysis of the Purified Conjugate

The characterization and purity of the isolated conjugate was characterized by either mass spec or SDS-PAGE. The purity of the conjugate was assessed by analytical HPLC using anion exchange chromatography method-2.

Scheme-16: Fab-siRNA Conjugate Generation

Step 1: Antibody Digestion with Pepsin

Antibody was buffer exchanged with pH 4.0, 20 mM sodium acetate/acetic acid buffer and made up to 5 mg/ml concentration. Immobilized pepsin (Thermo Scientific, Prod #20343) was added and incubated for 3 hours at 37° C. The reaction mixture was filtered using 30 kDa MWCO Amicon spin filters and pH 7.4 PBS. The retentate was collected and purified using size exclusion chromatography to isolate F(ab′)2. The collected F(ab′)2 was then reduced by 10 equivalents of TCEP and conjugated with SMCC-C6-siRNA-PEG5 at room temperature in pH 7.4 PBS. Analysis of reaction mixture on SAX chromatography showed Fab-siRNA conjugate along with unreacted Fab and siRNA-PEG. See FIG. 103.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1. Fractions containing DAR1 and DAR2 Fab-siRNA conjugates were separated, concentrated and buffer exchanged with pH 7.4 PBS.

Step-3: Analysis of the Purified Conjugate

The characterization and purity of the isolated conjugate was assessed by SDS-PAGE and analytical HPLC using anion exchange chromatography method-2.

Purification and Analytical Methods

Anion Exchange Chromatography Method-1.

Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5 mm ID×15 cm, 13 um

Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 mM TRIS, 1.5 M NaCl, pH 8.0; Flow Rate: 6.0 ml/min

Gradient:

	a.	% A	% B	Column Volume
	b.	100	0	1.00
	c.	60	40	18.00
	d.	40	60	2.00
	e.	40	60	5.00
	f.	0	100	2.00
	g.	100	0	2.00

Anion Exchange Chromatography Method-2

Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm

Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80% 10 mM TRIS pH 8, 20% ethanol, 1.5 M NaCl; Flow Rate: 1.0 ml/min

Gradient:

	a.	Time	% A	% B
	b.	0.0	90	10
	c.	3.00	90	10
	d.	11.00	40	60
	e.	13.00	40	60
	f.	15.00	90	10
	g.	20.00	90	10

Anion Exchange Chromatography Method-3

Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm

Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80% 10 mM TRIS pH 8, 20% ethanol, 1.5 M NaCl

Flow Rate: 0.75 ml/min

Gradient:

	a.	Time	% A	% B
	b.	0.0	90	10
	c.	3.00	90	10
	d.	11.00	40	60
	e.	23.00	40	60
	f.	25.00	90	10
	g.	30.00	90	10

Size Exclusion Chromatography Method-1

Column: TOSOH Biosciences, TSKgelG3000SW XL, 7.8×300 mm, 5 μM

Mobile phase: 150 mM phosphate buffer

Flow Rate: 1.0 ml/min for 20 mins

siRNA Synthesis

All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA.

Each siRNA passenger strand contains two conjugation handles, C6-NH2 and C6-SH, one at each end of the strand. The passenger strand with C6-NH2 handle at 5′ end contains C6-SH at its 3′ end and the strand that contains C6-NH₂ handle at 3′ end contains C6-SH at its 5′ end. Both conjugation handles are connected to siRNA passenger strand via inverted abasic phosphodiester or phosphorothioate.

A representative structure of siRNA with C6-NH₂ conjugation handle at the 5′ end and C6-SH at 3′end of the passenger strand is shown in FIG. 104.

ASC Architectures Described in Examples 10-41

ASC Architecture-1:

Antibody-Lys-SMCC-S-3′-Passenger strand. This conjugate was generated by antibody lysine-SMCC conjugation to thiol at the 3′ end of passenger strand.

ASC Architecture-2:

Antibody-Cys-SMCC-3′-Passenger strand. This conjugate (see FIG. 106) was generated by antibody inter-chain cysteine conjugation to SMCC at the 3′ end of passenger strand.

ASC Architecture-3:

Antibody-Lys-SMCC-S-5′-passenger strand. This conjugate (see FIG. 107) was generated by antibody lysine-SMCC conjugation to C6-thiol at the 5′ end of passenger strand.

ASC Architecture-4:

Antibody-Cys-SMCC-5′-passenger strand. This conjugate (see FIG. 108) was generated by antibody inter-chain cysteine conjugation to SMCC at the 5′ end of passenger strand.

ASC Architecture-5:

Antibody-Lys-PEG-5′-passenger strand. This conjugate (see FIG. 109) was generated by antibody PEG-TCO conjugation to tetrazine at the 5′ end of passenger strand.

ASC Architecture-6:

Antibody-Lys-PEG-5′-passenger strand. This conjugate (see FIG. 110) was generated by antibody PEG-TCO conjugation to tetrazine at the 5′ end of passenger strand.

ASC Architecture-7:

Antibody-Cys-PEG-5′-passenger strand without inverted abasic at 5′ end. This conjugate (see FIG. 111) was generated using procedure similar to architecture-2. The antibody was conjugated directly to the amine on passenger strand 5′ end sugar.

Zalutumumab (EGFR-Ab)

Zalutumumab is a fully human IgG1κ monoclonal antibody directed against the human epidermal growth factor receptor (EGFR). It is produced in the Chinese Hamster Ovary cell line DJT33, which has been derived from the CHO cell line CHO-K1SV by transfection with a GS vector carrying the antibody genes derived from a human anti-EGFR antibody producing hybridoma cell line (2F8). Standard mammalian cell culture and purification technologies are employed in the manufacturing of zalutumumab.

The theoretical molecular weight (MW) of zalutumumab without glycans is 146.6 kDa. The experimental MW of the major glycosylated isoform of the antibody is 149 kDa as determined by mass spectrometry. Using SDS-PAGE under reducing conditions the MW of the light chain was found to be approximately 25 kDa and the MW of the heavy chain to be approximately 50 kDa. The heavy chains are connected to each other by two inter-chain disulfide bonds, and one light chain is attached to each heavy chain by a single inter-chain disulfide bond. The light chain has two intra-chain disulfide bonds and the heavy chain has four intra-chain disulfide bonds. The antibody is N-linked glycosylated at Asn305 of the heavy chain with glycans composed of N-acetyl-glucosamine, mannose, fucose and galactose. The predominant glycans present are fucosylated bi-antennary structures containing zero or one terminal galactose residue. The charged isoform pattern of the IgG1κ antibody has been investigated using imaged capillary IEF, agarose IEF and analytical cation exchange HPLC. Multiple charged isoforms are found, with the main isoform having an isoelectric point of approximately 8.7.

The major mechanism of action of zalutumumab is a concentration dependent inhibition of EGF-induced EGFR phosphorylation in A431 cancer cells. Additionally, induction of antibody-dependent cell-mediated cytotoxicity (ADCC) at low antibody concentrations has been observed in pre-clinical cellular in vitro studies.

Panitumumab (EGFR2-Ab)

Panitumumab is a clinically approved, fully human IgG2 subclass monoclonal antibody specific to the epidermal growth factor receptor (EGFR). Panitumumab has two gamma heavy chains and two kappa light chains. Glycosylated panitumumab has a total molecular weight of approximately 147 kDa. Panitumumab is expressed as a glycoprotein with a single consensus N-linked glycosylation site located on the heavy chain. Panitumumab is produced from Chinese Hamster Ovary (CHO) cells and purified by a series of chromatography steps, viral inactivation step, viral filtration step and ultrafiltration/diafiltration steps.

Panitumumab acts as a competitive antagonist at the ligand binding site of EGFR to inhibit binding and signaling mediated by EGF and transforming growth factor α, the natural ligands for this receptor. The affinity of binding panitumumab to the EGFR was determined be 3.5 and 5.7×10⁻¹²M in recombinant EGFR using BIAcore methods. Inhibition of binding of EGF was shown in A431 cells, a human epidermal carcinoma cell line that expresses EGFR. Intracellular acidification, phosphorylation and internalization of the EGFR were blocked in a dose-dependent manner by panitumumab in A431 cells. Panitumumab was also shown to inhibit cell growth in vitro and in vivo in the same cell line.

Herceptin (EGFR3-Ab)

Herceptin is a clinically approved, humanized IgG1 subclass monoclonal antibody specific to the epidermal growth factor receptor2 (EGFR2) also known as Her2. Herceptin has human Fc γ1 isotype along with kappa light chains.

PSMA-Ab

PSMA-Ab is a humanized IgG1 subclass monoclonal antibody specific to prostate specific membrane antigen (PSMA).

ASGR1-Ab

ASGR mAb-Sino103 is a rabbit IgG monoclonal antibody that binds mouse asialoglycoprotein receptor1 (ASGPR1). It is supplied by Sino Biologicals Inc. (Cat #50083-R103).

ASGR2-Ab

ASGR mAb-R&D is a rat IgG_2A subclass monoclonal antibody that binds mouse asialoglycoprotein receptor1 (ASGPR1). It is purified by protein A or G from hybridoma culture supernatant and supplied by R&D Systems (Cat # MAB2755)

siRNA-TriGalNAc Conjugate

The siRNA triGalNAc conjugate was synthesized using Lys-Lys dipeptide. Protected triGalNAc was coupled with dipeptide PEG linker and purified. After the removal of carboxylic acid protection group on the triGalNAc-dipeptide was conjugated to the 5′ end of siRNA passenger strand.

Example 10: 2016-PK-163-LNCap

siRNA Design and Synthesis

EFGR: A 2l mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to obtain the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate modifications were used to reduce immunogenicity and were comparable to those used in the active siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

The AXBYC conjugate used in groups 3-4 were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9. The AXB and AXCYB conjugates were made as described in Example 9.

In Vivo Study Design

Groups (n=5) of female SCID SHO mice bearing subcutaneous flank LNCaP tumors 100-350 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control groups (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Treatment groups 1-6 were dosed at 1.0 or 0.5 mg/kg (based on the weight of siRNA) as per the study design below. All groups (treatments and controls) were administered a dose volume of 5 mL/kg. Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose. Table 22 describes the study design in more detail. 50 mg pieces of tumor and liver, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 22
			siRNA		Dose		Harvest
			Dose		Volume	# of	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)
1	PSMA-Ab(Cys)-EGFR (n = 1)	5	1	IV	5.0	1	96
2	PSMA-Ab(Cys)-EGFR (n = 1)	5	0.5	IV	5.0	1	96
3	PSMA-Ab(Cys)-EGFR-PEG5k (n = 1)	5	1	IV	5.0	1	96
4	PSMA-Ab(Cys)-EGFR-PEG5k (n = 1)	5	0.5	IV	5.0	1	96
5	PSMA-Ab(Cys)-PEG5k-EGFR (n = 1)	5	1	IV	5.0	1	96
6	PSMA-Ab(Cys)-PEG5k-EGFR (n = 1)	5	0.5	IV	5.0	1	96
7	PSMA-Ab(Cys)-scramble-PEG5k	5	1	IV	5.0	1	96
	(n = 1)
8	PBS Control	5	—	IV	5.0	1	96
	Total # of Animals:	40
SCID SHO mice with LNCaP tumors

The orientation of the siRNA and PEG relative to the PSMA-Ab was explored in an in vivo mouse tumor model. As illustrated in FIG. 50A, having the siRNA in between the PSMA-Ab and the PEG5k (PSMA-Ab(Cys)-EGFR-PEG5k or the AXBYC format) resulted in higher levels of EGFR mRNA knockdown in the tumor relative to the alternative conjugate where PEG5k is in between the PSMA-Ab and the siRNA (PSMA-Ab(Cys)-PEG5k-EGFR or AXCYB format). This approach (AXBYC) also resulted in higher levels of EGFR mRNA knockdown in the tumor relative to the conjugate without PEG5K (PSMA-Ab(Cys)-EGFR or AXB format).

The orientation of the siRNA and PEG relative to the PSMA-Ab was also explored relative to the tissue PK profiles. Tissue concentrations were measured pmol/g and then converted to pmol/mL by assuming the density of tissue equals 1 g/mL (a concentration of 1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue). As illustrated in FIG. 50B, having the siRNA in between the PSMA-Ab and the PEG5k (AXBYC) resulted in higher levels of siRNA delivery to the tumor relative to the alternative conjugate where PEG5k is in between the PSMA-Ab and the siRNA (AXCYB). This approach (AXBYC) resulted in higher levels of EGFR siRNA delivery to the tumor relative to the conjugate without PEG5K (AXB).

In a mouse LNCaP subcutaneous xenograph model, it was demonstrated that the AXBYC format for the antibody siRNA conjugate resulted in higher levels of siRNA accumulation in the tumor tissue and a greater magnitude of EGFR mRNA knockdown, relative to the AXCYB and AXB formats. The LNCap tumor expresses human PSMA, resulting in tumor tissue specific accumulation of the PSMA targeted siRNA conjugates after i.v. administration, receptor mediate uptake and siRNA facilitated knockdown of the target gene.

Example 11: 2016-PK-202-LNCap

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate modifications were used to reduce immunogenicity and were comparable to those used in the active siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

The AXBYC conjugate used in groups 3-5 and 7 was made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9. The AXB (groups 1-2) and AXCYB (group 6) conjugates were made as described in Example 9.

In Vivo Study Design

Groups (n=5) of female SCID SHO mice bearing subcutaneous flank LNCaP tumors 100-350 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control groups (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Treatment groups 1-6 were dosed at 1.0 or 0.5 mg/kg (based on the weight of siRNA) as per the study design below. All groups (treatments and controls) were administered a dose volume of 5 mL/kg. Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose. Table 23 describes the study design in more detail. 50 mg pieces of tumor and liver, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 23
			siRNA		Dose		Harvest
			Dose		Volume	# of	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)
1	PSMA-Ab(Cys)-EGFR (n = 1)	5	1	IV	5.0	1	96
2	PSMA-Ab(Cys)-EGFR (n = 1)	5	0.5	IV	5.0	1	96
3	PSMA-Ab(Cys)-EGFR-PEG5k (n = 1)	5	1	IV	5.0	1	96
4	PSMA-Ab(Cys)-EGFR-PEG5k (n = 1)	5	0.5	IV	5.0	1	96
5	PSMA-Ab(Cys)-EGFR-PEG5k (n = 1)	5	0.25	IV	5.0	1	96
6	PSMA-Ab(Cys)-PEG5k-EGFR (n = 1)	5	0.5	IV	5.0	1	96
7	PSMA-Ab(Cys)-scramble-PEG5k (n = 1)	5	1	IV	5.0	1	96
8	PBS Control	5	—	IV	5.0	1	96
	Total # of Animals:	40
SCID SHO mice with LNCaP tumors

The orientation of the siRNA and PEG relative to the PSMA-Ab was also explored in an in vivo mouse tumor model. As illustrated in FIG. 51A, having the siRNA in between the PSMA-Ab and the PEG5k (PSMA-Ab(Cys)-EGFR-PEG5k or AXBYC format)) resulted in higher levels of EGFR mRNA knockdown in the tumor relative to the alternative conjugate where PEG5k is in between the PSMA-Ab and the siRNA (PSMA-Ab(Cys)-PEG5k-EGFR or AXCYB format). This approach (AXBYC) also resulted in higher levels of EGFR mRNA knockdown in the tumor relative to the conjugate without PEG5K (PSMA-Ab(Cys)-EGFR or AXB format).

The orientation of the siRNA and PEG relative to the PSMA-Ab was also explored relative to the tissue PK profiles. Tissue concentrations were measured pmol/g and then converted to pmol/mL by assuming the density of tissue equals 1 g/mL (a concentration of 1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue). As illustrated in FIG. 51B, having the siRNA in between the PSMA-Ab and the PEG5k (PSMA-Ab(Cys)-EGFR-PEG5k or AXBYC) resulted in higher levels of siRNA delivery to the tumor relative to the alternative conjugate where PEG5k is in between the PSMA-Ab and the siRNA (PSMA-Ab(Cys)-PEG5k-EGFR or AXCYB). This approach (AXBYC) also resulted in higher levels of EGFR siRNA delivery to the tumor relative to the conjugate without PEG5K (PSMA-Ab(Cys)-EGFR or AXB).

In a mouse LNCaP subcutaneous xenograph model, it was demonstrated that the AXBYC format for the antibody siRNA conjugate results in higher levels of siRNA accumulation in the tumor tissue and a greater magnitude of EGFR mRNA knockdown, relative to the AXCYB and AXB formats. The LNCap tumor expresses human PSMA, resulting in tumor tissue specific accumulation of the PSMA targeted siRNA conjugates after i.v. administration, receptor mediate uptake and siRNA facilitated knockdown of the target gene.

