NUCLEIC ACID-POLYPEPTIDE COMPOSITIONS AND USES THEREOF

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 11, 2018, is named 45532-707_301_SL.txt and is 615,711 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 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-C₆alkyl group. In some embodiments, Y is a C₁-C₆alkyl group. In some embodiments, X is a homobifunctional linker or a heterobifunctional linker, optionally conjugated to a C₁-C₆alkyl group. In some embodiments, Y is a homobifunctional 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-C₆alkyl group. In some embodiments, L is a homobifunctional 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-C₆alkyl group. In some embodiments, Y is a C₁-C₆alkyl group. In some embodiments, X is a homobifunctional linker or a heterobifunctional linker, optionally conjugated to a C₁-C₆alkyl group. In some embodiments, Y is a homobifunctional 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-C₆alkyl group. In some embodiments, L is a homobifunctional 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 of which:

FIG. 1A-FIG. 1C 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-PEG20 kDa-EGFR.

FIG. 6 illustrates a SDS-PAGE analysis of EGFR antibody-PEG20 kDa-EGFR conjugate.

FIG. 7 illustrates an analytical chromatogram of EGFR antibody-PEG10 kDa-EGFR siRNA.

FIG. 8 shows an analytical chromatogram of EGFR antibody-PEG5 kDa-EGFR siRNA.

FIG. 9 shows a SDS PAGE analysis of EGFR antibody-PEG10 kDa-EGFR siRNA and EGFR antibody-PEG5 kDa-EGFR siRNA conjugates.

FIG. 10 illustrates the overlay of EGFR antibody-PEG1 kDa-EGFR siRNA conjugates with siRNA loading of 1, 2 and 3.

FIG. 11 shows a HPLC chromatogram of EGFR antibody-KRAS-PEG5 kDa.

FIG. 12 shows a HPLC chromatogram of Panitumumab-KRAS-PEG5 kDa.

FIG. 13 shows a HPLC chromatogram of EGFR antibody-S-S-siRNA-PEG5 kDa (DAR=1).

FIG. 14 shows a HPLC chromatogram of EGFR antibody-PEG24-Melittin (loading=˜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-PEG1 kDa-INF7 (Peptide loading=˜1).

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

FIG. 19 shows INF7-PEG1 kDa-(EGFR antibody-KRAS-PEG5 kDa).

FIG. 20 illustrates Melittin-PEG1 kDa-(EGFR antibody-KRAS-PEG5 kDa).

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.

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 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 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 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

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 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 III

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; and

n is an integer between 0 and 10; and

wherein 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 (IIIb): 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-L-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; and

wherein 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 FIG. 1. In some instances, a molecule (e.g., a therapeutic molecule) described herein is a molecule as illustrated in FIG. 1A. In some cases, a molecule (e.g., a therapeutic molecule) described herein is a molecule as illustrated in FIG. 1B. In additional cases, a molecule (e.g., a therapeutic molecule) described herein is a molecule as illustrated in FIG. 1C.

In some embodiments, a molecule (e.g., a therapeutic molecule) is a molecule as illustrated:

The

as illustrated above 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, MY H11/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, 5380 (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, A767-5768 (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, 51042N, R1100S, H1129Y, T1141S, 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), 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, A767-5768 (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, 51042N, 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, L701I, 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, M807I, 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, L701I, 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, M807I, 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, 1140T, D162E, K170M, V199I, 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, 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, 1140T, D162E, K170M, V199I, 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, 1102 (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), L279I, 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, M10431, 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, 1102 (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), L279I, 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, M10431, 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, M681, R103K, H135N, L140S, S167C, G252W, R270W, K290N, E301V, 1304R, R321Q, V369I, T417M, N442K, E470K, E497D, P507S, 1512M, 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, T58I, P59L, A76V, F115L, F138S, A141S, V145I, 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, V145I, 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, R871, 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 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.

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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.

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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.

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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.

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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, carboxyalkylaminoalkyl 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.

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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.

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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.

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

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

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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 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-methoxy ethyl (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 (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 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 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, 157901:5. 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(261:5100-5109 (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)′₃fragments, 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)′₃fragments, 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 divalent 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 (R05520985; 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 mAb²from 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 (R05520985; 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 mAb²from 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)′₃fragments, 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)′₃fragment. 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)′₃fragments or a triabody. In some instances, A is a trispecific F(ab)′₃fragment. 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, F1120041, 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, F1120041, 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 FIG. 1. 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 hydroxyethyl 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):

embedded image

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, m 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. 1.

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

Name
Origin
Amino Acid Sequence
ID 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
DPKGDPKGVTVTVTVTVTGKGDPKPD
2066
β-sheet

amphipathic

C105Y
1-antitrypsin
CSIPPEVKFNKPFVYLI
2067
—

Transportan
Galanin and mastoparan
GWTLNSAGYLLGKINLKALAALAKKIL
2068
Primary

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 1

amphipathic

α-helical

CADY
PPTG1 peptide
GLWRALWRLLRSLWRLLWRA
2072
Secondary

amphipathic

α-helical

GALA
Synthetic peptide
WEAALAEALAEALAEHLAEALAEALE
2073
Secondary

ALAA

amphipathic

α-helical

INF
Influenza HA2 fusion
GLFEAIEGFIENGWEGMIDGWYGC
2074
Secondary

peptide

amphipathic

α-helical/

pH-dependent

membrane

active peptide

HA2E5-TAT
Influenza HA2 subunit
GLFGAIAGFIENGWEGMIDGWYG
2075
Secondary

of influenza virus X31

amphipathic

strain fusion peptide

α-helical/

pH-dependent

membrane

active peptide

HA2-
Influenza HA2 subunit
GLFGAIAGFIENGWEGMIDGRQIKIW
2076
pH-dependent

penetratin
of influenza virus X31
FQNRRMKW

membrane

strain fusion peptide
KK-amide

active peptide

HA-K4
Influenza HA2 subunit
GLFGAIAGFIENGWEGMIDG-SSKKK
2077
pH-dependent

of influenza virus X31
K

membrane

strain fusion peptide

active peptide

HA2E4
Influenza HA2 subunit
GLFEAIAGFIENGWEGMIDGGGYC
2078
pH-dependent

of influenza virus X31

membrane

strain fusion peptide

active peptide

H5WYG
HA2 analogue
GLFHAIAHFIHGGWHGLIHGWYG
2079
pH-dependent

membrane

active peptide

GALA-INF3-
INF3 fusion peptide
GLFEAIEGFIENGWEGLAEALAEALE
2080
pH-dependent

(PEG)6-NH

ALAA-(PEG)6-NH2

membrane

active peptide

CM18-
Cecropin-A-Melittin_2-12
KWKLFKKIGAVLKVLTTG-
2081
pH-dependent

(CM₁₈) fusion peptide
YGRKKRRQRRR

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:

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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, amodiaquins (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 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

c is an integer between 0 and 1; and

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 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 IIa

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

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

In some instances, the endosomolytic moiety conjugate is according to Formula (IIb):

A-X-B-L-D Formula IIb

wherein,

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 is a sequence as illustrated in Table 62.

In some instances, the endosomolytic moiety conjugate is according to Formula (IIc):

A-X-B-Y-C_c-L-D Formula IIc

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

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

In some instances, the endosomolytic moiety conjugate is according to Formula (IId):

A-L-D-X-B-Y-C_c Formula IId

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

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

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-C₆alkyl group (e.g., a C₅, C₄, C₃, C₂, or C₁alkyl 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 homobifunctional linker. Exemplary homobifunctional 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)methyl)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-nitrobenzoyloxy succinimide (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 (ρNPDP), 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 ρ-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 ρ-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 carboxy late, 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 aminoa cid 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 heterobifunctional linker, such as a heterobifunctional 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-C₆alkyl group. In some cases, X is a C₁-C₆alkyl group, such as for example, a C₅, C₄, C₃, C₂, or C₁alkyl group. In some cases, the C₁-C₆alkyl group is an unsubstituted C₁-C₆alkyl 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 homobifunctional linker or a heterobifunctional linker described supra. In some cases, X includes a heterobifunctional linker. In some cases, X includes sMCC. In other instances, X includes a heterobifunctional linker optionally conjugated to a C₁-C₆alkyl group. In other instances, X includes sMCC optionally conjugated to a C₁-C₆alkyl group. In additional instances, X does not include a homobifunctional 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-C₆alkyl group. In some instances, Y is a homobifunctional linker or a heterobifunctional linker described supra. In some instances, Y is a homobifunctional 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-C₆alkyl group. In some instances, L is a homobifunctional linker or a heterobifunctional linker described supra. In some instances, L is a homobifunctional 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, GALS, 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, GALS, 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 rHIgM12B7;

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-1a, 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, polyvinylpyrollidone (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 position
sense strand
SEQ
antisense
SEQ

Id #
in NM_033360.2
sequence (5′-3′)
ID NO:
strand (5′-3′)
ID NO:

182
182-200
AUGACUGAAUAUAAACUUGTT
16
CAAGUUUAUAUUCAGUCAUTT
17

183
183-201
UGACUGAAUAUAAACUUGUTT
18
ACAAGUUUAUAUUCAGUCATT
19

197
197-215
CUUGUGGUAGUUGGAGCUGTT
20
CAGCUCCAACUACCACAAGTT
21

224
224-242
GGCAAGAGUGCCUUGACGATT
22
UCGUCAAGGCACUCUUGCCTT
23

226
226-244
CAAGAGUGCCUUGACGAUATT
24
UAUCGUCAAGGCACUCUUGTT
25

227
227-245
AAGAGUGCCUUGACGAUACTT
26
GUAUCGUCAAGGCACUCUUTT
27

228
228-246
AGAGUGCCUUGACGAUACATT
28
UGUAUCGUCAAGGCACUCUTT
29

232
232-250
UGCCUUGACGAUACAGCUATT
30
UAGCUGUAUCGUCAAGGCATT
31

233
233-251
GCCUUGACGAUACAGCUAATT
32
UUAGCUGUAUCGUCAAGGCTT
33

236
236-254
UUGACGAUACAGCUAAUUCTT
34
GAAUUAGCUGUAUCGUCAATT
35

237
237-255
UGACGAUACAGCUAAUUCATT
36
UGAAUUAGCUGUAUCGUCATT
37

245
245-263
CAGCUAAUUCAGAAUCAUUTT
38
AAUGAUUCUGAAUUAGCUGTT
39

266
266-284
GUGGACGAAUAUGAUCCAATT
40
UUGGAUCAUAUUCGUCCACTT
41

269
269-287
GACGAAUAUGAUCCAACAATT
42
UUGUUGGAUCAUAUUCGUCTT
43

270
270-288
ACGAAUAUGAUCCAACAAUTT
44
AUUGUUGGAUCAUAUUCGUTT
45

TABLE 3

KRAS siRNA Sequences with Chemical Modification

siRNA sequence with

siRNA sequence with

sequence
chemical modification

chemical modification

position in
sense strand sequence
SEQ
antisense strand sequence
SEQ

Id #
NM_033360.2
(5′-3′)
ID NO:
(5′-3′)
ID NO:

182
182-200
auGfaCfuGfaAfuAfuAfaA
46
CfAfaGfuUfuAfuAfuUfcAfgUf
47

fcUfuGfdTsdT

cAfudTsdT

183
183-201
ugAfcUfgAfaUfaUfaAfaC
48
AfCfaAfgUfuUfaUfaUfuCfaGf
49

fuUfgUfdTsdT

uCfadTsdT

197
197-215
cuUfgUfgGfuAfgUfuGfgA
50
CfAfgCfuCfcAfaCfuAfcCfaCf
51

fgCfuGfdTsdT

aAfgdTsdT

224
224-242
ggCfaAfgAfgUfgCfcUfuG
52
UfCfgUfcAfaGfgCfaCfuCfuUf
53

faCfgAfdTsdT

gCfcdTsdT

226
226-244
caAfgAfgUfgCfcUfuGfaC
54
UfAfuCfgUfcAfaGfgCfaCfuCf
55

fgAfuAfdTsdT

uUfgdTsdT

227
227-245
aaGfaGfuGfcCfuUfgAfcG
56
GfUfaUfcGfuCfaAfgGfcAfcUf
57

faUfaCfdTsdT

cUfudTsdT

228
228-246
agAfgUfgCfcUfuGfaCfgA
58
UfGfuAfuCfgUfcAfaGfgCfaCf
59

fuAfcAfdTsdT

uCfudTsdT

232
232-250
ugCfcUfuGfaCfgAfuAfcA
60
UfAfgCfuGfuAfuCfgUfcAfaGf
61

fgCfuAfdTsdT

gCfadTsdT

233
233-251
gcCfuUfgAfcGfaUfaCfaG
62
UfUfaGfcUfgUfaUfcGfuCfaAf
63

fcUfaAfdTsdT

gGfcdTsdT

236
236-254
uuGfaCfgAfuAfcAfgCfuA
64
GfAfaUfuAfgCfuGfuAfuCfgUf
65

faUfuCfdTsdT

cAfadTsdT

237
237-255
ugAfcGfaUfaCfaGfcUfaA
66
UfGfaAfuUfaGfcUfgUfaUfcGf
67

fuUfcAfdTsdT

uCfadTsdT

245
245-263
caGfcUfaAfuUfcAfgAfaU
68
AfAfuGfaUfuCfuGfaAfuUfaGf
69

fcAfuUfdTsdT

cUfgdTsdT

266
266-284
guGfgAfcGfaAfuAfuGfaU
70
UfUfgGfaUfcAfuAfuUfcGfuCf
71

fcCfaAfdTsdT

cAfcdTsdT

269
269-287
gaCfgAfaUfaUfgAfuCfcA
72
UfUfgUfuGfgAfuCfaUfaUfuCf
73

faCfaAfdTsdT

gUfcdTsdT

270
270-288
acGfaAfuAfuGfaUfcCfaA
74
AfUfuGfuUfgGfaUfcAfuAfuU
75

fcAfaUfdTsdT

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
sequence of total

Id
pos. in
23mer target
SEQ

#
NM_005228.3
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

hs Id
Sequence position
sense strand
SEQ
antisense strand
SEQ

#
in NM_005227.3
sequence (5′-3′)
ID NO:
sequence (5′-3′)
ID 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

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

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

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

hs Id
position in
sense strand
SEQ
antisense strand
SEQ

#
NM_005228.3
sequence (5′-3′)
ID NO:
sequence (5′-3′)
ID 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
agUfcCfcGfaGfcUfaGfcCfc
1214
CfCfgGfgGfcUfaGfcUfcGfgGf
1215

CfgGfdTsdT

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
gcCfcGfaGfuCfcCfcGfcCfu
1260
GfCfgAfgGfcGfgGfgAfcUfcGf
1261