Example 12: 2016-PK-219-WT

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082)). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate modifications were used to reduce immunogenicity and were comparable to those used in the active siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

The AXBYC conjugate used in groups 4-6 was made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9. The AXB (groups 1-3) and AXCYB (groups 7-9) and BYC (groups 10-12) conjugates were made as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with one intravenous (i.v.) tail vein injections of siRNA conjugates. Treatment groups received 0.5 mg/kg (based on the weight of siRNA) and all groups were administered a dose volume of 5.0 mL/kg. Table 24 illustrates the study design in more detail. Non-terminal blood samples were collected at 5, 30, and 180 minutes post-dose via puncture of the retro-orbital plexus and centrifuged to generate plasma for PK analysis. Mice were sacrificed by CO₂ asphyxiation at 24, 96, or 168 h post-dose. Terminal blood samples were collected via cardiac puncture and processed to generate plasma for PK analysis. Quantitation of plasma siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 24
			siRNA				Survival	Terminal
			Dose		# of	Dose	Bleed	Bleed
Group	Test Article	N	(mg/kg)	ROA	Doses	Schedule	(min)	(h)
1	EGFR-Ab(Cys)-EGFR	4	0.5	IV	1	t = 0	5	24
2	(n = 1)	4	0.5	IV	1	t = 0	30	96
3		4	0.5	IV	1	t = 0	180	168
4	EGFR-Ab(Cys)-EGFR-	4	0.5	IV	1	t = 0	5	24
5	PEG5k (n = 1)	4	0.5	IV	1	t = 0	30	96
6		4	0.5	IV	1	t = 0	180	168
7	EGFR-Ab(Cys)-PEG5k-	4	0.5	IV	1	t = 0	5	24
8	EGFR (n = 1)	4	0.5	IV	1	t = 0	30	96
9		4	0.5	IV	1	t = 0	180	168
10	EGFR Alone (aka	4	0.5	IV	1	t = 0	5	24
11	EGFR-PEG5k)	4	0.5	IV	1	t = 0	30	96
12		4	0.5	IV	1	t = 0	180	168
	Total # of Animals:	48
WT mice CD-1

In this in vivo PK experiment the orientation of the siRNA and PEG relative to the EGFR-Ab was explored to determine the behavior of the mAb-siRNA conjugate in plasma. As illustrated in FIG. 52, all the mAb-siRNA conjugates (AXB, AXBYC and AXCYB formats) had comparable plasma PK with approximately 10% of the siRNA remaining in the systemic circulation after 168 hours (7 days), compared to the siRNA-PEG5K (BYC format) which was rapidly cleared from the plasma.

The AXBYC format for the antibody siRNA conjugate has improved PK properties relative the siRNA-PEG conjugate (BYC) which was rapidly cleared from the plasma.

Example 13: 2016-PK-199-HCC827

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082)). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). The same base, sugar and phosphate modifications that were used for the active EGFR siRNA duplex were used in the negative control siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank HCC827 tumors 100-300 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control groups (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Treatment groups 1-3 and 4-6 were dosed at 1.0, 0.5 or 0.25 mg/kg (based on the weight of siRNA) as per the study design below. As described in Example 9, groups 1-3 contained the same targeting antibody, but groups 4-6 had a different EGFR targeting antibody, while the rest of the conjugate components (linker, siRNA and PEG) were identical. Group 7 received an antibody conjugate with a negative control siRNA sequence (scramble) as a control for groups 1. All groups (treatments and controls) were administered a dose volume of 5 mL/kg. Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose. Table 25 describes the study design in more detail. 50 mg pieces of tumor and liver, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 25
			siRNA		Dose		Harvest
			Dose		Volume	# of	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)
1	EGFR-Ab(Cys)-EGFR-PEG5k (n = 1)	5	1	IV	5.0	1	96
2	EGFR-Ab(Cys)-EGFR-PEG5k (n = 1)	5	0.5	IV	5.0	1	96
3	EGFR-Ab(Cys)-EGFR-PEG5k (n = 1)	5	0.25	IV	5.0	1	96
4	EGFR2-Ab(Cys)-EGFR-PEG5k	5	1	IV	5.0	1	96
	(n = 1)
5	EGFR2-Ab(Cys)-EGFR-PEG5k	5	0.5	IV	5.0	1	96
	(n = 1)
6	EGFR2-Ab(Cys)-EGFR-PEG5k	5	0.25	IV	5.0	1	96
	(n = 1)
7	EGFR-Ab(Cys)-scramble-PEG5k	5	1	IV	5.0	1	96
	(n = 1)
8	PBS Control	5	—	IV	5.0	1	96
	Total # of Animals:	40
nu/nu mice with HCC827 tumors

siRNA concentrations were determined 96 hours in the tumor and liver after a single i.v. injection at 1.0, 0.5 and 0.25 mg/kg. Tissue concentrations were measured pmol/g and then converted to pmol/mL by assuming the density of tissue equals 1 g/mL. In FIG. 53A, a concentration of 1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue. As illustrated in FIG. 53A, both antibody conjugates were capable of delivering higher levels of siRNA to the tumor relative to the liver, and a dose response was observed. The EGFR antibody conjugate was capable of delivering more siRNA to the tumor tissue, at all the doses tested, relative to the EGFR2 antibody. See FIG. 53B. Both conjugates were capable of EGFR gene specific mRNA knockdown at 96 hours post-administration. The control conjugate that contained the scrambled siRNA and the PBS vehicle control did not produce significant EGFR gene specific mRNA knockdown.

As highlighted in FIG. 54, biological activity was demonstrated with the A-X—B—Y—C conjugate with a range of different antibodies and siRNA cargos that are capable of in vivo biological activity in a range of different tissue targets. In this example, it was demonstrated that tumor specific accumulation of 2 conjugates targeted with two different EGFR antibodies conjugated to an siRNA designed to down regulate EGFR mRNA. The HCC827 tumor expresses high levels of human EGFR and both conjugates have a human specific EGFR antibody to target the siRNA, resulting in tumor tissue specific accumulation of the conjugates. Receptor mediate uptake resulted in siRNA mediated knockdown of the target gene.

Example 14: 2016-PK-236-HCC827

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate modifications were used to reduce immunogenicity and were comparable to those used in the active siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank HCC827 tumors 100-300 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control group 6 (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Treatment groups 1-3 were dosed at 1.0, 0.5 or 0.25 mg/kg (based on the weight of siRNA), groups 4 and 5 at 1.0 mg/kg, as per the study design below. As described in Example 9, groups 1-3 contained the same targeting antibody, but groups 4 had a different EGFR targeting antibody, while the rest of the conjugate components (linker, siRNA and PEG) were identical. Group 6 received an antibody conjugate with a negative control siRNA sequence (scramble) as a control for groups 5. All groups (treatments and controls) were administered a dose volume of 5 mL/kg. Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose. Table 26 describes the study design in more detail. 50 mg pieces of tumor and liver, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt).

TABLE 26
			siRNA		Dose		Harvest
			Dose		Volume	# of	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)
1	EGFR3-Ab(Cys)-EGFR-	5	1	IV	5.0	1	96
	PEG5k (n = 1)
2	EGFR3-Ab(Cys)-EGFR-	5	0.5	IV	5.0	1	96
	PEG5k (n = 1)
3	EGFR3-Ab(Cys)-EGFR-	5	0.25	IV	5.0	1	96
	PEG5k (n = 1)
4	EGFR-Ab(Cys)-EGFR-	5	1	IV	5.0	1	96
	PEG5k (n = 1)
5	EGFR-Ab(Cys)-scramble-	5	1	IV	5.0	1	96
	PEG5k (n = 1)
6	PBS Control	5	—	IV	5.0	1	96
	Total # of Animals:	30
nu/nu mice with HCC827 tumors

In this in vivo PD experiment, it was demonstrated that dose dependent EGFR gene specific mRNA knockdown (FIG. 55) at 96 hour's post-administration with a third example of an EGFR antibody targeting agent (EGFR3). The control conjugate that contained the scrambled siRNA and the PBS vehicle control did not produce significant EGFR gene specific mRNA knockdown.

As highlighted in FIG. 54, it was demonstrated that biological activity with the A-X—B—Y—C conjugate with a range of different antibodies and siRNA cargos that are capable of in vivo biological activity in a range of different tissue targets. In this example, it was demonstrated that tumor specific down regulation of EGFR mRNA using a third EGFR antibody targeting ligand. The HCC827 tumor expresses human EGFR and both conjugates have a human specific EGFR antibody (EGFR and EGFR3) to target the siRNA, resulting in tumor tissue specific accumulation of the conjugates. Receptor mediate uptake resulted in siRNA mediated knockdown of the target gene.

Example 15: 2016-PK-234-HCC827

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence (5′ to 3′) of the guide/antisense strand was TCUCGUGCCUUGGCAAACUUU (SEQ ID NO: 2117) and it was design to be complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR. Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate modifications were used to reduce immunogenicity and were comparable to those used in the active siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank HCC827 tumors 100-300 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control group 10 (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Treatment groups 1-3, 4-6 and 7-9 were dosed at 1.0, 0.5 or 0.25 mg/kg (based on the weight of siRNA), as per the study design below. As described in Example 9, groups 1-3 contained the same targeting antibody (EGFR3) but groups 4-9 had a different EGFR targeting antibody, while the rest of the conjugate components (linker, siRNA and PEG) were identical. Group 7-9 received an antibody conjugate with a negative control siRNA sequence (scramble) as a control for groups 1-6. All groups (treatments and controls) were administered a dose volume of 5 mL/kg. Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose. Table 27 describes the study design in more detail. 50 mg pieces of tumor and liver, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 27
			siRNA		Dose		Harvest
			Dose		Volume	# of	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)
1	EGFR3-Ab(Cys)-N3′-EGFR-5′S-PEG5k	5	1	IV	5.0	1	96
	(n = 1)
2	EGFR3-Ab(Cys)-N3′-EGFR-5′S-PEG5k	5	0.5	IV	5.0	1	96
	(n = 1)
3	EGFR3-Ab(Cys)-N3′-EGFR-5′S-PEG5k	5	0.25	IV	5.0	1	96
	(n = 1)
4	EGFR-Ab(Cys)-N5′-EGFR-3′S-PEG5k	5	1	IV	5.0	1	96
	(n = 1)
5	EGFR-Ab(Cys)-N5′-EGFR-3′S-PEG5k	5	0.5	IV	5.0	1	96
	(n = 1)
6	EGFR-Ab(Cys)-N5′-EGFR-3′S-PEG5k	5	0.25	IV	5.0	1	96
	(n = 1)
7	EGFR-Ab(Cys)-N5′-scramble-3′S-	5	1	IV	5.0	1	96
	PEG5k (n = 1)
8	EGFR-Ab(Cys)-N5′-scramble-3′S-	5	0.5	IV	5.0	1	96
	PEG5k (n = 1)
9	EGFR-Ab(Cys)-N5′-scramble-3′S-	5	0.25	IV	5.0	1	96
	PEG5k (n = 1)
10	PBS Control	5	—	IV	5.0	1	96
	Total # of Animals:	50
nu/nu mice with HCC827 tumors

siRNA concentrations were determined 96 hours in the tumor and liver after a single i.v. injection at 1.0, 0.5 and 0.25 mg/kg. Tissue concentrations were measured pmol/g and then converted to pmol/mL by assuming the density of tissue equals 1 g/mL. In FIG. 56A, a concentration of 1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue. As illustrated in FIG. 56A, both antibody conjugates were capable of delivering higher levels of siRNA to the tumor relative to the liver, and a dose response was observed. Both conjugates were capable of EGFR gene specific mRNA knockdown at 96 hours post-administration relative to the scramble and vehicle control. See FIG. 56B.

As highlighted in FIG. 54, it was demonstrated biological activity with the A-X—B—Y—C conjugate with a range of different antibodies and siRNA cargos that are capable of in vivo biological activity in a range of different tissue targets. In this example, it was demonstrated tumor specific accumulation of 2 conjugates targeted with two different EGFR antibodies conjugated to an siRNA designed to down regulate EGFR mRNA. The HCC827 tumor expresses high levels of human EGFR and both conjugates have a human specific EGFR antibody to target the siRNA, resulting in tumor tissue specific accumulation of the conjugates. Receptor mediate uptake resulted in siRNA mediated knockdown of the target gene

Example 16: 2016-PK-237-HCC827

siRNA Design and Synthesis

KRAS: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human KRAS. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 237 for the human mRNA transcript for KRAS (UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-SH at the 3′ end, which was connected to siRNA passenger strand via phosphodiester-inverted abasic-phosphorothioate linker. The C6-SH was connected through the phosphodiester, see Example 9 for the chemical structure. In addition, the 5′ end of the passenger strand had the inverted abasic removed and the antibody was conjugated directly to the amine on passenger strand 5′ end sugar on a T base using a procedure similar to architecture 2, see Example 9.

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

Conjugates in groups 1-3 were made and purified as a DAR1 (n=1) using ASC architecture-7, as described in Example 9.

Conjugates in groups 4-6 were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank HCC827 tumors 100-300 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control group 7 (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Treatment groups 1-3, 4-6 were dosed at 1.0, 0.5 or 0.25 mg/kg (based on the weight of siRNA), as per the study design below. As described in Example 9, groups 1-6 contained the same targeting antibody (EGFR) but groups 1-3 had an siRNA designed to downregulate KRAS and groups 4-6 had an siRNA designed to downregulate EGFR. All groups (treatments and controls) were administered a dose volume of 5 mL/kg. Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose. Table 28 describes the study design in more detail. 50 mg pieces of tumor and liver, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in the methods section. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves. Plasma concentrations of the antibody component of the conjugate were determined using an ELISA assay.

TABLE 28
			siRNA		Dose		Survival	Terminal	Harvest
			Dose		Volume	# of	Bleed	Bleed	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(min)	(h)	(h)
1	EGFR-Ab(Cys)-KRAS-PEG5k	5	0.5	IV	5.0	1	0.25	72	72
	(n = 1)
2	EGFR-Ab(Cys)-KRAS-PEG5k	5	0.5	IV	5.0	1	3	96	96
	(n = 1)
3	EGFR-Ab(Cys)-KRAS-PEG5k	5	0.5	IV	5.0	1	24	168	168
	(n = 1)
4	EGFR-Ab(Cys)-EGFR-PEG5k	5	0.5	IV	5.0	1	0.25	72	72
	(n = 1)
5	EGFR-Ab(Cys)-EGFR-PEG5k	5	0.5	IV	5.0	1	3	96	96
	(n = 1)
6	EGFR-Ab(Cys)-EGFR-PEG5k	5	0.5	IV	5.0	1	24	168	168
	(n = 1)
7	PBS Control	5	—	IV	5.0	1	—	—	96
	Total # of Animals:	35
nu/nu mice with HCC827 tumors

siRNA concentrations were determined 96 hours in the tumor and liver after a single i.v. injection at 1.0, 0.5 and 0.25 mg/kg. Tissue concentrations were measured pmol/g and then converted to pmol/mL by assuming the density of tissue equals 1 g/mL. In FIG. 57A and FIG. 57B, a concentration of 1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue. As illustrated in FIG. 57A and FIG. 57B, both antibody conjugates were capable of delivering higher levels of siRNA to the tumor relative to the liver. The conjugate that contained the siRNA designed to downregulate KRAS was capable of KRAS gene specific mRNA knockdown (FIG. 57C) at 96 hours post-administration relative to the conjugate that contained the siRNA designed to down regulate EGFR or the PBS vehicle control. Both antibody conjugate constructs had similar PK properties (see FIG. 58A and FIG. 58B) indicating the alternative conjugation strategy used on the 5′ guide strand for the antibody had no impact on this biological parameter.

As highlighted in FIG. 54, it was demonstrated biological activity with the A-X—B—Y—C conjugate with a range of different antibodies and siRNA cargos that are capable of in vivo biological activity in a range of different tissue targets. In this example it was demonstrated tumor specific accumulation and siRNA mediated mRNA knockdown of a EGFR antibody conjugated to an siRNA designed to down regulate KRAS mRNA. The HCC827 tumor expresses high levels of human EGFR and the conjugate has a human specific EGFR antibody to target the siRNA, resulting in tumor tissue specific accumulation of the conjugates. Receptor mediate uptake resulted in siRNA mediated knockdown of the KRAS gene.