CfgCfdTsdT

gGfcdTsdT

164
164-182
aaCfgCfcAfcAfaCfcAfcCfg
1262
GfCfgCfgGfuGfgUfuGfuGfgCf
1263

CfgCfdTsdT

gUfudTsdT

165
165-183
acGfcCfaCfaAfcCfaCfcGfc
1264
UfGfcGfcGfgUfgGfuUfgUfgGf
1265

GfcAfdTsdT

cGfudTsdT

166
166-184
cgCfcAfcAfaCfcAfcCfgCfg
1266
GfUfgCfgCfgGfuGfgUfuGfuGf
1267

CfaCfdTsdT

gCfgdTsdT

168
168-186
ccAfcAfaCfcAfcCfgCfgCfa
1268
CfCfgUfgCfgCfgGfuGfgUfuGf
1269

CfgGfdTsdT

uGfgdTsdT

169
169-187
caCfaAfcCfaCfcGfcGfcAfc
1270
GfCfcGfuGfcGfcGfgUfgGfuUf
1271

GfgCfdTsdT

gUfgdTsdT

170
170-188
acAfaCfcAfcCfgCfgCfaCfg
1272
GfGfcCfgUfgCfgCfgGfuGfgUf
1273

GfcCfdTsdT

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
UfCfuGfgAfgGfcUfgAfgAfaAf
1329

AfgAfdTsdT

aUfgdTsdT

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
UfGfgAfuGfgUfcUfuUfaAfgAf
1385

CfcAfdTsdT

aGfgdTsdT

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
AfCfaUfaAfcCfaGfcCfaCfcUf
1399

UfgUfdTsdT

cCfudTsdT

495
495-513
ggAfgGfuGfgCfuGfgUfuAfu
1400
GfAfcAfuAfaCfcAfgCfcAfcCf
1401

GfuCfdTsdT

uCfcdTsdT

496
496-514
gaGfgUfgGfcUfgGfuUfaUfg
1402
GfGfaCfaUfaAfcCfaGfcCfaCf
1403

UfcCfdTsdT

cUfcdTsdT

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
UfGfaUfgAfuCfuGfcAfgGfuUf
1415

UfcAfdTsdT

uUfcdTsdT

551
551-569
aaAfaCfcUfgCfaGfaUfcAfu
1416
CfUfgAfuGfaUfcUfgCfaGfgUf
1417

CfaGfdTsdT

uUfudTsdT

553
553-571
GaaCfcUfgCfaGfaUfcAfuCf
1418
CfUfcUfgAfuGfaUfcUfgCfaGf
1419

afaGfdTsdT

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
UfAfgUfuAfgAfuAfaGfaCfuGf
1447

CfuAfdTsdT

cUfadTsdT

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
GfCfaUfcAfuAfgUfuAfgAfuAf
1455

UfgCfdTsdT

aGfadTsdT

609
609-627
cuUfaUfcUfaAfcUfaUfgAfu
1456
UfGfcAfuCfaUfaGfuUfaGfaUf
1457

GfcAfdTsdT

aAfgdTsdT

610
610-628
uuAfuCfuAfaCfuAfuGfaUfg
1458
UfUfgCfaUfcAfuAfgUfuAfgAf
1459

CfaAfdTsdT

uAfadTsdT

611
611-629
uaUfcUfaAfcUfaUfgAfuGfc
1460
UfUfuGfcAfuCfaUfaGfuUfaGf
1461

AfaAfdTsdT

aUfadTsdT

612
612-630
auCfuAfaCfuAfuGfaUfgCfa
1462
AfUfuUfgCfaUfcAfuAfgUfuAf
1463

AfaUfdTsdT

gAfudTsdT

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
AfGfuCfcGfgUfuUfuAfuUfuGf
1473

AfcUfdTsdT

cAfudTsdT

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
AfGfgGfuUfgUfuGfcUfgAfaCf
1529

CfcUfdTsdT

cGfcdTsdT

692
692-710
ggUfuCfaGfcAfaCfaAfcCfc
1530
GfCfaGfgGfuUfgUfuGfcUfgAf
1531

UfgCfdTsdT

aCfcdTsdT

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
UfUfgGfgAfcAfgCfuUfgGfaUf
1591

CfaAfdTsdT

cAfcdTsdT

817
817-835
gaUfcCfaAfgCfuGfuCfcCfa
1592
CfAfuUfgGfgAfcAfgCfuUfgGf
1593

AfuGfdTsdT

aUfcdTsdT

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
AfGfaUfgAfuUfuUfgGfuCfaGf
1609

UfcUfdTsdT

uUfudTsdT

872
872-890
aaCfuGfaCfcAfaAfaUfcAfu
1610
CfAfgAfuGfaUfuUfuGfgUfcAf
1611

CfuGfdTsdT

gUfudTsdT

873
873-891
acUfgAfcCfaAfaAfuCfaUfc
1612
AfCfaGfaUfgAfuUfuUfgGfuCf
1613

UfgUfdTsdT

aGfudTsdT

877
877-895
acCfaAfaAfuCfaUfcUfgUfg
1614
GfGfgCfaCfaGfaUfgAfuUfuUf
1615

CfcCfdTsdT

gGfudTsdT

878
878-896
ccAfaAfaUfcAfuCfuGfuGfc
1616
UfGfgGfcAfcAfgAfuGfaUfuUf
1617

CfcAfdTsdT

uGfgdTsdT

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
GfGfgGfuUfgUfaGfaGfcAfuGf
1683

CfcCfdTsdT

aGfudTsdT

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
17,3

CfaGfdTsdT

gGfudTsdT

1087
1087-1105
ccCfgAfgGfgCfaAfaUfaCfa
1704
AfGfcUfgUfaUfuUfgCfcCfuCf
17,5

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
GfGfaGfgUfgCfaGfuUfuUfuGf
1877