Example 17: 2016-PK-187-Hep3B

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate modifications were used to reduce immunogenicity and were comparable to those used in the active siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank Hep-3B2 1-7 tumors 100-300 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control group 5 (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Treatment groups 1-3 were dosed at 1.0, 0.5 or 0.25 mg/kg (based on the weight of siRNA), group 4 (scramble control) was dosed at 1.0 mg/kg, as per the study design below. Group 4 received an antibody conjugate with a negative control siRNA sequence (scramble) as a control for group 1. All groups (treatments and controls) were administered a dose volume of 5 mL/kg. Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose. Table 29 describes the study design in more detail. 50 mg pieces of tumor and liver, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 29
			siRNA		Dose		Harvest
			Dose		Volume	# of	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)
1	EGFR-Ab(Cys)-EGFR-PEG5k (n = 1)	5	1	IV	5.0	1	96
2	EGFR-Ab(Cys)-EGFR-PEG5k (n = 1)	5	0.5	IV	5.0	1	96
3	EGFR-Ab(Cys)-EGFR-PEG5k (n = 1)	5	0.25	IV	5.0	1	96
4	EGFR-Ab(Cys)-scramble-PEG5k	5	1	IV	5.0	1	96
	(n = 1)
5	PBS Control	5	—	IV	5.0	1	96
	Total # of Animals:	25
nu/nu mice with Hep3B tumors

siRNA concentrations were determined 96 hours in the tumor and liver after a single i.v. injection at 1.0, 0.5 and 0.25 mg/kg. Tissue concentrations were measured pmol/g and then converted to pmol/mL by assuming the density of tissue equals 1 g/mL. In FIG. 59A, a concentration of 1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue. As illustrated in FIG. 59A, the antibody conjugate was capable of delivering siRNA to the tumor. The conjugate was capable of EGFR gene specific mRNA knockdown (FIG. 59B) at 96 hours post-administration relative to the conjugate that contained the negative control siRNA sequence or the PBS vehicle control.

As highlighted in FIG. 54, it was demonstrated biological activity with the A-X—B—Y—C conjugate with a range of different antibodies and siRNA cargos that are capable of in vivo biological activity in a range of different tissue targets. In this example it was demonstrated tumor specific accumulation and siRNA mediated mRNA knockdown of an EGFR antibody conjugated to an siRNA designed to down regulate EGFR mRNA. The Hep-3B2 1-7 tumor cells express human EGFR and the conjugate has a human specific EGFR antibody to target the siRNA, resulting in tumor tissue specific accumulation of the conjugates. Receptor mediate uptake resulted in siRNA mediated knockdown of the EGFR gene.

Example 18: 2016-PK-257-WT

siRNA Design and Synthesis

R1442: N5-CTNNB1-3'S

CTNNB1: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human CTNNB1. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 1248 for the human mRNA transcript for CTNNB1 (UAAUGAGGACCUAUACUUAUU; SEQ ID NO: 2095). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to the siRNA passenger strand via phosphodiester-inverted abasic-phosphorothioate linker. The C6-NH₂ and C6-SH were connected through the phosphodiester, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

The antibody conjugate was made and purified as a DAR1 (n=1) using ASC architecture-1, as described in Example 9. The tri-GalNAc-CTNNB1 conjugate was made as described in Example 9.

In Vivo Study Design

Groups 1-3 (n=4) of wild-type female CD-1 mice were treated with one intravenous (i.v.) tail vein injections of siRNA conjugates, the GalNAc targeted control was doses by subcutaneous injection. Treatment groups 1-3 received doses of 2.0 1.0 and 0.5 mg/kg (based on the weight of siRNA) and the GalNAc targeted control conjugate was doses at 2 mg/kg. All groups were administered a dose volume of 5.0 mL/kg. Table 30 illustrates the study design in more detail. 50 mg pieces of liver were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt).

TABLE 30
			siRNA		Dose		Harvest
			Dose		Volume	# of	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)
1	ASGR1-Ab(Lys)-CTNNB1-	4	2	IV	5.0	1	96
	PEG5k (n = 1)
2	ASGR1-Ab(Lys)-CTNNB1-	4	1	IV	5.0	1	96
	PEG5k (n = 1)
3	ASGR1-Ab(Lys)-CTNNB1-	4	0.5	IV	5.0	1	96
	PEG5k (n = 1)
4	3Ga1NAc-CTNNB1 Control	5	2	s.c.	5.0	1	96
5	PBS Control	5	—	IV	5.0	1	96
	Total # of Animals:	22	WT mice (CD-1)

CTNNB1 gene knockdown was determined 96 hours post administration. As illustrated in FIG. 60, the GalNac-conjugated siRNA was capable of gene specific knockdown after a single s.c injection, as has been well described by others in the field. The same siRNA conjugated to an ASGR antibody was also capable of CTNNB1 gene specific downregulation and in a dose dependent manner.

As highlighted in FIG. 54, it was demonstrated biological activity with the A-X—B—Y—C conjugate with a range of different antibodies and siRNA cargos that are capable of in vivo biological activity in a range of different tissue targets. In this example it was demonstrated liver delivery with an ASGR antibody conjugated to an siRNA designed to down regulate CTNNB1 mRNA. Mouse Liver cells express the asialoglycoprotein receptor (ASGR) and the conjugate has a mouse specific ASGR antibody to target the siRNA, resulting in siRNA mediated knockdown of the CTNNB1 in the liver.

Example 19: 2016-PK-253-WT

siRNA Design and Synthesis

KRAS: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human KRAS. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 237 for the human mRNA transcript for KRAS (UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphodiester-inverted abasic-phosphorothioate linker. The C6-NH2 and C6-SH were connected through the phosphodiester, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

The antibody conjugate was made and purified as a DAR1 (n=1) using ASC architecture-1, as described in Example 9. The tri-GalNAc-CTNNB1 conjugate was made as described in Example 9.

In Vivo Study Design

Groups 1-3 (n=4) of wild-type female CD-1 mice were treated with one intravenous (i.v.) tail vein injections of siRNA conjugates, the GalNAc targeted control was doses by subcutaneous injection. Treatment groups 1-3 received doses of 2.0 1.0 and 0.5 mg/kg (based on the weight of siRNA) and the GalNAc targeted control conjugate was doses at 2 mg/kg. All groups were administered a dose volume of 5.0 mL/kg. Table 31 illustrates the study design in more detail. 50 mg pieces of liver were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt).

TABLE 31
			siRNA				Harvest
			Dose		Dose Volume	# of	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)
1	ASGR2-Ab(Lys)-KRAS-	4	2	IV	5.0	1	96
	PEG5k (n = 1)
2	ASGR2-Ab(Lys)-KRAS-	4	1	IV	5.0	1	96
	PEG5k (n = 1)
3	ASGR2-Ab(Lys)-KRAS-	4	0.5	IV	5.0	1	96
	PEG5k (n = 1)
4	3Ga1NAc-KRAS Control	5	2	s.c.	5.0	1	96
5	PBS Control	5	—	IV	5.0	1	96
	Total # of Animals:	22	WT mice (CD-1)

KRAS gene knockdown was determined 96 hours post administration. As illustrated in FIG. 61, the GalNac-conjugated siRNA was capable of gene specific knockdown after a single s.c injection, as has been well described by others in the field. The same siRNA conjugated to an ASGR antibody was also capable of KRAS gene specific downregulation and in a dose dependent manner.

As highlighted in FIG. 54, it was demonstrated biological activity with the A-X—B—Y—C conjugate with a range of different antibodies and siRNA cargos that are capable of in vivo biological activity in a range of different tissue targets. In this example it was demonstrated liver delivery with an ASGR antibody conjugated to an siRNA designed to down regulate KRAS mRNA. Mouse Liver cells express the asialoglycoprotein receptor (ASGR) and the conjugate has a mouse specific ASGR antibody to target the siRNA, resulting in siRNA mediated knockdown of the KRAS in the liver

Example 20: 2016-PK-129-WT-plasma

siRNA Design and Synthesis

KRAS: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human KRAS. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 237 for the human mRNA transcript for KRAS (UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-1, as described in Example 9.

In Vivo Study Design

Groups (n=3) of wild-type female CD-1 mice were treated with one intravenous (i.v.) tail vein injections of siRNA conjugates. Treatment groups received 0.5 mg/kg (based on the weight of siRNA) and all groups were administered a dose volume of 5.0 mL/kg. Table 32 illustrates the study design in more detail. Non-terminal blood samples were collected at 5, 30, and 180 minutes post-dose via puncture of the retro-orbital plexus and centrifuged to generate plasma for PK analysis. Mice were sacrificed by CO₂ asphyxiation at 24, 96, or 168 h post-dose. Terminal blood samples were collected via cardiac puncture and processed to generate plasma for PK analysis. Quantitation of plasma siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves. Plasma concentrations of antibody were determined using an ELISA assay.

TABLE 32
					Dose		Survival	Terminal
			siRNA Dose		Volume	# of	Bleed	Bleed
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(min)	(h)
1	EGFR2-Ab(Lys)-	3	0.5	IV	5.0	1	5	24
2	KRAS-PEG5k (N = 1)	3	0.5	IV	5.0	1	30	96
3		3	0.5	IV	5.0	1	180	168
4	PSMA-Ab(Lys)-	3	0.5	IV	5.0	1	5	24
5	EGFR-PEG5k (N = 1)	3	0.5	IV	5.0	1	30	96
6		3	0.5	IV	5.0	1	180	168
	Total # of Animals:	18	WT mice CD-1

In this in vivo PK experiment the plasma clearance of two different conjugates was explored. As illustrated in FIG. 62, both the mAb-siRNA conjugates had comparable plasma PK when comparing the plasma levels of the siRNA (KRAS vs EGFR) or the antibody (EGFR2 vs PSMA).

As highlighted in FIG. 54, it was demonstrated biological activity with the A-X—B—Y—C conjugate with a range of different antibodies and siRNA cargos that are capable of in vivo biological activity in a range of different tissue targets. In this example it was demonstrated that two different conjugates with different antibody targeting ligands and different siRNA cargos have comparable plasma PK properties.

Example 21: 2016-PK-123-LNCaP

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) or DAR2 (n=2) using ASC architecture-1, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female SCID SHO mice bearing subcutaneous flank LNCaP tumors 100-350 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control groups (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Treatment groups were dosed as per the study design in Table 33. All groups (treatments and controls) were administered a dose volume of 5.71 mL/kg. Mice were sacrificed by CO₂ asphyxiation at 72 hours post-dose. 50 mg pieces of tumor and liver, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PP1B (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 33
					Dose
			siRNA Dose		Volume	# of	Harvest
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	Time (h)
1	PSMA-Ab(Lys)-EGFR-PEG5k	5	2	IV	5.71	1	72
2	(n = 1)	5	1	IV	5.71	1	72
3		5	0.5	IV	5.71	1	72
4	PSMA-Ab(Lys)-EGFR-PEG5k	5	4	IV	5.71	1	72
5	(n = 2)	5	2	IV	5.71	1	72
6		5	1	IV	5.71	1	72
7	PSMA-Ab(Lys)-Scramble-PEG5k	5	2	IV	5.71	1	72
	(n = 1)
8	EGFR siRNA Alone	5	2	IV	5.71	1	72
9	Vehicle	5	—	IV	5.71	1	72
	Total # of Animals:	45	SCID SHO mice with LNCaP tumors

siRNA concentrations were determined 72 hours in the tumor and liver after a single i.v. injection, see FIG. 63A. As illustrated in FIG. 63A, the antibody conjugate with a drug to antibody ratio of 1 (n=1) was capable of delivering siRNA to the tumor in a dose dependent manner, at levels greater than measured in the liver and produced EGFR gene specific mRNA knockdown relative to the scrambled and PBS controls. This is in contrast to the antibody conjugate with a drug to antibody ratio of 2 (n=2), which achieved lower concentrations of siRNA in the tumor at an equivalent dose, liver and tumor concentrations which were of the same magnitude and a lower levels of EGFR knockdown. The unconjugated siRNA had poor tumor and liver accumulation and no measurable EGFR mRNA knockdown. FIG. 63B illustrates relative percentage of EGFR mRNA in LNCaP Tumor.

As highlighted in FIG. 54, it was demonstrated biological activity with the A-X—B—Y—C conjugate with a range of different antibodies and siRNA cargos that are capable of in vivo biological activity in a range of different tissue targets. In this example it was demonstrated that the DAR1 conjugate is able to achieve greater siRNA tumor concentrations, relative to the DAR 2 and unconjugated siRNA. In addition, the DAR1 conjugate is able to achieve greater levels of siRNA mediate knockdown of EGFR, relative to the DAR 2 and unconjugated siRNA.

Example 22: 2016-PK-258-WT

siRNA Design and Synthesis

HPRT: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human HPRT. The sequence of the guide/antisense strand was AUAAAAUCUACAGUCAUAGUU (SEQ ID NO: 2102) and design to be complementary to the gene sequence starting a base position 425 for the human mRNA transcript for HPRT. Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphodiester-inverted abasic-phosphorothioate linker. The C6-NH₂ and C6-SH were connected through the phosphodiester, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). The same base, sugar and phosphate modifications that were used for the active EGFR siRNA duplex were used in the negative control siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

Conjugates in groups 1-3 and 7-9 were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9. Conjugates in groups 4-6 were made and purified as a DAR1 (n=1) using ASC architecture-1, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with one intravenous (i.v.) tail vein injections of siRNA conjugates, while the control group (n=4) of the same mice received one i.v. injection of PBS as a vehicle control. Table 34 illustrates the study design in more detail. 50 mg pieces of tissue, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 34
			siRNA		Dose		Harvest
			Dose		Volume	# of	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)
1	Anti-B cell Ab(Cys)-	4	3	IV	5.0	1	96
	HPRT-PEG5k (n = 1)
2	Anti-B cell Ab(Cys)-	4	1	IV	5.0	1	96
	HPRT-PEG5k (n = 1)
3	Anti-B cell Ab(Cys)-	4	0.3	IV	5.0	1	96
	HPRT-PEG5k (n = 1)
4	Anti-B cell Ab(Lys)-	4	3	IV	5.0	1	96
	HPRT-PEG5k (n = 1)
5	Anti-B cell Ab(Lys)-	4	1	IV	5.0	1	96
	HPRT-PEG5k (n = 1)
6	Anti-B cell Ab(Lys)-	4	0.3	IV	5.0	1	96
	HPRT-PEG5k (n = 1)
7	Anti-B cell Ab(Cys)-	4	3	IV	5.0	1	96
	scramble-PEG5k (n = 1)
8	Anti-B cell Ab(Cys)-	4	1	IV	5.0	1	96
	scramble-PEG5k (n = 1)
9	Anti-B cell Ab(Cys)-	4	0.3	IV	5.0	1	96
	scramble-PEG5k (n = 1)
10	PBS Control	4	—	IV	5.0	1	96
	Total # of Animals:	77	WT mice (CD-1)

As illustrated on FIG. 64A-FIG. 64C, after a single i.v. administration of an ASC dose dependent knockdown of HPRT in heart muscle, gastroc skeletal muscle and liver were measured. There was no measurable knockdown of HPRT in the lung tissue (FIG. 64D). In addition, dose dependent accumulation of the siRNA in all four tissue compartments was observed (FIG. 64E). There was no significant difference in the biological activity (KD and tissue concentration) between the lysine and cysteine conjugates.

As highlighted in FIG. 54, it was demonstrated biological activity with the A-X—B—Y—C conjugate with a range of different antibodies and siRNA cargos that are capable of in vivo biological activity in a range of different tissue targets. In this example it was demonstrated that an anti-B cell antibody can be used to target an siRNA to heart muscle, gastroc skeletal muscle and liver and achieve gene specific downregulation of the reporter gene HPRT. There was no measurable difference in the biological activity of the ASC constructs when a lysine or cysteine conjugation strategy was use to attach to the antibody.

Example 23: 2016-PK-254-WT

siRNA Design and Synthesis

HPRT: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human HPRT. The sequence of the guide/antisense strand was AUAAAAUCUACAGUCAUAGUU (SEQ ID NO: 2102) and design to be complementary to the gene sequence starting a base position 425 for the human mRNA transcript for HPRT. Base, sugar and phosphate modifications that are well described in the field of RNAi were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphodiester-inverted abasic-phosphorothioate linker. The C6-NH2 and C6-SH were connected through the phosphodiester, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). The same base, sugar and phosphate modifications that were used for the active EGFR siRNA duplex were used in the negative control siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with one intravenous (i.v.) tail vein injections of siRNA conjugates, while the control group (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Table 35 illustrates the study design in more detail. 50 mg pieces of tissue, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 35
			siRNA
			Dose		Dose Volume		Harvest
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	# of Doses	Time (h)
1	Anti-B cell Fab(Cys)-	4	10	IV	5.1	1	96
	HPRT-PEG5k (n = 1)
2	Anti-B cell Fab(Cys)-	4	3	IV	5.1	1	96
	HPRT-PEG5k (n = 1)
3	Anti-B cell Fab(Cys)-	4	1	IV	5.1	1	96
	HPRT-PEG5k (n = 1)
4	Anti-B cell Fab(Cys)-	4	10	IV	5.1	1	96
	scramble-PEG5k (n = 1)
5	Anti-B cell Fab(Cys)-	4	3	IV	5.1	1	96
	scramble-PEG5k (n = 1)
6	Anti-B cell Fab(Cys)-	4	1	IV	5.1	1	96
	scramble-PEG5k (n = 1)
7	PBS Control	5	—	IV	5.1	1	96
	Total # of Animals:	29	WT mice (CD-1)

As illustrated on FIG. 65A-FIG. 65C, after a single i.v. administration of an ASC containing an anti-B cell Fab targeting ligand, dose dependent knockdown of HPRT in heart muscle, gastroc skeletal muscle and liver were measured. There was no measurable knockdown of HPRT in the lung tissue (FIG. 65D). In addition, dose dependent accumulation of the siRNA in all four tissue compartments was observed (FIG. 65E).

As highlighted in FIG. 54, biological activity was demonstrated with the A-X—B—Y—C conjugate with a range of different antibodies and siRNA cargos that are capable of in vivo biological activity in a range of different tissue targets. In this example it was demonstrated that an anti-B cell Fab is used to target an siRNA to heart muscle, gastroc skeletal muscle and liver and achieve gene specific downregulation of the reporter gene HPRT.

Example 24: 2016-PK-245-WT

siRNA Design and Synthesis

CTNNB1: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human CTNNB1. The sequence of the guide/antisense strand was TUUCGAAUCAAUCCAACAGUU (SEQ ID NO: 2096), design to target the gene sequence starting a base position 1797 for the human mRNA transcript for CTNNB1. Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphodiester-inverted abasic-phosphorothioate linker. The C6-NH₂ and C6-SH were connected through the phosphodiester, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

Conjugates in groups 3-4 were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9. Conjugates in groups 1-2 were made and purified as a DAR1 (n=1) using ASC architecture-1, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with one intravenous (i.v.) tail vein injections of siRNA conjugates, while the control group (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Table 36 illustrates the study design in more detail. 50 mg pieces of tissue, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 36
					Dose
			siRNA Dose		Volume	# of	Harvest
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	Time (h)
1	Anti-B cell Ab(Lys)-CTNNB1-	4	3	IV	5.0	1	96
	PEG5k (n = 1)
2	Anti-B cell Ab(Lys)-CTNNB1-	4	1	IV	5.0	1	96
	PEG5k (n = 1)
3	Anti-B cell Ab(Cys)-CTNNB1-	4	3	IV	5.0	1	96
	PEG5k (n = 1)
4	Anti-B cell Ab(Cys)-CTNNB1-	4	1	IV	5.0	1	96
	PEG5k (n = 1)
5	PBS Control	5	—	IV	5.0	1	96
	Total # of Animals:	21	WT mice (CD-1)

As illustrated on FIG. 66A and FIG. 66B, after a single i.v. administration of an ASC containing an anti-B cell antibody targeting ligand (anti-B cell-Ab), HPRT knockdown and dose dependent tissue siRNA accumulation in heart muscle were elicited. As illustrated on FIG. 66C and FIG. 66D, after a single i.v. administration of an ASC containing an anti-B cell antibody targeting ligand, HPRT knockdown and dose dependent tissue siRNA accumulation in gastroc skeletal muscle were elicited. There was no significant difference in the biological activity (KD and tissue concentration) between the lysine and cysteine conjugates.

As highlighted in FIG. 54, it was demonstrated biological activity with the A-X—B—Y—C conjugate with a range of different antibodies and siRNA cargos that are capable of in vivo biological activity in a range of different tissue targets. In this example, it was demonstrated that an anti-B cell antibody is used to target an siRNA to heart muscle and gastroc skeletal muscle and achieve gene specific downregulation of CTNNB1 mRNA.

Example 25: 2016-PK-160-LNCaP

siRNA Design and Synthesis

AR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human AR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 2822 for the human mRNA transcript for AR (Guide strand sequence: GAGAGCUCCAUAGUGACACUU; SEQ ID NO: 2108). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate modifications were used to reduce immunogenicity and were comparable to those used in the active siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-1, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female SCID SHO mice bearing subcutaneous flank LNCaP tumors 100-350 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control group (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. The table below describes the study design. Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose. 50 mg pieces of tumor and liver, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 37
			siRNA		Dose		Harvest
			Dose		Volume	# of	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)
1	ANT4044(Lys)-AR-	5	1	IV	5.0	1	96
	PEG5k (n = 1)
2	ANT4044(Lys)-AR-	5	0.5	IV	5.0	1	96
	PEG5k (n = 1)
3	ANT4044(Lys)-AR-	5	0.25	IV	5.0	1	96
	PE5k (n = 1)
4	ANT4044(Lys)-scramble-	5	1	IV	5.0	1	96
	PEG5k (n = 1)
5	PBS Control	5	—	IV	5.0	1	96
	Total # of Animals:	30	castrated SCID SHO mice with LNCaP tumors

As illustrated in FIG. 67A, after a single i.v. administration of an ASC containing a PSMA antibody targeting ligand and siRNA designed to downregulate AR, AR knockdown in the LNCaP tumor tissue at all the doses tested relative to the scrambled control was elicited. As illustrated FIG. 67B, there was measurable accumulation of siRNA in the tumor tissue and at levels higher than those measured in the liver tissue.

As highlighted in FIG. 54, it was demonstrated biological activity with the A-X—B—Y—C conjugate with a range of different antibodies and siRNA cargos that are capable of in vivo biological activity in a range of different tissue targets. In this example, it was demonstrated delivery to an LNCaP prostate tumor with a PSMA antibody conjugated to an siRNA designed to down regulate AR mRNA. LNCaP cells express human PSMA on cell surface, the conjugate has a human specific PSMA antibody that binds to the antigen and allows internalization of the siRNA, resulting in siRNA mediated knockdown of AR in the tumor tissue.

Example 26: In Vitro Uptake and Knockdown in B Cells

siRNA Design and Synthesis

HPRT: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human HPRT. The sequence of the guide/antisense strand was AUAAAAUCUACAGUCAUAGUU (SEQ ID NO: 2102) and design to be complementary to the gene sequence starting a base position 425 for the human mRNA transcript for HPRT. Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphodiester-inverted abasic-phosphorothioate linker. The C6-NH₂ and C6-SH were connected through the phosphodiester, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vitro Study Design

Mouse spleens were harvested and kept in PBS with 100 u/ml penicillin and streptomycin on ice. Spleens were smashed with clean glass slides, cut into small pieces, homogenized with 18G needles, and filtered (70 um nylon membrane). Dead cells were removed with the dead cell removal kit from Milteny biotec (Catalog #130-090101) according to manufacturer instruction. To isolate mouse B cells, B cell isolation kit Milteny biotec (Catalog #130-090-862) was used following manufacturer instruction. Briefly, live spleen cells were resuspended with 200 μl of MACS buffer per mouse spleen. Non-B cells were depleted with biotin-conjugated monoclonal antibodies against CD43 (Ly48), CD4, and Ter-119, coupled with anti-biotin magnetic microbeads. From one mouse spleen, 30 million live B cells can be obtained. To activate isolated mouse B cells (2×10⁶/ml in 10% FBS RPMI-1640 with 100 u/ml penicillin and streptomycin), a cocktail of 10 μg/ml LPS, 5n/ml anti-IgM, 1 μg/ml anti-CD40, 0.05 mg/ml IL-4, and 0.05 μg/ml INFγ was added. After four hours of activation, ASCs (1 pM to 10 nM) were added to 10⁶ cells per well in 24 (0.5 ml media) or 12 (1 ml media) well plates. After 48 hours of ASC treatments, cells were harvested and isolated RNAs were analyzed for mRNA knockdown.

TABLE 38
Group Test Article
1     Anti-B cell Ab(Cys)-HPRT-PEG5k (n = 1)
2     Anti-B cell Ab (Cys)-scramble-PEG5k (n = 1)
3     Anti-B cell Fab(Cys)-HPRT-PEG5k (n = 1)
4     Anti-B cell Fab(Cys)-scramble-PEG5k (n = 1)
5     Anti-B cell Ab
6     Vehicle Control

In this in vitro experiment in activated primary mouse B cells, the ability of an anti-B cell antibody and Fab ASCs to deliver an siRNA design to downregulate Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was measured. As illustrated in FIG. 68A, the Fab conjugate was able to downregulate HPRT relative to the vehicle or scramble controls. As illustrated in FIG. 68B, the antibody conjugate was able to downregulate HPRT relative to the antibody, vehicle, and scramble controls.

As highlighted in FIG. 54, it was demonstrated biological activity with the A-X—B—Y—C conjugate with a range of different antibodies and siRNA cargos that are capable of in vivo biological activity in a range of different tissue targets. In this example, it was demonstrated delivery to an activated mouse B cell with a mouse anti-B cell antibody or anti-B cell Fab conjugated to an siRNA designed to down regulate HPRT mRNA. Activated mouse B cells recognize and internalize the antibody-siRNA conjugate via surface receptors that recognize the anti-B cell antibody, resulting in siRNA mediated knockdown of HPRT.

Example 27: 2016-PK-249-WT

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

KRAS: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 237 for the human mRNA transcript for KRAS (UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

The conjugate for groups 1-2 were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9. The conjugate for groups 3-4 were made and purified as a DAR2 (n=2) using ASC architecture-4, as described in Example 9. The conjugate for groups 5-6 were made and purified as a DAR1 (n=1) using ASC architecture-5, as described in Example 9. The conjugate for groups 7-8 were made and purified as a DAR2 (n=2) using ASC architecture-5, as described in Example 9. The conjugate for groups 9-10 were made and purified as a DAR1 (n=1) using ASC architecture-6, as described in Example 9. The conjugate for groups 11-12 were made and purified as a DAR2 (n=2) using ASC architecture-6, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with one intravenous (i.v.) tail vein injections of siRNA conjugates (groups 1-12) or antibody alone (groups 13-14). Table 39 illustrates the study design. Non-terminal blood samples were collected at 0.25, and 3 hours post-dose via puncture of the retro-orbital plexus and centrifuged to generate plasma for PK analysis. Mice were sacrificed by CO₂ asphyxiation at 24 and 72 hours post-dose. Terminal blood samples were collected via cardiac puncture and processed to generate plasma for PK analysis. Quantitation of plasma siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves. Plasma concentrations of antibody were determined using an ELISA assay.

TABLE 39
				Dose
			siRNA Dose	Volume	# of	Survival	Terminal
Gr	Test Article	N	(mg/kg)	(mL/kg)	Doses	Bleed (h)	Bleed (h)
1	EGFR-Ab(Cys)-EGFR-	4	0.5	5.0	1	0.25	24
	PEG5k (n = 1)
2	EGFR-Ab(Cys)-EGFR-	4	0.5	5.0	1	3	72
	PEG5k (n = 1)
3	EGFR-Ab(Cys)-EGFR-	4	0.5	5.0	1	0.25	24
	PEG5k (n = 2)
4	EGFR-Ab(Cys)-EGFR-	4	0.5	5.0	1	3	72
	PEG5k (n = 2)
5	EGFR-Ab (Lys-DHPz)-	4	0.5	5.0	1	0.25	24
	KRAS-PEG5k (n = 1)
6	EGFR-Ab (Lys-DHPz)-	4	0.5	5.0	1	3	72
	KRAS-PEG5k (n = 1)
7	EGFR-Ab (Lys-DHPz)-	4	0.5	5.0	1	0.25	24
	KRAS-PEG5k (n = 2)
8	EGFR-Ab (Lys-DHPz)-	4	0.5	5.0	1	3	72
	KRAS-PEG5k (n = 2)
9	EGFR-Ab(Asn297-DHPz)-	4	0.125	5.0	1	0.25	24
	KRAS-PEG5k (n = 1)
10	EGFR-Ab(Asn297-DHPz)-	4	0.125	5.0	1	3	72
	KRAS-PEG5k (n = 1)
11	EGFR-Ab(Asn297-DHPz)-	4	0.125	5.0	1	0.25	24
	KRAS-PEG5k (n = 2)
12	EGFR-Ab(Asn297-DHPz)-	4	0.125	5.0	1	3	72
	KRAS-PEG5k (n = 2)
13	EGFR-Ab	4	0.5	5.0	1	0.25	24
14	EGFR-Ab	4	0.5	5.0	1	3	72
	Total # of Animals:	56	WT mice CD -1

In this in vivo PK study it was demonstrated the utility of site specific conjugation. As illustrated in FIG. 69A, the DAR1 (n=1) test article (group 9) had a comparable siRNA plasma clearance profile to the two controls (groups 1 and 5), with approximately 10% of the siRNA remaining in the plasma after 168 hours. All the DAR2 (n=2) conjugates had much faster clearance of the siRNA from the plasma relative to the DAR1 conjugates. As illustrated in FIG. 69B, the DAR1 (n=1) test article (group 9) had a comparable EGFR-Ab plasma clearance profile to the two controls (groups 1 and 5). All the DAR2 (n=2) conjugates had much faster clearance of the antibody from the plasma relative to the DAR1 conjugates.

In the above Examples, it was demonstrated the use of lysine and cysteine conjugation strategies to attach the siRNA to the antibody. In this example, it was demonstrated the utility of a site specific conjugation strategy and demonstrate the conjugate has comparable PK properties to non-specific conjugation strategies.

Example 28: 2016-PK-180-HCC827

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate modifications were used to reduce immunogenicity and were comparable to those used in the active siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank HCC827 tumors 100-300 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control group (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Table 40 describes the study design. Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose. 50 mg pieces of tumor and liver, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of plasma and tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma and tissue concentrations using the linear equations derived from the standard curves.

TABLE 40
			siRNA Dose		Dose Volume	# of	Harvest
Gr	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	Time (h)
1	EGFR-Ab(Cys)-EGFR-	5	1	IV	5.0	1	96
	PEG5k (n = 1)
2	EGFR-Ab(Cys)-EGFR-	5	0.5	IV	5.0	1	96
	PEG5k (n = 1)
3	EGFR-Ab(Cys)-EGFR-	5	0.25	IV	5.0	1	96
	PEG5k (n = 1)
4	EGFR-Ab(Cys)-ECL-	5	1	IV	5.0	1	96
	EGFR-PEG5k (n = 1)
5	EGFR-Ab(Cys)-ECL-	5	0.5	IV	5.0	1	96
	EGFR-PEG5k (n = 1)
6	EGFR-Ab(Cys)-ECL-	5	0.25	IV	5.0	1	96
	EGFR-PEG5k (n = 1)
7	EGFR-Ab(Cys)-EGFR-	5	1	IV	5.0	1	96
	SS-PEG5k (n = 1)
8	EGFR-Ab(Cys)-EGFR-	5	0.5	IV	5.0	1	96
	SS-PEG5k (n = 1)
9	EGFR-Ab(Cys)-EGFR-	5	0.25	IV	5.0	1	96
	SS-PEG5k (n = 1)
10	EGFR-Ab(Cys)-ECL-	5	1	IV	5.0	1	96
	EGFR-SS-PEG5k (n = 1)
11	EGFR-Ab(Cys)-ECL-	5	0.5	IV	5.0	1	96
	EGFR-SS-PEG5k (n = 1)
12	EGFR-Ab(Cys)-ECL-	5	0.25	IV	5.0	1	96
	EGFR-SS-PEG5k (n = 1)
15	EGFR-Ab(Cys)-scramble-	5	1	IV	5.0	1	96
	PEG5k (n = 1)
16	PBS Control	5	—	IV	5.0	1	96
	Total # of Animals:	80	nu/nu mice with HCC827 tumors

In this in vivo PK study, replacing the SMCC linker between the antibody and siRNA with an enzymatically cleavable linker and the introduction of a cleavable disulfide linker between the siRNA and PEG, or the combination of both were tested. As illustrated in FIG. 70A, all the linker combination were capable of EGFR mRNA knockdown in the HCC827 tumor cells relative to the scrambled control. As illustrated in FIG. 70B, all the linker combinations produced comparable siRNA tissue accumulation in the tumor and liver. As illustrated in FIG. 70C, all the conjugates were capable of maintaining high levels of siRNA in the plasma, with approximately 10% remaining in the plasma after 168 hours.

In this AXBYC example, it was demonstrated that different linker combinations (“X” and/or “Y”) can be used to conjugate the siRNA to the antibody and PEG.