UfcCfdTsdT

aAfgdTsdT

1321
1321-1339
uuCfaAfaAfaCfuGfcAfcCfu
1878
UfGfgAfgGfuGfcAfgUfuUfuU
1879

CfcAfdTsdT

fgAfadTsdT

1322
1322-1340
ucAfaAfaAfcUfgCfaCfcUfc
1880
AfUfgGfaGfgUfgCfaGfuUfuUf
1881

CfaUfdTsdT

uGfadTsdT

1323
1323-1341
caAfaAfaCfuGfcAfcCfuCfc
1882
GfAfuGfgAfgGfuGfcAfgUfuU
1883

AfuCfdTsdT

fuUfgdTsdT

1324
1324-1342
aaAfaAfcUfgCfaCfcUfcCfa
1884
UfGfaUfgGfaGfgUfgCfaGfuUf
1885

UfcAfdTsdT

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
GfCfaGfgAfuGfuGfgAfgAfuCf
1899

UfgCfdTsdT

gCfcdTsdT

1346
1346-1364
gcGfaUfcUfcCfaCfaUfcCfu
1900
GfGfcAfgGfaUfgUfgGfaGfaUf
1901

GfcCfdTsdT

cGfcdTsdT

1347
1347-1365
cgAfuCfuCfcAfcAfuCfcUfg
1902
CfGfgCfaGfgAfuGfuGfgAfgAf
1903

CfcGfdTsdT

uCfgdTsdT

1348
1348-1366
gaUfcUfcCfaCfaUfcCfuGfc
1904
CfCfgGfcAfgGfaUfgUfgGfaGf
1905

CfgGfdTsdT

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
AfUfuUfcCfuUfuAfcGfgUfuUf
1953

AfaUfdTsdT

uCfadTsdT

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
Code
Target Sequence
ID NO:
NM_000044.3
Exon
Species

XD-01817K1
17

CAAAGGUUCUCUGCUAGACGACA

1956
1987-2005
1
h

XD-01827K1
27

UCUGGGUGUCACUAUGGAGCUCU

1957
2819-2837
2
h

XD-01828K1
28

CUGGGUGUCACUAUGGAGCUCUC

1958
2820-2838
2
h

XD-01829K1
29

GGGUGUCACUAUGGAGCUCUCAC

1959
2822-2840
2
h

XD-01821K1
21
UACUACAACUUUCCACUGGCUCU
1960
2207-2225
1
h

XD-01825K1
25
AAGCUUCUGGGUGUCACUAUGGA
1961
2814-2832
2
h, m

XD-01862K1
26
CUUCUGGGUGUCACUAUGGAGCU
2962
2817-2835
2
h

TABLE 8

β-catenin Target Sequences

Generic

R #
name
Gene
Target sequences

R-1146
1797mfm
CTNNB1
CUGUUGGAUUGAUU
SEQ ID
UUUCGAAUCAAUCC
SEQ ID

CGAAAUU
NO: 1963
AACAGUU
NO: 1964

R-1147
1870mfm
CTNNB1
ACGACUAGUUCAGU
SEQ ID
AAGCAACUGAACUA
SEQ ID

UGCUUUU
NO: 1965
GUCGUUU
NO: 1966

TABLE 9

β-catenin and β-catenin associated siRNA Sequences

Sense Strand

Antisense Strand

Generic

Sequence (5′-3′)
SEQ
Sequence (5′-3′)
SEQ

R #
name
Gene
Passenger Strand (PS)2
ID NO:
Guide Strand (GS)3
ID NO:

R-1146
1797mfm
CTNNB1
iBcsuGfuUfgGfaUfuGfa
1967
usUfsusCfgAfaUfcAfa
1968

UfuCfgAfaAfusuiB

UfcCfaAfcAfgusu

R-1147
1870mfm
CTNNB1
iBascGfaCfuAfgUfuCfa
1969
asAfsgsCfaAfcUfgAfa
1970

GfuUfgCfuUfusuiB

CfuAfgUfcGfuusu

R-1150
PA1746
1746
GCUCAAAGCAAUUUCUACAd
1971
UGUAGAAAUUGCUUUGAGC
1972

TsdT

dTsdT

R-1151
PA2328
2328
GGAUGAAACACAAAAGGUAd
1973
UACCUUUUGUGUUUCAUCC
1974

TsdT

dTsdT

R-1152
PA2522
2522
UGUCAGAGUUACUGUUUCAd
1975
UGAAACAGUAACUCUGACA
1976

TsdT

dTsdT

R-1153
PA3484
3484
AGCAAGAACAGAAAUAAAAd
1977
UUUUAUUUCUGUUCUUGCU
1978

TsdT

dTsdT

R-1154
PA5018
5018
CUAGUUCAUUUCAAAAUUAd
1979
UAAUUUUGAAAUGAACUAG
1980

TsdT

dTsdT

R-1155
PB183
183
CAAGUUCACAAUUACCCAAd
1981
UUGGGUAAUUGUGAACUUG
1982

TsdT

dTsdT

R-1156
PB272
272
GCUUGAAGAUGAAACACGAd
1983
UCGUGUUUCAUCUUCAAGC
1984

TsdT

dTsdT

R-1157
PB862
862
AGAUCAAGAAAAUGUAUGAd
1985
UCAUACAUUUUCUUGAUCU
1986

TsdT

dTsdT

R-1158
PB948
948
CCAAAGAAAACACGAAUUAd
1987
UAAUUCGUGUUUUCUUUGG
1988

TsdT

dTsdT

R-1159
PB1520
1520
CUUCGAUAAGAUUAUUGAAd
1989
UUCAAUAAUCUUAUCGAAG
1990

TsdT

dTsdT

R-1160
Myc953U
953
AGGAACUAUGACCUCGACUd
1991
AGUCGAGGUCAUAGUUCCU
1992

TsdT

dTsdT

R-1161
Myc622U
622
ACGACGAGACCUUCAUCAAd
1993
UUGAUGAAGGUCUCGUCGU
1994

TsdT

dTsdT

R-1162
Myc1370U
1370
AAGAUGAGGAAGAAAUCGAd
1995
UCGAUUUCUUCCUCAUCUU
1996

TsdT

dTsdT

R-1163
Myc1364U
1364
AGGAAGAAAUCGAUGUUGUd
1997
ACAACAUCGAUUUCUUCCU
1998

TsdT

dTsdT

R-1164
Myc1711U
1711
AGCUUUUUUGCCCUGCGUGd
1999
CACGCAGGGCAAAAAAGCU
2000

TsdT

dTsdT

R-1165
Myc1769U
1769
AGGUAGUUAUCCUUAAAAAd
2001
UUUUUAAGGAUAACUACCU
2002

TsdT

dTsdT

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

Symbol
ID
Name
Target Sequence (97-mer)
ID NO:

PIK3CA
5290
PIK3CA_1746
TGCTGTTGACAGTGAGCGCCAGCTCAAAGCAATTT
2003

CTACATAGTGAAGCCACAGATGTATGTAGAAATTG

CTTTGAGCTGTTGCCTACTGCCTCGGA

PIK3CA
5290
PIK3CA_2328
TGCTGTTGACAGTGAGCGAAAGGATGAAACACAAA
2004

AGGTATAGTGAAGCCACAGATGTATACCTTTTGTG

TTTCATCCTTCTGCCTACTGCCTCGGA

PIK3CA
5290
PIK3CA_2522
TGCTGTTGACAGTGAGCGCCATGTCAGAGTTACTG
2005

TTTCATAGTGAAGCCACAGATGTATGAAACAGTAA

CTCTGACATGATGCCTACTGCCTCGGA

PIK3CA
5290
PIK3CA_3555
TGCTGTTGACAGTGAGCGCAACTAGTTCATTTCAA
2006

AATTATAGTGAAGCCACAGATGTATAATTTTGAAA

TGAACTAGTTTTGCCTACTGCCTCGGA

PIK3CA
5290
PIK3CA_3484
TGCTGTTGACAGTGAGCGCACAGCAAGAACAGAAA
2007

TAAAATAGTGAAGCCACAGATGTATTTTATTTCTG

TTCTTGCTGTATGCCTACTGCCTCGGA

PIK3CB
5291
PIK3CB_862
TGCTGTTGACAGTGAGCGACAAGATCAAGAAAATG
2008

TATGATAGTGAAGCCACAGATGTATCATACATTTT

CTTGATCTTGCTGCCTACTGCCTCGGA

PIK3CB
5291
PIK3CB_183
TGCTGTTGACAGTGAGCGCAGCAAGTTCACAATTA
2009

CCCAATAGTGAAGCCACAGATGTATTGGGTAATTG

TGAACTTGCTTTGCCTACTGCCTCGGA

PIKC3B
5291
PIK3CB_1520
TGCTGTTGACAGTGAGCGCCCCTTCGATAAGATTA
2010

TTGAATAGTGAAGCCACAGATGTATTCAATAATCT

TATCGAAGGGATGCCTACTGCCTCGGA

PIKC3B
5291
PIK3CB_272
TGCTGTTGACAGTGAGCGAGAGCTTGAAGATGAAA
2011

CACGATAGTGAAGCCACAGATGTATCGTGTTTCAT

CTTCAAGCTCCTGCCTACTGCCTCGGA

PIK3CB
5291
PIK3CB_948
TGCTGTTGACAGTGAGCGACACCAAAGAAAACACG
2012

AATTATAGTGAAGCCACAGATGTATAATTCGTGTT

TTCTTTGGTGGTGCCTACTGCCTCGGA

*Species is Homo sapiens.