Example 29: 2016-PK-162-LNCaP

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate modifications were used to reduce immunogenicity and were comparable to those used in the active siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups 1-7 (n=5) of female SCID SHO mice bearing subcutaneous flank LNCaP tumors 100-350 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control group 8 (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. The table below describes the study design. Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose. 50 mg pieces of tumor and liver, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 41
			siRNA		Dose		Harvest
			Dose		Volume	# of	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)
1	PSMA-Ab(Lys)-SS-	5	1	IV	5.0	1	96
	EGFR-PEG5k (n = 1)
2	PSMA-Ab(Lys)-SS-	5	0.5	IV	5.0	1	96
	EGFR-PEG5k (n = 1)
3	PSMA-Ab(Cys)-ECL-	5	1	IV	5.0	1	96
	EGFR-PEG5k (n = 1)
4	PSMA-Ab(Cys)-ECL-	5	0.5	IV	5.0	1	96
	EGFR-PEG5k (n = 1)
5	PSMA-Ab(Cys)-EGFR-	5	1	IV	5.0	1	96
	PEG5k (n = 1) FRESH
6	PSMA-Ab(Cys)-EGFR-	5	1	IV	5.0	1	96
	PEG5k (n = 1) FROZEN
7	PSMA-Ab(Cys)-svcramble-	5	1	IV	5.0	1	96
	PEG5k (n = 1)
8	PBS Control	5	—	IV	5.0	1	96
Total # of Animals:	40	SCID SHO mice with LNCaP tumors

In this in vivo PK study, a disulfide (SS), enzymatically cleavable (ECL) or SMCC linker was used between the antibody and siRNA. As illustrated in graph 1 on slide 42, the tumor tissue accumulation of the siRNA was reduced when the cleavable disulfide leaker was used instead of the ECL or SMCC linkers. As illustrated on graph 2 on slide 42, the ECL linker strategy produced EGFR mRNA knockdown in the LNCaP tumor cells relative to the scrambled control. However, the SS linker failed to produce EGFR mRNA knockdown in the LNCaP tumor cells relative to the scrambled control. In addition to these linker experiments, the feasibility of −80° C. storage of the ASC was examined. The Formulation was snap-frozen in liquid nitrogen at 5 mg/ml antibody concentration, thawed at room temperature after 30 days storage at −80° C. and diluted to the required dosing concentration prior to administration. As illustrated on graph 3 on slide 42, the construct stored at −80° C., thawed prior to administration, retained its ability to produce EGFR mRNA knockdown in the LNCaP tumor cells relative to the scrambled control.

In this AXBYC example, it was demonstrated that an ECL linker (“X”) can be used to conjugate the antibody to the siRNA and that an ASC can be stored at −80° C. for 1 month and thawed prior to administration.

Example 30: 2016-PK-181-HCC827

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications that are well described in the field of RNAi were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate modifications were used to reduce immunogenicity and were comparable to those used in the active siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank HCC827 tumors 100-300 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control group (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Table 42 describes the study design. Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose. 50 mg pieces of tumor and liver, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in the methods section. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into tissue concentrations using the linear equations derived from the standard curves.

TABLE 42
					Dose
			siRNA		Volume	# of	Harvest
Group	Test Article	N	Dose (mg/kg)	ROA	(mL/kg)	Doses	Time (h)
1	EGFR-Ab(Cys)-EGFR-	5	1	IV	5.0	1	96
	PEG5k (n = 1)
2	EGFR-Ab(Cys)-EGFR-	5	0.5	IV	5.0	1	96
	PEG5k (n = 1)
3	EGFR-Ab(Cys)-SS-EGFR-	5	1	IV	5.0	1	96
	PEG5k (n = 1)
4	EGFR-Ab(Cys)-SS-EGFR-	5	0.5	IV	5.0	1	96
	PEG5k (n = 1)
6	EGFR-Ab(Cys)-scramble-	5	1	IV	5.0	1	96
	PEG5k (n = 1)
7	EGFR-Ab(Cys)-scramble-	5	0.5	IV	5.0	1	96
	PEG5k (n = 1)
8	PBS Control	5	—	IV	5.0	1	96
	Total # of Animals:	80	nu/nu mice with HCC827 tumors

In this in vivo PK study, a disulfide or SMCC linker was used between the antibody and siRNA. As illustrated in FIG. 72A, the tumor tissue accumulation of the siRNA was reduced when the cleavable disulfide leaker was used instead of the more stable SMCC linker. As illustrated in FIG. 72B, both linker strategies were capable of producing EGFR mRNA knockdown in the HCC827 tumor cells relative to the scrambled control.

In this AXBYC example, it was demonstrated the use of a cleavable disulfide linker (“X”) between the antibody and siRNA.

Example 31: 2016-PK-220-WT

siRNA Design and Synthesis

KRAS: A 2l mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human KRAS. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 237 for the human mRNA transcript for KRAS (Guide strand sequence: UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with one intravenous (i.v.) tail vein injections of siRNA conjugates. Treatment groups received 0.5 mg/kg (based on the weight of siRNA) and all groups were administered a dose volume of 5.0 mL/kg. Table 43 illustrates the study design in more detail. Non-terminal blood samples were collected at 5, 30, and 180 minutes post-dose via puncture of the retro-orbital plexus and centrifuged to generate plasma for PK analysis. Mice were sacrificed by CO₂ asphyxiation at 24, 96, or 168 hours post-dose. Terminal blood samples were collected via cardiac puncture and processed to generate plasma for PK analysis. Quantitation of plasma siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves. Plasma concentrations of antibody were determined using an ELISA assay.

TABLE 43
			siRNA		Dose		Survival	Terminal
			Dose		Volume	# of	Bleed	Bleed
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(min)	(h)
1	EGFR-Ab(Lys)-SPDP-	4	0.5	IV	5.0	1	5	24
2	KRAS-PEG5k (n = 1)	4	0.5	IV	5.0	1	30	96
3		4	0.5	IV	5.0	1	180	168
4	EGFR-Ab(Cys)-SPDP-	4	0.5	IV	5.0	1	5	24
5	KRAS-PEG5k (n-1)	4	0.5	IV	5.0	1	30	96
6		4	0.5	IV	5.0	1	180	168
7	EGFR-Ab(Cys)-	4	0.5	IV	5.0	1	5	24
8	SMPT-KRAS-	4	0.5	IV	5.0	1	30	96
9	PEG5k (n = 1)	4	0.5	IV	5.0	1	180	168
10	EGFR-Ab(Cys)-	4	0.5	IV	5.0	1	5	24
11	SS(methyl)-	4	0.5	IV	5.0	1	30	96
12	KRAS-PEG5k (n-1)	4	0.5	IV	5.0	1	180	168
13	EGFR-Ab(Cys)-	4	0.5	IV	5.0	1	5	24
14	SS(dimethyl)-KRAS-	4	0.5	IV	5.0	1	30	96
15	PEG5k (n = 1)	4	0.5	IV	5.0	1	180	168
	Total # of Animals:	60	WT mice CD-1

In this in vivo PK study, different disulfide linkers were explored, with varying degrees of steric hindrance, to understand how the rate of disulfide cleavage impacts ASC plasma PK. As illustrated in FIG. 73A, the clearance of the siRNA from the plasma was modulated by varying the degree of steric hindrance of the disulfide linker. FIG. 73B illustrates the clearance of the antibody zalutumumab from the plasma.

In this example, it was demonstrated biological activity with a range of different AXBYC conjugates in which a range of different disulfide linkers (“X”) can be used to conjugate the siRNA to the antibody.

Example 32: 2016-PK-256-WT

siRNA Design and Synthesis

KRAS: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human KRAS. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 237 for the human mRNA transcript for KRAS (Guide strand sequence: UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with one intravenous (i.v.) tail vein injections of siRNA conjugates. Treatment groups received 0.5 mg/kg (based on the weight of siRNA) and all groups were administered a dose volume of 5.0 mL/kg. Table 44 illustrates the study design in more detail. Non-terminal blood samples were collected at 0.25, 3, and 24 hours post-dose via puncture of the retro-orbital plexus and centrifuged to generate plasma for PK analysis. Mice were sacrificed by CO₂ asphyxiation at 72, 96, or 168 h post-dose. Terminal blood samples were collected via cardiac puncture and processed to generate plasma for PK analysis. Quantitation of plasma siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves. Plasma concentrations of antibody were determined using an ELISA assay.

TABLE 44
			siRNA		Dose		Survival	Terminal
			Dose		Volume	# of	Bleed	Bleed
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)	(h)
1	EGFR-Ab(Cys)-SMCC-	4	0.5	IV	5.0	1	0.25	72
	KRAS-PEG5k (n = 1)
2	EGFR-Ab(Cys)-SMCC-	4	0.5	IV	5.0	1	3	96
	KRAS-PEG5k (n = 1)
3	EGFR-Ab(Cys)-SMCC-	4	0.5	IV	5.0	1	24	168
	KRAS-PEG5k (n = 1)
4	EGFR-Ab(Cys)-CBTF-	4	0.5	IV	5.0	1	0.25	72
	KRAS-PEG5k (n = 1)
5	EGFR-Ab(Cys)-CBTF-	4	0.5	IV	5.0	1	3	96
	KRAS-PEG5k (n = 1)
6	EGFR-Ab(Cys)-CBTF-	4	0.5	IV	5.0	1	24	168
	KRAS-PEG5k (n = 1)
7	EGFR-Ab(Cys)-MBS-	4	0.5	IV	5.0	1	0.25	72
	KRAS-PEG5k (n = 1)
8	EGFR-Ab(Cys)-MBS-	4	0.5	IV	5.0	1	3	96
	KRAS-PEG5k (n = 1)
9	EGFR-Ab(Cys)-MBS-	4	0.5	IV	5.0	1	24	168
	KRAS-PEG5k (n = 1)
	Total # of Animals:	60	WT mice CD-1

In this in vivo PK study a range of different linkers between the antibody and siRNA were tested to determine the effect on plasma clearance. As illustrated on the graph on slide 45, all the conjugates were capable of maintaining high levels of siRNA in the plasma, with greater than 10% remaining in the plasma after 168 hours.

In this example, it was demonstrated biological activity with a range of different AXBYC conjugates in which a range of different linkers (“Y”) can be used to conjugate the siRNA to the antibody while maintaining the improved plasma kinetics over those historically observed for unconjugated siRNA.

Example 33: 2016-PK-237-HCC827

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (Guide strad sequence: ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA.

Two different passenger strands were made containing two conjugation handles (C6-NH₂ and C6-SH) in two different orientations (S5′-EGFR-3′N and N5′-EGFR-3'S). In the N5′-EGFR-3'S passenger strand both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure. In the S5′-EGFR-3′N passenger strand both conjugation handles were connected to siRNA passenger strand via phosphodiester-inverted abasic-phosphorothioate linker. The C6-NH2 and C6-SH were connected through the phosphodiester, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

The conjugate for groups 1-3 was made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9. The conjugate for groups 4-6 was made and purified as a DAR1 (n=1) using ASC architecture-2, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank HCC827 tumors 100-300 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control group (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Table 45 describes the study design. Mice were sacrificed by CO₂ asphyxiation at 72, 96, and 168 hours post-dose. 50 mg pieces of tumor and liver, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue and plasma siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 45
			siRNA		Dose		Survival	Terminal	Harvest
			Dose		Volume	# of	Bleed	Bleed	Time
Gr	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(min)	(h)	(h)
1	EGFR-Ab(Cys)-S5’-	5	0.5	IV	5.0	1	0.25	72	72
	EGFR-3’N-PEG5k
	(n = 1)
2	EGFR-Ab(Cys)-S5’-	5	0.5	IV	5.0	1	3	96	96
	EGFR-3’N-PEG5k
	(n = 1)
3	EGFR-Ab(Cys)-S5’-	5	0.5	IV	5.0	1	24	168	168
	EGFR-3’N-PEG5k
	(n = 1)
4	EGFR-Ab(Cys)-N5’-	5	0.5	IV	5.0	1	0.25	72	72
	EGFR-3’S-PEG5k
	(n = 1)
5	EGFR-Ab(Cys)-N5’-	5	0.5	IV	5.0	1	3	96	96
	EGFR-3'S-PEG5k
	(n = 1)
6	EGFR-Ab(Cys)-N5’-	5	0.5	IV	5.0	1	24	168	168
	EGFR-3’S-PEG5k
	(n = 1)
7	PBS Control	5	—	IV	5.0	1	—	—	96
	Total # of Animals:	65	nu/nu mice with HCC827 tumors

In this in vivo PK study the biological outcome of changes in the orientation of the conjugation site of the antibody and PEG (5′ or 3′) onto the siRNA were evaluated. In addition, the biological outcome of using a lysine or cysteine to attach the linker to the antibody was evaluated As illustrated FIG. 75A, both orientations of siRNA produced comparable EGFR tumor knockdown. As illustrated FIG. 75B and FIG. 75C, both orientations produced comparable siRNA tissue accumulation in the tumor and liver. As illustrated in FIG. 75D, both orientations produce a comparable plasma clearance kinetics.

As highlighted in FIG. 54, it was demonstrated biological activity with the A-X—B—Y—C conjugate with a range of different antibodies and siRNA cargos that are capable of in vivo biological activity in a range of different tissue targets. In this example, it was demonstrated that the antibody can be conjugated onto the 5′ and 3′ ends of the passenger strand of the siRNA and while maintaining the biological activity of the EGFR siRNA and tissue distribution.

Example 34: 2016-PK-259-WT

siRNA Design and Synthesis

HPRT: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human HPRT. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 425 for the human mRNA transcript for HPRT (guide strand sequence: UUAAAAUCUACAGUCAUAGUU; SEQ ID NO: 2104). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. Two different passenger strands were made containing two conjugation handles (C6-NH2 and C6-SH) in two different orientations (S5′-HPRT-3′N and N5′-HPRT-3'S). Both conjugation handles were connected to siRNA passenger strand via phosphodiester-inverted abasic-phosphorothioate linker. The C6-NH2 and C6-SH were connected through the phosphodiester, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

The conjugate for groups 1-3 was made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9. The conjugate for groups 4-6 was made and purified as a DAR1 (n=1) using ASC architecture-2, as described in Example 9. The conjugate for groups 7-9 was made and purified as a DAR1 (n=1) using ASC architecture-1, as described in Example 9. The conjugate for groups 10-12 was made and purified as a DAR1 (n=1) using ASC architecture-3, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with one intravenous (i.v.) tail vein injections of siRNA conjugates, while the control group (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Table 46 illustrates the study design in more detail. 50 mg pieces of tissue, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 46
			siRNA		Dose		Harvest
			Dose		Volume	# of	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)
1	Anti-B cell Ab(Cys)-N5′-HPRT-3′S-	4	3	IV	5.0	1	96
	PEG5k (n = 1)
2	Anti-B cell Ab(Cys)-N5′-HPRT-3′S-	4	1	IV	5.0	1	96
	PEG5k (n = 1)
3	Anti-B cell Ab(Cys)-N5′-HPRT-3′S-	4	0.3	IV	5.0	1	96
	PEG5k (n = 1)
4	Anti-B cell Ab(Cys)-N3′-HPRT-5′S-	4	3	IV	5.0	1	96
	PEG5k (n = 1)
5	Anti-B cell Ab(Cys)-N3′-HPRT-5′S-	4	1	IV	5.0	1	96
	PEG5k (n = 1)
6	Anti-B cell Ab(Cys)-N3′-HPRT-5′S-	4	0.3	IV	5.0	1	96
	PEG5k (n = 1)
7	Anti-B cell Ab(Lys)-S3′-HPRT-5′N-	4	2	IV	5.0	1	96
	PEG5k (n = 1)
8	Anti-B cell Ab(Lys)-S3′-HPRT-5′N-	4	0.75	IV	5.0	1	96
	PEG5k (n = 1)
9	Anti-B cell Ab(Lys)-S3′-HPRT-5′N-	4	0.25	IV	5.0	1	96
	PEG5k (n = 1)
10	Anti-B cell Ab(Lys)-S5′-HPRT-3′N-	4	2	IV	5.0	1	96
	PEG5k (n = 1)
11	Anti-B cell Ab(Lys)-S5′-HPRT-3′N-	4	0.75	IV	5.0	1	96
	PEG5k (n = 1)
12	Anti-B cell Ab(Lys)-S5′-HPRT-3′N-	4	0.25	IV	5.0	1	96
	PEG5k (n = 1)
13	PBSControl	5	—	IV	5.0	1	96
	Total # of Animals:	53	WT mice (CD-1)

In the in vivo PK study the biological outcome of changes in the orientation of the conjugation site of the antibody and PEG (5′ or 3′) onto the siRNA were evaluated. In addition, the biological outcome of using a lysine or cysteine to attach the linker to the antibody was evaluated. As illustrated in FIG. 76A-FIG. 76D, all the combinations of making the antibody conjugates produced comparable HPRT knockdown in the four tissue compartments measured. As illustrated in FIG. 77A-FIG. 77D, all the combinations of making the antibody conjugates produced comparable siRNA tissue accumulation in the different compartments measured.

In this example, it was demonstrated that a variety of different conjugation strategies to the siRNA and antibody can be used in the A-X—B—Y—C format while maintaining the biological activity of the HPRT siRNA and tissue distribution.

Example 35: 2016-PK-267-WT

siRNA Design and Synthesis

CTNNB1: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human CTNNB1. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 1797 for the human mRNA transcript for CTNNB1 (guide strand sequence: UUUCGAAUCAAUCCAACAGUU; SEQ ID NO: 2098). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA.

Two different passenger strands were made containing two conjugation handles (C6-NH2 and C6-SH) in two different orientations (S5′-CTNNB1-3′N and N5′-CTNNB1-3'S). Both conjugation handles were connected to siRNA passenger strand via phosphodiester-inverted abasic-phosphorothioate linker. The C6-NH2 and C6-SH were connected through the phosphodiester, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

The conjugate for groups 1-3 was made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9. The conjugate for groups 4-6 was made and purified as a DAR1 (n=1) using ASC architecture-3, as described in Example 9. The conjugate for groups 7-9 was made and purified as a DAR1 (n=1) using ASC architecture-2, as described in Example 9. The conjugate for groups 10-12 was made and purified as a DAR1 (n=1) using ASC architecture-1, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with one intravenous (i.v.) tail vein injections of siRNA conjugates, while the control group (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Table 47 illustrates the study design in more detail. 50 mg pieces of tissue, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt).

TABLE 47
			siRNA		Dose		Harvest
			Dose		Volume	# of	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)
1	Anti-B cell Ab(Cys)-N5′-	4	3	IV	5.0	1	96
	CTNNB1-3′S-PEG5k (n = 1)
2	Anti-B cell Ab(Cys)-N5′-	4	1	IV	5.0	1	96
	CTNNB1-3′S-PEG5k (n = 1)
3	Anti-B cell Ab(Cys)-N5′-	4	0.3	IV	5.0	1	96
	CTNNB1-3′S-PEG5k (n = 1)
4	Anti-B cell Ab(Lys)-S5′-	4	3	IV	5.0	1	96
	CTNNB1-3′N-PEG5k (n = 1)
5	Anti-B cell Ab(Lys)-S5′-	4	1	IV	5.0	1	96
	CTNNB1-3′N-PEG5k (n = 1)
6	Anti-B cell Ab(Lys)-S5′-	4	0.3	IV	5.0	1	96
	CTNNB1-3′N-PEG5k (n = 1)
7	Anti-B cell Ab(Cys)-N3′-	4	3	IV	5.0	1	96
	CTNNB1-5′S-PEG5k (n = 1)
8	Anti-B cell Ab(Cys)-N3′-	4	1	IV	5.0	1	96
	CTNNB1-5′S-PEG5k (n = 1)
9	Anti-B cell Ab(Cys)-N3′-	4	0.3	IV	5.0	1	96
	CTNNB1-5′S-PEG5k (n = 1)
10	Anti-B cell Ab(Lys)-S3′-	4	3	IV	5.0	1	96
	CTNNB1-5′N-PEG5k (n = 1)
11	Anti-B cell Ab(Lys)-S3′-	4	1	IV	5.0	1	96
	CTNNB1-5′N-PEG5k (n = 1)
12	Anti-B cell Ab(Lys)-S3′-	4	0.3	IV	5.0	1	96
	CTNNB1-5′N-PEG5k (n = 1)
13	PBS Control	5	—	IV	5.0	1	96
	Total # of Animals:	53	WT mice (CD-1)

In this in vivo PK study, the biological outcome of changes in the orientation of the conjugation site of the antibody and PEG (5′ or 3′) onto the siRNA and the biological outcome of using a lysine or cysteine to attach the linker to the antibody were evaluated. As illustrated in FIG. 78A-FIG. 78D, all the combinations of making the antibody conjugates produced comparable CTNNB1 knockdown in the four tissue compartments measured.

In this example, it was demonstrated that a variety of different conjugation strategies to the siRNA and antibody can be used in the A-X—B—Y—C format while maintaining the biological activity of the CTNNB1 siRNA.

Example 36: 2016-PK-188-PK

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with one intravenous (i.v.) tail vein injections of siRNA conjugates. Treatment groups received 0.5 mg/kg (based on the weight of siRNA) and all groups were administered a dose volume of 5.0 mL/kg. Table 48 illustrates the study design in more detail. Non-terminal blood samples were collected at 5, 30, and 180 minutes post-dose via puncture of the retro-orbital plexus and centrifuged to generate plasma for PK analysis. Mice were sacrificed by CO₂ asphyxiation at 24, 96, or 168 h post-dose. Terminal blood samples were collected via cardiac puncture and processed to generate plasma for PK analysis. Quantitation of plasma siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 48
				Dose		Survival	Terminal
				Volume	# of	Bleed	Bleed
Gr	Test Article	N	ROA	(mL/kg)	Doses	(min)	(h)
1	EGFR-Ab(Cys)-EGFR-PEG5k (n = 1)	4	IV	5.0	1	5	24
2		4	IV	5.0	1	30	96
3		4	IV	5.0	1	180	168
4	EGFR-Ab(Cys)-ECL-EGFR-PEG5k	4	IV	5.0	1	5	24
5	(n = 1)	4	IV	5.0	1	30	96
6		4	IV	5.0	1	180	168
7	EGFR-Ab(Cys)-EGFR-SS-PEG5k	4	IV	5.0	1	5	24
8	(n = 1)	4	IV	5.0	1	30	96
9		4	IV	5.0	1	180	168
10	EGFR-Ab(Cys)-ECL-EGFR-SS-	4	IV	5.0	1	5	24
11	PEG5k	4	IV	5.0	1	30	96
12	(n = 1)	4	IV	5.0	1	180	168
	Total # of Animals:	48	WT mice CD-1

As illustrated in FIG. 79, all the ASC with the different cleavable linker configurations achieved equivalent plasma PK profiles, with approximately 10% of the siRNA remaining 168 hours after administration.

In this example, it was demonstrated biological activity with a range of A-X—B—Y—C conjugates in which a variety of different linker strategies (component X and Y) were used to conjugate the PEG and antibody to the siRNA passenger strand.

Example 37: 2016-PK-201-LNCaP

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate modifications were used to reduce immunogenicity and were comparable to those used in the active siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups 1-7 (n=5) of female SCID SHO mice bearing subcutaneous flank LNCaP tumors 100-350 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control group 8 (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Table 49 describes the study design. Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose. 50 mg pieces of tumor and liver, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 49
							Har-
			siRNA		Dose		vest
			Dose		Volume	# of	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)
1	PSMA-Ab(Cys)-	5	1	IV	5.0	1	96
	EGFR-SS-PEG5k
	(n = 1)
2	PSMA-Ab(Cys)-	5	0.5	IV	5.0	1	96
	EGFR-SS-PEG5k
	(n = 1)
3	PSMA-Ab(Cys)-	5	1	IV	5.0	1	96
	EGFR-ECL-
	PEG5k (n = 1)
4	PSMA-Ab(Cys)-	5	0.5	IV	5.0	1	96
	EGFR-ECL-
	PEG5k (n = 1)
5	PSAM-Ab(Cys)-	5	1	IV	5.0	1	96
	EGFR-PEG5k
	(n = 1)
6	PSAM-Ab(Cys)-	5	0.5	IV	5.0	1	96
	EGFR-PEG5k
	(n = 1)
7	PSMA-Ab(Cys)-	5	1	IV	5.0	1	96
	scramble-PEG5k
	(n = 1)
8	PBS Control	5	—	IV	5.0	1	96
	Total # of Animals:	40	SCID SHO mice with LNCaP tumors

As illustrated in FIG. 80A, a variety of different linkers were used between the siRNA and PEG, after i.v administration of a single dose of siRNA measurable tumor tissue EGFR downregulation was achieved relative to the negative control siRNA sequence or PBS controls. In addition, as illustrated in FIG. 80B, the different linker configurations resulted in tumor siRNA accumulation at higher levels than the other tissue samples measured (liver, spleen, lung and kidney).

In this example, it was demonstrated biological activity with a range of A-X—B—Y—C conjugates in which a variety of different linkers strategies (component Y) were used to conjugate the PEG to the siRNA passenger strand.

Example 38: 2016-PK-198-HCC827

siRNA Design and Synthesis

EFGR: A 2l mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate modifications were used to reduce immunogenicity and were comparable to those used in the active siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups 1-15 (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank HCC827 tumors 100-300 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control group 16 (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Table 50 describes the study design. Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose. 50 mg pieces of tumor and liver, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 50
							Har-
			siRNA		Dose		vest
			Dose		Volume	# of	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)
1	EGFR-Ab(Cys)-	5	1	IV	5.0	1	96
	EGFR-PEG2k
	(n = 1)
2	EGFR-Ab(Cys)-	5	0.5	IV	5.0	1	96
	EGFR-PEG2k
	(n = 1)
3	EGFR-Ab(Cys)-	5	0.25	IV	5.0	1	96
	EGFR-PEG2k
	(n = 1)
4	EGFR-Ab(Cys)-	5	1	IV	5.0	1	96
	EGFR-dPEG48
	(n = 1)
5	EGFR-Ab(Cys)-	5	0.5	IV	5.0	1	96
	EGFR-dPEG48
	(n = 1)
6	EGFR-Ab(Cys)-	5	0.25	IV	5.0	1	96
	EGFR-dPEG48
	(n = 1)
7	EGFR-Ab(Cys)-	5	1	IV	5.0	1	96
	EGFR-dPEG24
	(n = 1)
8	EGFR-Ab(Cys)-	5	0.5	IV	5.0	1	96
	EGFR-dPEG24
	(n = 1)
9	EGFR-Ab(Cys)-	5	0.25	IV	5.0	1	96
	EGFR-dPEG24
	(n = 1)
10	EGFR-Ab(Cys)-	5	1	IV	5.0	1	96
	EGFR-dPEG12
	(n = 1)
11	EGFR-Ab(Cys)-	5	0.5	IV	5.0	1	96
	EGFR-dPEG12
	(n = 1)
12	EGFR-Ab(Cys)-	5	0.25	IV	5.0	1	96
	EGFR-dPEG12
	(n = 1)
13	EGFR-Ab(Cys)-	5	1	IV	5.0	1	96
	EGFR-PEG5k
	(n = 1)
14	PSMA-Ab(Cys)-	5	1	IV	5.0	1	96
	EGFR-PEG5k
	(n = 1)
15	EGFR-Ab(Cys)-	5	1	IV	5.0	1	96
	scramble-
	PEG5k (n = 1)
16	PBS Control	5	—	IV	5.0	1	96
	Total # of Animals:	80	nu/nu mice with HCC827 tumors

As illustrated in FIG. 81A, all the ASC with the different configurations of linear PEG length achieved dose dependent EGFR mRNA knockdown in the HCC827 tumor cells, relative to the negative control siRNA sequence (scramble) and PBS controls. As illustrated in FIG. 81B, all the ASC with the different configurations in linear PEG length achieved equivalent dose dependent siRNA tumor tissue accumulation. In addition to low liver, lung, kidney and spleen accumulation relative to tumor.

In this example, it was demonstrated biological activity with a range of A-X—B—Y—C conjugates in which a variety of different PEG (component C) lengths were used.

Example 39: 2016-PK-194-WT

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with one intravenous (i.v.) tail vein injections of siRNA conjugates. Treatment groups received 0.5 mg/kg (based on the weight of siRNA) and all groups were administered a dose volume of 5.0 mL/kg. Table 51 illustrates the study design in more detail. Non-terminal blood samples were collected at 5, 30, and 180 minutes post-dose via puncture of the retro-orbital plexus and centrifuged to generate plasma for PK analysis. Mice were sacrificed by CO₂ asphyxiation at 24, 96, or 168 h post-dose. Terminal blood samples were collected via cardiac puncture and processed to generate plasma for PK analysis. Quantitation of plasma siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 51
					Dose		Survival	Terminal
			siRNA Dose		Volume	# of	Bleed	Bleed
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(min)	(h)
1	EGFR-Ab(Cys)-	4	0.5	IV	5.0	1	5	24
2	EGFR-PEG2k (n = 1)	4	0.5	IV	5.0	1	30	96
3		4	0.5	IV	5.0	1	180	168
4	EGFR-Ab(Cys)-	4	0.5	IV	5.0	1	5	24
5	EGFR-dPEG48 (n = 1)	4	0.5	IV	5.0	1	30	96
6		4	0.5	IV	5.0	1	180	168
7	EGFR-Ab(Cys)-	4	0.5	IV	5.0	1	5	24
8	EGFR-dPEG24 (n = 1)	4	0.5	IV	5.0	1	30	96
9		4	0.5	IV	5.0	1	180	168
10	EGFR-Ab(Cys)-	4	0.5	IV	5.0	1	5	24
11	EGFR-dPEG12 (n = 1)	4	0.5	IV	5.0	1	30	96
12		4	0.5	IV	5.0	1	180	168
	Total # of Animals:	48	WT mice CD-1

As illustrated on slide 54, all the ASC with the different linear PEG lengths achieved equivalent plasma PK profiles, with approximately 10% of the siRNA remaining 168 hours after administration.

In this example, it was demonstrated equivalent plasma PK properties with a range of A-X—B—Y—C conjugates in which a variety of different PEG (component C) lengths were used.

Example 40: 2016-PK-195-WT

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with one intravenous (i.v.) tail vein injections of siRNA conjugates. Treatment groups received 0.5 mg/kg (based on the weight of siRNA) and all groups were administered a dose volume of 5.0 mL/kg. Table 52 illustrates the study design in more detail. Non-terminal blood samples were collected at 5, 30, and 180 minutes post-dose via puncture of the retro-orbital plexus and centrifuged to generate plasma for PK analysis. Mice were sacrificed by CO₂ asphyxiation at 24, 96, or 168 h post-dose. Terminal blood samples were collected via cardiac puncture and processed to generate plasma for PK analysis. Quantitation of plasma siRNA concentrations were determined using a stern-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 52
			siRNA		Dose		Survival	Terminal
			Dose		Volume	# of	Bleed	Bleed
Gr	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(min)	(h)
1	EGFR-Ab(Cys)-EGFR-PEG10k	4	0.5	IV	5.0	1	5	24
2	(n = 1)	4	0.5	IV	5.0	1	30	96
3		4	0.5	IV	5.0	1	180	168
4	EGFR-Ab(Cys)-EGFR-	4	0.5	IV	5.0	1	5	24
5	(dPEG24)3 (n = 1)	4	0.5	IV	5.0	1	30	96
6		4	0.5	IV	5.0	1	180	168
7	EGFR-Ab(Cys)-EGFR-	4	0.5	IV	5.0	1	5	24
8	(dPEG12)3 (n = 1)	4	0.5	IV	5.0	1	30	96
9		4	0.5	IV	5.0	1	180	168
10	EGFR-Ab(Cys)-EGFR-(dPEG4)3	4	0.5	IV	5.0	1	5	24
11	(n = 1)	4	0.5	IV	5.0	1	30	96
12		4	0.5	IV	5.0	1	180	168
	Total #of Animals:	48	WT mice CD-1

As illustrated in FIG. 83, all the ASC with the different PEG configurations (length and branching) achieved equivalent plasma PK profiles, with approximately 10% of the siRNA remaining 168 hours after administration.

In this example, it was demonstrated equivalent plasma PK properties with a range of A-X—B—Y—C conjugates in which a variety of different PEG (component C) lengths and branching were used.

Example 41: 2016-PK-236-HCC827

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human EGFR. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 333 for the human mRNA transcript for EGFR (ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphorothioate-inverted abasic-phosphorothioate linker, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). Base, sugar and phosphate modifications were used to reduce immunogenicity and were comparable to those used in the active siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups 1-12 (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank HCC827 tumors 100-300 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control group 13 (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Table 53 describes the study design. Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose. 50 mg pieces of tumor and liver, were collected and snap-frozen in liquid nitrogen. mRNA knockdown in target tissue was determined using a comparative qPCR assay as described in Example 2. Total RNA was extracted from the tissue, reverse transcribed and mRNA levels were quantified using TaqMan qPCR, using the appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control, results were calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ΔCt) is calculated and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt). Quantitation of tissue siRNA concentrations were determined using a stem-loop qPCR assay as described in Example 2. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA reverse transcription kit using a sequence-specific stem-loop RT primer. The cDNA from the RT step was then utilized for real-time PCR and Ct values were transformed into plasma or tissue concentrations using the linear equations derived from the standard curves.