TABLE 11

PIK3CA and PIK3CB siRNA Sequences

Gene
Gene

SEQ

SEQ

Symbol
ID
Name
siRNA Guide
ID NO:
siRNA passenger
ID 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 start

SEQ

SEQ

position
Guide strand
ID NO:
Passenger strand
ID NO:

EGFR
333
ACUCGUGCCUUGGCAAACUUU
2082
AGUUUGCCAAGGCACGAGUUU
2083

R1246

EGFR
333
ACUCGUGCCUUGGCAAACUUU
2084
AGUUUGCCAAGGCACGAGUUU
2085

R1195

EGFR
333
ACUCGUGCCUUGGCAAACUUU
2086
AGUUUGCCAAGGCACGAGUUU
2087

R1449

KRAS
237
UGAAUUAGCUGUAUCGUCAUU
2088
TGACGAUACAGCUAAUUCAUU
2089

R1450

KRAS
237
UGAAUUAGCUGUAUCGUCAUU
2090
UGACGAUACAGCUAAUUCAUU
2091

R1443

KRAS
237
UGAAUUAGCUGUAUCGUCAUU
2092
UGACGAUACAGCUAAUUCAUU
2093

R1194

CTNNB1
1248
UAAGUAUAGGUCCUCAUUAUU
2094
UAAUGAGGACCUAUACUUAUU
2095

R1442

CTNNB1
1797
TUUCGAAUCAAUCCAACAGUU
2096
CUGUUGGAUUGAUUCGAAAUU
2097

R1404

CTNNB1
1797
UUUCGAAUCAAUCCAACAGUU
2098
CUGUUGGAUUGAUUCGAAAUU
2099

R1441

CTNNB1
1797
UUUCGAAUCAAUCCAACAGUU
2100
CUGUUGGAUUGAUUCGAAAUU
2101

R1523

HPRT
425
AUAAAAUCUACAGUCAUAGUU
2102
CUAUGACUGUAGAUUUUAUUU
2103

R1492

HPRT
425
UUAAAAUCUACAGUCAUAGUU
2104
CUAUGACUGUAGAUUUUAAUU
2105

R1526

HPRT
425
UUAAAAUCUACAGUCAUAGUU
2106
CUAUGACUGUAGAUUUUAAUU
2107

R1527

AR
2822
GAGAGCUCCAUAGUGACACUU
2108
GUGUCACUAUGGAGCUCUCUU
2109

R1245

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 Biosy stems) 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 Biosy stems) 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
Antisense
UGAAUUAGCUGUAUCGUCAUU
2033

KRAS
RT
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGAT
2034

ACGACAATGACG

KRAS
Forward
GGCGGCTGAATTAGCTGTATCGT
2035

KRAS
Reverse
AGTGCAGGGTCCGAG
2036

KRAS
Probe
(6FAM)-TGGATACGACAATGAC-(NFQ-MGB)
2037

EGFR
Antisense
ACUCGUGCCUUGGCAAACUUU
2038

EGFR
RT
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGAT
2039

ACGACAAAGTTTG

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)-TCACTTTGT(Zen)GGATGAGTATGACCCTACG-
2045 and 2114

(IABkFQ)

KRAS
Human
Forward
GTGCCTTGACGATACAGCTAAT
2046

KRAS
Human
Reverse
CCAAGAGACAGGTTTCTCCATC
2047

KRAS
Human
Probe
(6FAM)-CCAACAATA(Zen)GAGGATTCCTACAGGAAGCA-
2048 and 2115

(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 (0.1-5×10⁶cells) 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-1740™) 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 phosphoramidite 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

siRNA
Strand
Sequence (5′-3′)
observed
ID 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

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. 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, PEG1 kDa, PEG5 kDa and PEG10 kDa were separated based on the siRNA loading. Conjugates with PEG20 kDa 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-PEG1 kDa-EGFR
9.2
10.0
10.6

EGFR-Ab-PEG5 kDa-EGFR
8.7
9.3
ND

EGFR-Ab-PEG10 kDa-EGFR
8.6
8.8 to 10; mix of DAR 2-3

EGFR-Ab-PEG20 kDa-EGFR
8.6; Mixture of DAR of 1-3

Holo-anti-B cell
9.2

9.5

Ab-PEG20 kDa-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:

%
%
Column

a.
A
B
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-PEG20 kDa-EGFR are illustrated in FIG. 5 and FIG. 6. FIG. 5 shows the analytical HPLC of EGFR antibody-PEG20 kDa-EGFR. FIG. 6 shows a SDS-PAGE analysis of EGFR antibody-PEG20 kDa-EGFR conjugate. The analytical chromatogram of EGFR antibody-PEG10 kDa-EGFR is illustrated in FIG. 7. The analytical data for EGFR antibody-PEG5 kDa-EGFR are illustrated in FIG. 8 and FIG. 9. FIG. 8 shows the analytical chromatogram of EGFR antibody-PEG5 kDa-EGFR. FIG. 9 shows SDS PAGE analysis of EGFR antibody-PEG10 kDa-EGFR and EGFR antibody-PEG5 kDa-EGFR conjugates. The analytical data for EGFR antibody-PEG1 kDa-EGFR conjugates with different siRNA loading is illustrated in FIG. 10.

Example 4: Synthesis, Purification and Analysis of Antibody-siRNA-PEG Conjugates

Step 1: Antibody Conjugation with SMCC Linker Followed by SH-KRAS-PEG5 kDa

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. 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-PEG5 kDa 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-ICRAS-PEG5kDa
9.2

EGFR-Ab-S-S-KRAS-
9.0

PEG5kDa

Holo-anti-B cell Ab-ICRAS-
9.2
9.7
10.1

PEGkDa

Panitumumab-KRAS-
9.2
9.7
10.2

PEG5kDa

The HPLC chromatogram of EGFR Antibody-KRAS-PEG5 kDa is illustrated in FIG. 11. The HPLC chromatogram of Panitumumab-KRAS-PEG5 kDa is as shown in FIG. 12.