TABLE 53
			siRNA		Dose		Harvest
			Dose		Volume	# of	Time
Group	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)
1	EGFR-Ab(Cys)-EGFR-PEG10k (n = 1)	5	1	IV	5.0	1	96
2	EGFR-Ab(Cys)-EGFR-PEG10k (n = 1)	5	0.5	IV	5.0	1	96
3	EGFR-Ab(Cys)-EGFR-PEG10k (n = 1)	5	0.25	IV	5.0	1	96
4	EGFR-Ab(Cys)-EGFR-(dPEG24)3 (n = 1)	5	1	IV	5.0	1	96
5	EGFR-Ab(Cys)-EGFR-(dPEG24)3 (n = 1)	5	0.5	IV	5.0	1	96
6	EGFR-Ab(Cys)-EGFR-(dPEG24)3 (n = 1)	5	0.25	IV	5.0	1	96
7	EGFR-Ab(Cys)-EGFR-(dPEG12)3 (n = 1)	5	1	IV	5.0	1	96
8	EGFR-Ab(Cys)-EGFR-(dPEG12)3 (n = 1)	5	0.5	IV	5.0	1	96
9	EGFR-Ab(Cys)-EGFR-(dPEG12)3 (n = 1)	5	0.25	IV	5.0	1	96
10	EGFR-Ab(Cys)-EGFR-(dPEG4)3 (n = 1)	5	1	IV	5.0	1	96
11	EGFR-Ab(Cys)-EGFR-PEG5k (n = 1)	5	1	IV	5.0	1	96
12	EGFR-Ab(Cys)-scramble-PEG5k (n = 1)	5	1	IV	5.0	1	96
13	PBS Control	5	—	IV	5.0	1	96
	Total # of Animals:	65	nu/nu mice with HCC827 tumors

As illustrated in FIG. 84, all the ASC with the different configurations of PEG (length and branching) achieved equivalent EGFR mRNA knockdown in the HCC827 tumor cells to the construct with the linear PEG5K at the 1 mg/kg dose. Those constructs tested in a dose response format, showed dose dependent knockdown of EGFR mRNA. As illustrated in FIG. 85, all the ASC with the different variations in linear PEG length and PEG branching achieved equivalent siRNA tumor tissue accumulation to the construct with the linear PEG5K at the 1 mg/kg dose. In addition to low liver accumulation relative to tumor, those constructs tested in a dose response format, showed dose dependent tumor tissue accumulation of siRNA.

In this example, it was demonstrated biological activity with a range of A-X—B—Y—C conjugates in which a variety of different PEG (component C) lengths and branching were used.

Example 42: In Vitro Knockdown with ASCs with PEG Polymers

siRNA Design and Synthesis

HPRT: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human HPRT. The sequence of the guide/antisense strand was AUAAAAUCUACAGUCAUAGUU (SEQ ID NO: 2082) and design to be complementary to the gene sequence starting a base position 425 for the human mRNA transcript for HPRT. Base, sugar and phosphate modifications were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via phosphodiester-inverted abasic-phosphorothioate linker. The C6-NH₂ and C6-SH were connected through the phosphodiester, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was used. The sequence (5′ to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 2116). The same base, sugar and phosphate modifications that were used for the active EGFR siRNA duplex were used in the negative control siRNA. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

Conjugates in groups 1-3 made and purified as a DAR1 (n=1) using ASC architecture-1, as described in Example 9. Conjugates in groups 4-6 were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9.

In Vitro Study Design

Mouse spleens were harvested and kept in PBS with 100 u/ml penicillin and streptomycin on ice. Spleens were smashed with clean glass slides, cut into small pieces, homogenized with 18G needles, and filtered (70 um nylon membrane). Dead cells were removed with the dead cell removal kit from Milteny biotec (Catalog #130-090101) according to manufacturer instruction. To isolate mouse B cells, B cell isolation kit Milteny biotec (Catalog #130-090-862) was used following manufacturer instruction. Briefly, live spleen cells were resuspended with 200 μl of MACS buffer per mouse spleen. Non-B cells were depleted with biotin-conjugated monoclonal antibodies against CD43 (Ly48), CD4, and Ter-119, coupled with anti-biotin magnetic microbeads. From one mouse spleen, 30 million live B cells can be obtained. To activate isolated mouse B cells (2×10⁶/ml in 10% FBS RPMI-1640 with 100 u/ml penicillin and streptomycin), a cocktail of 10 μg/ml LPS, 5 μg/ml anti-IgM, 1 μg/ml anti-CD40, 0.05 μg/ml IL-4, and 0.05 μg/ml INFγ was added. After four hours of activation, ASCs (1 pM to 10 nM) were added to 10⁶ cells per well in 24 (0.5 ml media) or 12 (1 ml media) well plates. After 48 hours of ASC treatments, cells were harvested and isolated RNAs were analyzed for mRNA knockdown. See Table 54 for the study design.

TABLE 54
Group Test Article
1     Anti-B cell Ab(Lys)-S3′-HPRT-5′N-pOEGMA8K
2     Anti-B cell Ab(Lys)-S3′-HPRT-5′N-pHPMA5K
3     Anti-B cell Ab(Lys)-S3′-HPRT-5′N-pHPMA10K
4     Anti-B cell Ab(Cys)-N5′-HPRT-3′S-pMAA10K
5     Anti-B cell Ab(Cys)-N5′-HPRT-3′S-PEG5K
6     Anti-B cell Ab(Cys)-N5′-scramble-3′S-PEG5K

In this in vitro experiment in activated primary mouse B cells, the ability of an anti-B cell antibody ASCs to deliver an siRNA design to downregulate Hypoxanthine-guanine phosphoribosyltransferase (HPRT) with a range of alternative PEG polymers were measured. As illustrated in FIG. 86, the range of ASC with alternative PEGs were able to downregulate HPRT relative to the scramble control.

In this example, the biological activity was demonstrated with a range of A-X—B—Y—C conjugates in which a variety of polymer alternatives to PEG (component C) were used.

Example 43: PK-236-WT

siRNA Design and Synthesis

KRAS: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotide overhangs was designed against human KRAS. The sequence of the guide/antisense strand was complementary to the gene sequence starting a base position 237 for the human mRNA transcript for KRAS (Guide strand sequence: UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base, sugar and phosphate modifications that are well described in the field of RNAi were used to optimize the potency of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on solid phase using standard phospharamidite chemistry and purified over HPLC. The base at position 11 on the passenger strand had a Cy5 fluorescent label attached, as described in Example 9. Purified single strands were duplexed to get the double stranded siRNA. The passenger strand contained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles were connected to siRNA passenger strand via a phosphodiester-inverted abasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

Conjugates in groups 1-3 were made and purified as a DAR1 (n=1) using ASC architecture-4, as described in Example 9. Conjugates in groups 4-6 were made and purified as a DAR1 (n=1) using ASC architecture-4, but there was no PEG on the 3′ end of the passenger strand. Prior to conjugation, the 3′thiol was end-capped using N-ethylmaleimide. Conjugates in groups 7-9 were made and purified as a DAR1 (n=1) using ASC architecture-1, as described in Example 9. Conjugates in groups 10-12 made and purified as a DAR1 (n=1) using ASC architecture-1, but there was no PEG on the 5′ end of the passenger strand.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with one intravenous (i.v.) tail vein injections of siRNA conjugates. Treatment groups received 0.5 mg/kg (based on the weight of siRNA) and all groups were administered a dose volume of 5.0 mL/kg. Table 55 illustrates the study design in more detail. Non-terminal blood samples were collected at 0.25, 1, and 4 hours post-dose via puncture of the retro-orbital plexus and centrifuged to generate plasma for PK analysis. Mice were sacrificed by CO₂ asphyxiation at 24, 48, or 72 h post-dose. Terminal blood samples were collected via cardiac puncture and processed to generate plasma for PK analysis.

Plasma samples (K2 EDTA) were processed within 4 hours after harvesting. Plasma samples were diluted with matching mouse plasma (Bioreclamation) (2-400 fold) and the concentration of CY5-siRNA in these plasma samples quantified spectroscopically using a TECAN Infinite M200 Pro (Excitation 635 nm; Emission 675 nm). To release macromolecular interactions that might quench the CY5 fluorescence, all samples were diluted 2-fold into water containing 0.01% Tween 20 and 100 ug/ml heparin prior to quantification. To determine the amount of intact ASCs in these plasma samples, plasma samples were diluted with mouse plasma to 2-50 nM CY5-siRNA and incubated with Protein G Dynabeads (Thermofisher) loaded with 150 nM of a purified EGFR-Fc protein (Sino Biological). These binding reactions were incubated at RT for 1 hour. Beads were washed twice with PBS containing 0.01% Tween 20 and 0.05% BSA before ASCs bound to EGFR were eluted by incubation in 0.1 M citric acid (pH 2.7). The amount of CY5-siRNA contained in the input, unbound fraction, washes and bead eluate was quantified by fluorescence as stated above.

TABLE 55
			siRNA		Dose		Survival	Terminal
			Dose		Volume	# of	Bleed	Bleed
Gr	Test Article	N	(mg/kg)	ROA	(mL/kg)	Doses	(h)	(h)
1	EGFR-Ab(Cys)-N5′-Cy5.KRAS-	4	0.5	IV	5.0	1	0.25	24
	3′S-PEG5k (n = 1)
2	EGFR-Ab(Cys)-N5′-Cy5.KRAS-	4	0.5	IV	5.0	1	1	48
	3′S-PEG5k (n = 1)
3	EGFR-Ab(Cys)-N5′-Cy5.KRAS-	4	0.5	IV	5.0	1	4	72
	3′S-PEG5k (n = 1)
4	EGFR-Ab(Cys)-N5′-Cy5.KRAS-	4	0.5	IV	5.0	1	0.25	24
	3′S-NEM (n = 1)
5	EGFR-Ab(Cys)-N5′-Cy5.KRAS-	4	0.5	IV	5.0	1	1	48
	3′S-NEM (n = 1)
6	EGFR-Ab(Cys)-N5′-Cy5.KRAS-	4	0.5	IV	5.0	1	4	72
	3′S-NEM (n = 1)
7	EGFR-Ab(Lys)-S3′-Cy5.KRAS-	4	0.5	IV	5.0	1	0.25	24
	5′N-PEG5k (n = 1)
8	EGFR-Ab(Lys)-S3′-Cy5.KRAS-	4	0.5	IV	5.0	1	1	48
	5′N-PEG5k (n = 1)
9	EGFR-Ab(Lys)-S3′-Cy5.KRAS-	4	0.5	IV	5.0	1	4	72
	5′N-PEG5k (n = 1)
10	EGFR-Ab(Lys)-S3′-Cy5.KRAS-	4	0.5	IV	5.0	1	0.25	24
	5′NH2 (n = 1)
11	EGFR-Ab(Lys)-S3′-Cy5.KRAS-	4	0.5	IV	5.0	1	1	48
	5′NH2 (n = 1)
12	EGFR-Ab(Lys)-S3′-Cy5.KRAS-	4	0.5	IV	5.0	1	4	72
	5′NH2 (n = 1)
	Total # of Animals:	96	WT mice CD-1

In this in vivo PK study, the in vivo plasma stability of two AXBYC conjugates (cysteine and lysine conjugation to the EGFR-Ab) relative to two AXB conjugates were compared. As illustrated in FIG. 87, the concentration of the siRNA was determined using two methods. The fluorescence of the plasma was measured directly and the siRNA concentration determined using a standard curve. Or a magnetic bead decorated with EGFR was used to bind the antibody conjugates and then the fluorescence of the sample was measured and the siRNA concentration determined using a standard curve. All data were plotted as a percentage of the injected dose. In both examples of the AXBYC conjugates (cysteine and lysine conjugation to the EGFR-Ab) improved plasma PK were observed relative to the corresponding AXB conjugate.

In this example, in vivo plasma PK for the Cys and Lys AXBYC conjugates compared to the matching control AXB conjugates was demonstrate.

Example 44: In Vivo Pharmacodynamics Study of a Cholesterol-KRAS Conjugate (PD-058)

Groups (n=5) of female NCr nu/nu mice bearing intrahepatic Hep 3B tumors one week after inoculation were treated with three intravenous (i.v.) tail vein injections (separated by 48 h) of cholesterol-siRNA conjugate, while control groups (n=5) of the same mice received three i.v. injections of PBS as a vehicle control on the same dosing schedule. Treatment groups that received chol-KRAS were dosed at 10, 4, or 2 mg/kg. All groups (treatments and controls) were administered a dose volume of 6.25 mL/kg. Table 56 describes the study design in more detail and gives a cross-reference to the conjugate synthesis and characterization. Mice were sacrificed by CO₂ asphyxiation at 72 h post-final dose. 50 mg pieces of tumor-bearing liver were collected and snap-frozen in liquid nitrogen. mRNA knockdown analysis and siRNA quantitation were performed as described in Examples 2-7.

TABLE 56
Study design for a Cholesterol-KRAS Conjugate (PD-058) with a
cross-reference to the synthesis and characterization of the conjugates tested.
			siRNA Dose			Cross-reference for synthesis and
Group	Test Article	N	(mg/kg)	ROA	# of Doses	characterization
1	Chol-KRAS	5	10	iv	3	General experimental (Example 2)
2	Chol-KRAS	5	4	iv	3	General experimental (Example 2)
3	Chol-KRAS	5	2	iv	3	General experimental (Example 2)
4	Vehicle (PBS)	5		iv	3

The chol-KRAS conjugate was assessed for mRNA knockdown in a 3-dose study with a dose response. As illustrated in FIG. 35, within the mouse liver tissue there was a clear dose-response for mouse KRAS mRNA knockdown. The lowest dose of 2 mg/kg resulted in 45% knockdown of mouse KRAS, while the highest dose of 10 mg/kg resulted in 65% knockdown of mouse KRAS in this 3-dose format. However, there were not enough human tumor cells in the mouse liver at the time of harvest to detect a signal from human KRAS (potentially due to model development issues, it appeared that not enough human cells were inoculated to produce fast-growing tumors). As such, it was not possible to measure the knockdown in tumor.

Example 45: In Vivo Pharmacokinetics Study of a Cholesterol-siRNA Conjugate (PK-063)

Groups (n=3) of wild-type female CD-1 mice were treated with either one or two intravenous (i.v.) tail vein injections of chol-siRNA conjugate. Treatment groups received chol-KRAS at 10 mg/kg (based on the weight of siRNA) and the 2-dose groups received the second dose 48 h after the first dose. All groups were administered a dose volume of 6.25 mL/kg. Table 57 illustrates the study design in more detail and gives a cross-reference to the conjugate synthesis and characterization. Non-terminal blood samples were collected at 2, 15, 60 or 120 minutes post-final dose via puncture of the retro-orbital plexus and centrifuged to generate plasma for PK analysis. Mice were sacrificed by CO₂ asphyxiation at 4, 24, 96, or 144 h post-final dose. Terminal blood samples were collected via cardiac puncture and processed to generate plasma for PK analysis. 50 mg pieces of tumor, liver, kidney, and lung were collected and snap-frozen in liquid nitrogen. mRNA knockdown analysis and siRNA quantitation were performed as described in Examples 2-7.

TABLE 57
Study design for a Cholesterol-siRNA Conjugate (PK-063) with a
cross-reference to the synthesis and characterization of the conjugates tested.
			siRNA			Survival	Terminal	Harvest	Cross-reference
	Test		Dose		# of	Bleed	Bleed	Time	to synthesis and
Group	Article	N	(mg/kg)	ROA	Doses	(min)	(h)	(h)	characterization
1	Chol-	3	10	IV	1	2	4	4	General
2	KRAS	3	10	IV	1	15	24	24	experimental
3		3	10	IV	1	60	96	96	(Example 2)
4		3	10	IV	1	120	144	144
5	Chol-	3	10	IV	2	2	4	4	General
6	KRAS	3	10	IV	2	15	24	24	experimental
7		3	10	IV	2	60	96	96	(Example 2)
8		3	10	IV	2	120	144	144

The pharmacokinetic behavior of chol-siRNA was assessed in a single-dose format compared to a 2-dose format. As illustrated from FIG. 36, the plasma PK profiles for the first dose and a second dose following 48 h later are nearly identical. The mechanism for clearance from plasma has not saturated from the first dose and the second dose behaves similarly. The tissue PK for 3 major tissues (the liver, kidneys, and lungs) was similarly assessed. As illustrated from FIG. 37, chol-KRAS was delivered to liver in the highest concentrations, with kidneys and lungs having approximately 10-fold lower siRNA concentrations compared to liver. For all three tissues, the siRNA concentrations following the second doses were higher than the siRNA concentrations following the first dose, demonstrating that there is accumulation of siRNA in tissues when doses of chol-siRNA are spaced by 48 h.

In Vivo Study a Cholesterol-siRNA Conjugate (PK-067).

Groups (n=3) of female NCr nu/nu mice bearing subcutaneous flank H358 tumors 100-150 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control groups (n=4) of the same mice received one i.v. injection of PBS as a vehicle control. Treatment groups that received cholesterol-siRNA conjugates were dosed at 5 mg/kg (based on the weight of siRNA). Some treatment groups also received cholesterol-peptide conjugates at specified molar peptide:siRNA ratios, where all chol-siRNA and chol-peptide conjugates were mixed together in solution and co-injected. All groups (treatments and controls) were administered a dose volume of 5 mL/kg. Table 58 shows the study design in more detail and gives a cross-reference to the conjugate synthesis and characterization. Mice were sacrificed by CO₂ asphyxiation at 24, 72, or 144 h post-dose. 50 mg pieces of tumor, liver, kidneys, and lungs were collected and snap-frozen in liquid nitrogen. mRNA knockdown analysis and siRNA quantitation were performed as described in Examples 2-7.

TABLE 58
Study design for a Cholesterol-siRNA Conjugate (PK-067) with a
cross-reference to the conjugate synthesis and characterization
				mol
				EEP/
			siRNA	mol			Harvest	Cross-reference
			Dose	siRNA		# of	Time	to synthesis and
Group	Test Article	N	(mg/kg)	Ratio	ROA	Doses	(h)	characterization
1	chol-KRAS	3	5	—	IV	1	24	General
2		3	5	—	IV	1	72	experimental
3		3	5	—	IV	1	144	(Example 2)
4	chol-KRAS +	3	5	1	IV	1	24	General
5	chol-Melittin	3	5	1	IV	1	72	experimental
6		3	5	1	IV	1	144	(Example 2)
7	chol-KRAS +	3	5	3	IV	1	24	General
8	chol-Melittin	3	5	3	IV	1	72	experimental
9		3	5	3	IV	1	144	(Example 2)
10	chol-KRAS +	3	5	10	IV	1	24	General
11	chol-Melittin	3	5	10	IV	1	72	experimental
12		3	5	10	IV	1	144	(Example 2)
13	chol-KRAS +	3	5	1	IV	1	24	General
14	chol-INF7	3	5	1	IV	1	72	experimental
15		3	5	1	IV	1	144	(Example 2)
16	chol-KRAS +	3	5	3	IV	1	24	General
17	chol-INF7	3	5	3	IV	1	72	experimental
18		3	5	3	IV	1	144	(Example 2)
19	chol-KRAS +	3	5	10	IV	1	24	General
20	chol-INF7	3	5	10	IV	1	72	experimental
21		3	5	10	IV	1	144	(Example 2)
22	Vehicle (PBS)	4	—	—	IV	1	24	General
23		4	—	—	IV	1	72	experimental
24		4	—	—	IV	1	144	(Example 2)
	Total # of	75	nu/nu mice with H358
	Animals:		tumors

Endosomolytic moieties (EEPs) such as INF7 and melittin were conjugated to cholesterol, mixed with chol-siRNA, and then co-injected into mice to demonstrate an increase in siRNA potency due to the improved endosomal escape. First, the effect of adding the EEPs on the siRNA concentration in various tissues was assessed. As illustrated in FIG. 38A, the addition of chol-INF7 at any of the molar ratios of EEP:siRNA did not affect the siRNA tumor PK. However, as illustrated in FIG. 38B, the addition of chol-melittin at a 1:1 ratio did not affect the tumor PK but the addition of chol-melittin at a 3:1 EEP:siRNA ratio decreased the amount of siRNA in tumor. As illustrated in FIG. 39, neither chol-INF7 nor chol-melittin had much of an impact on the liver PK. Similarly, as illustrated in FIGS. 40 and 41, the chol-INF7 and chol-melittin also did not have much of an impact on the PK profile in kidneys and lungs. Finally, the effect of the chol-EEP conjugates on mRNA KD was assessed and, as shown in FIG. 42, the baseline level of knockdown for chol-KRAS alone was approximately 50%. The addition of 1:1 chol-melittin or 3:1 chol-INF7 improves the knockdown at each time point, due to improved endosomal escape.

In Vivo Study a Cholesterol-siRNA Conjugate (PK-076).

Groups (n=5) of female NCr nu/nu mice bearing subcutaneous flank H358 tumors 100-150 mm³ in volume were treated with three intravenous (i.v.) tail vein injections of siRNA conjugate separated by 48 h, while control groups (n=5) of the same mice received three i.v. injections of PBS as a vehicle control on the same dosing schedule. Treatment groups that received cholesterol-siRNA conjugates were dosed at 5 mg/kg (based on the weight of siRNA). Some treatment groups also received cholesterol-peptide conjugates at specified molar peptide:siRNA ratios, where all chol-siRNA and chol-peptide conjugates were mixed together in solution and co-injected. All groups (treatments and controls) were administered a dose volume of 5 mL/kg. Table 59 describes the study design in more detail and gives a cross-reference to the conjugate synthesis and characterization. Mice were sacrificed by CO₂ asphyxiation at 24 or 96 h post-dose. 50 mg pieces of tumor, liver, kidneys, and lungs were collected and snap-frozen in liquid nitrogen. mRNA knockdown analysis and siRNA quantitation were performed as described in Examples 2-7.

TABLE 59
Study design for a Cholesterol-siRNA Conjugate (PK-076) with a
cross-reference to the synthesis and characterization of the conjugates tested.
				EEP/
			siRNA	siRNA			Harvest	Cross-reference to
			Dose	Ratio		# of	Time	synthesis and
Group	Test Article	N	(mg/kg)	(mol/mol)	ROA	Doses	(h)	characterization
1	chol-KRAS	5	5	—	IV	3	24	General
2		5	5	—	IV	3	96	experimental
								(Example 2)
3	chol-KRAS +	5	5	1	IV	3	24	General
4	chol-melittin (1:1)	5	5	1	IV	3	96	experimental
								(Example 2)
5	chol-KRAS +	5	5	3	IV	3	24	General
6	chol-INF7 (3:1)	5	5	3	IV	3	96	experimental
								(Example 2)
7	Vehicle	5	—	—	IV	3	24
8		5	—	—	IV	3	96

The activity seen in the single-dose study with chol-siRNA and chol-EEP was followed up with a three dose study. The 3:1 ratio of EEP:siRNA was selected for INF7, and the 1:1 ratio was selected for melittin. As illustrated in FIG. 43 and FIG. 44, the addition of either chol-EEP to the chol-siRNA does not seem to greatly affect the tissue PK following three doses. As for the knockdown, FIG. 45 shows that addition of chol-melittin clearly improves tumor knockdown 24 h post-dose. It also shows that chol-melittin improves tumor knockdown at 96 h post-dose.

In Vivo Study a Cholesterol-siRNA Conjugate (PK-079).

Groups (n=5) of female NCr nu/nu mice bearing subcutaneous flank H358 tumors 100-150 mm³ in volume were treated with one intravenous (i.v.) tail vein injection of siRNA conjugate, while control groups (n=5) of the same mice received one i.v. injection of PBS as a vehicle control. Treatment groups that received EGFR antibody-siRNA-PEG conjugates were dosed at 0.5 mg/kg (based on the weight of siRNA) and groups that also received EGFR antibody-melittin had the dose of EGFR-Ab matched between EGFR antibody-siRNA and EGFR antibody-melittin. All groups (treatments and controls) were administered a dose volume of 5 mL/kg. Table 60 describes the study design in more detail and gives a cross-reference to the conjugate synthesis and characterization. Mice were sacrificed by CO₂ asphyxiation at 96 h post-dose. 50 mg pieces of tumor, liver, kidney, and lung were collected and snap-frozen in liquid nitrogen. mRNA knockdown analysis and siRNA quantitation were performed as described in Examples 2-7.

TABLE 60
Study design for a Cholesterol-siRNA Conjugate (PK-079) with a
cross-reference to the synthesis and characterization of the conjugates tested.
				siRNA:	melittin:
			siRNA	EGFR-	siRNA			Harvest	Cross-reference
			Dose	Ab Ratio	Ratio		# of	Time	to synthesis and
Group	Test Article	N	(mg/kg)	(mol/mol)	(mol/mol)	ROA	Doses	(h)	characterization
1	EGFR-Ab-	5	0.5	1	—	IV	1	96	Example 4
	PEG5k-EGFR
2	EGFR-Ab-	5	0.5	1	1:1	IV	1	96	Example 3 and 6
	PEG5k-EGFR +
	EGFR-Ab-
	melittin
3	EGFR-Ab-KRAS-	5	0.5	1	1:1	IV	1	96	Example 3 and 6
	PEG5k +
	EGFR-Ab-
	melittin
4	EGFR antibody	5	—	—	—	IV	1	96	General
	Alone								experimental
									(Example 2)
5	Vehicle	5	—	—	—	IV	1	96

The PK/PD relationship for EGFR antibody-siRNA conjugates to deliver siRNA to tumor and produce mRNA knockdown in tumor was evaluated for reproducibility. As illustrated in FIG. 46, once again a single i.v. dose of 0.5 mg/kg of EGFR antibody-siRNA conjugate was able to deliver approximate 100 nM concentrations of siRNA into tumor with both configurations of the conjugate. The addition of EGFR antibody-melittin did not appear to impact the tissue PK. Out of the four tissues analyzed, tumor had the highest concentration and liver the second highest, with kidneys and lungs showing low uptake of siRNA. As illustrated in FIG. 47, the strong siRNA delivery to tumor once again translated into approximately 50% knockdown of EGFR or KRAS in the tumors. Free EGFR-Ab, run as a control group, showed no mRNA knockdown as did the PBS control.

In Vivo Study a Cholesterol-siRNA Conjugate (PD-077).

Groups (n=11) of female NCr nu/nu mice bearing intrahepatic Hep3B tumors one week after inoculation were treated with nine intravenous (i.v.) or subcutaneous (s.c.) injections (TIW) of cholesterol-siRNA conjugate, while control groups (n=11) of the same mice received nine i.v. tail vein injections of PBS as a vehicle control (also dosed TIW). Treatment groups that received chol-CTNNB1 were dosed at 5 mg/kg. All groups (treatments and controls) were administered a dose volume of 6.25 mL/kg. Table 61 describes the study design in more detail and gives a cross-reference to the conjugate synthesis and characterization. Non-terminal blood samples were collected once per week via puncture of the retro-orbital plexus and processed to generate serum for alpha-Fetoprotein (AFP) measurement. Mice were sacrificed by CO₂ asphyxiation at 24 h post-final dose. 50 mg pieces of tumor-bearing liver were collected and snap-frozen in liquid nitrogen. mRNA knockdown analysis was performed as described above. AFP was quantified using the Human alpha-Fetoprotein DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions.

TABLE 61
Study design for a Cholesterol-siRNA Conjugate (PK-077) with a
cross-reference to the synthesis and characterization of the conjugates tested.
			siRNA				Terminal	Cross-reference
			Dose		# of	Survival	Bleed	to synthesis and
Group	Test Article	N	(mg/kg)	ROA	Doses	Bleed	(h)	characterization
5	Chol-CTNNB1	11	5	IV	9	Weekly	24	General
								experimental
								(Example 2)
8	Chol-CTNNB1	11	5	SC	9	Weekly	24	General
								experimental
								(Example 2)
11	Vehicle	11		IV	9	Weekly	24
Total # of Animals:	33

Since earlier studies demonstrated that it was possible for a single dose of chol-siRNA to generate knockdown in normal liver, it was hypothesized that knockdown could be achieved in orthotopic liver tumors as well. Mice were inoculated with intrahepatic Hep3B tumors that were allowed to grow for one week post-inoculation, and then these mice were administered 5 mg/kg doses of chol-CTNNB1 (either i.v. or s.c.) three times a week for three weeks (9 total doses). As illustrated in FIG. 48, the chol-CTNNB1 dosed s.c. was able to produce >50% mRNA knockdown at the harvest time point of 24 h post-final dose. In contrast, the chol-CTNNB1 siRNA that was dosed i.v. does not seem to show any mRNA knockdown at this time point (although some mice did not have any measurable human CTNNB1 signal, it was hard to determine if the loss of signal was related to knockdown or low tumor burden). The human Hep3B cells are also known to secrete human alpha-Fetoprotein (AFP), and it is known that the amount of secreted AFP correlates with the number of Hep3B cells. Thus, the concentration of AFP in serum is taken as a marker of tumor load in the mouse, and the increase in AFP over time correlates with tumor growth. As illustrated in FIG. 49, the chol-CTNNB1 dosed s.c. markedly reduced the AFP levels in those mice, which provides evidence that the CTNNB1 mRNA knockdown led to the inhibition of tumor growth.

Example 46. Liver PK/PD Study

Female wild-type CD-1 mice will be dosed with chol-siRNA-EEP conjugates at 5 mg/kg (based on the weight of siRNA). In these studies the siRNA used will be against the mouse Factor VII (FVII) such that FVII knockdown can be determined by measuring the FVII protein levels in plasma. Multiple EEPs (endosomolytic moieties) will be used to determine the peptide sequence that demonstrates optimal endosomal escape, resulting in the best knockdown of the FVII target gene relative to the control.

Example 47. Tumor PK/PD Study

Female NCr nu/nu mice bearing subcutaneous flank H358 tumors will be dosed with EGFR antibody-siRNA-EEP conjugates at 0.5 mg/kg (based on siRNA). Multiple EEPs (endosomolytic moieties) will be used to determine the peptide sequence that demonstrates optimal endosomal escape, resulting in the best knockdown of the target gene relative to the control.

Example 48. Formulation of an ABC Conjugate with Nanoparticles

An exemplary ABC conjugate is packaged into self-assembled nanoparticles using cyclodextrin polymers (10 kDa) and an excess of non-conjugated siRNAs (ED 40-60 nm, PDI 0.1-0.2). In these particles, the exemplary ABC conjugate maintains its ability to interact with the antibody target. The stability and target binding competency of the particles in circulation in vivo is regulated through modifications of the packaging siRNAs.

Nanoparticle Formation

Nanoparticles are prepared at a final siRNA concentration of 1.6 mg/mL. siRNA containing CY5-siRNA at a ratio of 1:20 is first diluted to 2× final concentration in water. Cyclodextrin polymer (CDP) is diluted to 2× final concentration necessary to achieve a nitrogen to phosphorus ratio (N:P) of 3:1 in 10 mM phosphate buffer at neutral pH. CDP is added quickly to siRNA and is further mixed by pipetting. Particles are incubated for at least 15 minutes before dosing or analysis.

In Vitro EGFR Binding

Nanoparticles containing various amount of the exemplary ABC conjugate are diluted into Fetal calf serum to a final concentration of 10 nM and are incubated for 1 h at RT with Protein G Dynabeads (Thermofisher) loaded with 150 nM of a purified EGFR-Fc protein (Sino Biological). Beads are washed twice with PBS containing 0.01% Tween 20 and 0.05% BSA before bead-bound nanoparticles are disrupted with water containing 0.01% Tween 20 and 100 ug/ml heparin. The amount of CY5-siRNA contained in the input, unbound fraction, washes and bead eluate is quantified by fluorescence using a TECAN Infinite M200 Pro (Excitation 635 nm; Emission 675 nm).

CY5-ASC Plasma Quantification

Quantification of nanoparticles in mouse plasma is performed as illustrated in Example 43. The CY5-siRNAs bound to EGFR beads are released by using heparin to compete the electrostatic interactions between CDP and siRNAs.

While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A pharmaceutical composition comprising: a molecule of Formula (I): A-X—B—Y—C   Formula Iwherein, A is an antibody or its binding fragments thereof;B consists of a double-stranded siRNA consisting of a passenger strand and a guide strand;C consists of a polymer; andX and Y are each independently a linker selected from the group consisting of a heterobifunctional linker, a homobifunctional linker, a maleimide group, a dipeptide moiety, a benzoic acid group or derivatives thereof, a C1-C6 alkyl group, and a combination thereof; anda pharmaceutically acceptable excipient;

2. The pharmaceutical composition of claim 1, wherein the guide strand hybridizes to a target region of a gene selected from the group consisting of KRAS, EGFR, AR, CTNNB1, PIK3CA, PIK3CB, MYC, and HPRT1.

3. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated for the treatment of a solid tumor.

4. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated for the treatment of breast cancer, lung cancer, ovarian cancer, prostate cancer, or skin cancer.

5. The pharmaceutical composition of claim 1, wherein the at least one 2′ modified nucleotide comprises 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, 2'-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified nucleotide.

6. The pharmaceutical composition of claim 1, wherein the at least one 2′ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA).

7. The pharmaceutical composition of claim 1, wherein the at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage.

8. The pharmaceutical composition of claim 1, wherein the at least one inverted abasic moiety is at at least one terminus.

9. The pharmaceutical composition of claim 1, wherein the guide strand comprises a sequence having at least 80% sequence identity to SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117.

10. The pharmaceutical composition of claim 1, wherein the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or binding fragment thereof.

11. The pharmaceutical composition of claim 1, wherein the antibody or binding fragment thereof is an anti-transferrin receptor antibody or binding fragment thereof.

12. The pharmaceutical composition of claim 1, wherein C is polyethylene glycol.

13. The pharmaceutical composition of claim 12, wherein C has a molecular weight from about 1000 Da to about 5000 Da.

14. The pharmaceutical composition of claim 12, wherein C has a molecular weight of about 1000 Da, 2000 Da, or 5000 Da.

15. The pharmaceutical composition of claim 1, wherein A-X is conjugated to the 5′ end of the passenger strand and Y—C is conjugated to the 3′ end of the passenger strand.

16. The pharmaceutical composition of claim 1, wherein Y—C is conjugated to the 5′ end of the passenger strand and A-X is conjugated to the 3′ end of the passenger strand.