Example 5: Synthesis, Purification and Analysis of Antibody-S-S-siRNA-PEG Conjugates

Step 1: Antibody Conjugation with SPDP Linker Followed by SH-siRNA-PEG5 kDa

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-PEG5 kDa 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.

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-PEG5 kDa (DAR=1) is as shown in FIG. 13.

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

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-PEG1 kDa-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-CONH₂was added at RT and rotated overnight. 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-PEG1 kDa-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

Step 1: Conjugation of PEG24 linker followed by SH-Cys-Peptide-CONH₂to 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. 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-CONH₂was 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-PEG1 kDa-(EGFR antibody-KRAS-PEG5 kDa). FIG. 20 shows Melittin-PEG1 kDa-(EGFR antibody-KRAS-PEG5 kDa).

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 mm³in 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 CO₂asphyxiation 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:

Cross-

EGFR-
melittin:

Ter-
Har-
reference

siRNA
Ab
siRNA

Survival
minal
vest
to synthesis

Test

Dose
Ratio
Ratio

Dose
Bleed
Bleed
Time
and char-

Group
Article
N
(mg/kg)
(mol/mol)
(mol/mol)
ROA
Schedule
(min)
(h)
(h)
acterization

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

11
Ab-
3
0.5
1.25
—
IV
t = 0
15
96
96
Example 3

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

23
Ab-
3
0.5
1.0
1:1
IV
t = 0
15
96
96
4 and 6

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

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. 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.

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.

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.

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.

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.

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.

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

Step 1: Antibody Reduction with TCEP

Step 2: Purification

Step-3: Analysis of the Purified Conjugate

Step 1: Antibody Conjugation with SPDP Linker Followed by SH-siRNA-PEG5 kDa

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-PEG5 kDa 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.

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.

Step 1: Antibody Reduction and Conjugation with Pyridyldithio-siRNA-PEG5 kDa

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-PEG5 kDa 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.

Step 2: Purification

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.

Step 1: Antibody Reduction and Conjugation with Maleimide-ECL-siRNA-PEG5 kDa

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-PEG5 kDa 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.

Step 2: Purification

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.

Step 1: Antibody Conjugation with NHS-PEG4-TCO Followed by Methyltetrazine-PEG4-siRNA-PEG5 kDa

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-PEG5 kDa 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.

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.

Step 1: Antibody Glycan Modification and Gal-N₃Addition

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.

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-PEG5 kDa 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

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.

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 phosphoramidite 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-NH₂and C6-SH, one at each end of the strand. The passenger strand with C6-NH₂handle at 5′ end contains C6-SH at its 3′ end and the strand that contains C6-NH₂handle 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-NH₂conjugation handle at the 5′ end and C6-SH at 3′end of the passenger strand.

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 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 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 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 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 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 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 yl 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_2Asubclass 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 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 phosphoramidite 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-NH₂at 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 phosphoramidite 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-NH₂at 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 mm³in 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 CO₂asphyxiation 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

Har-

Dose

Dose

vest

(mg/

Volume
# of
Time

Group
Test Article
N
kg)
ROA
(mL/kg)
Doses
(h)

1
PSMA-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR (n = 1)

2
PSMA-Ab(Cys)-
5
0.5
IV
5.0
1
96

EGFR (n = 1)

3
PSMA-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR-PEG5k

(n = 1)

4
PSMA-Ab(Cys)-
5
0.5
IV
5.0
1
96

EGFR-PEG5k

(n = 1)

5
PSMA-Ab(Cys)-
5
1
IV
5.0
1
96

PEG5k-EGFR

(n = 1)

6
PSMA-Ab(Cys)-
5
0.5
IV
5.0
1
96

PEG5k-EGFR

(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

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 phosphoramidite 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-NH₂at 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 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 mm³in 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 CO₂asphyxiation 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

Dose

siRNA

Vol-

Har-

Dose

ume

vest

(mg/

(mL/
# of
Time

Group
Test Article
N
kg)
ROA
kg)
Doses
(h)

1
PSMA-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR (n = 1)

2
PSMA-Ab(Cys)-
5
0.5
IV
5.0
1
96

EGFR (n = 1)

3
PSMA-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR-PEG5k

(n = 1)

4
PSMA-Ab(Cys)-
5
0.5
IV
5.0
1
96

EGFR-PEG5k

(n = 1)

5
PSMA-Ab(Cys)-
5
0.25
IV
5.0
1
96

EGFR-PEG5k

(n = 1)

6
PSMA-Ab(Cys)-
5
0.5
IV
5.0
1
96

PEG5k-EGFR

(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

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 phosphoramidite 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-NH₂at 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 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 CO₂asphyxiation 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)-
4
0.5
IV
1
t = 0
5
24

2
EGFR
4
0.5
IV
1
t = 0
30
96

3
(n-1)
4
0.5
IV
1
t = 0
180
168

4
EGFR-
4
0.5
IV
1
t = 0
5
24

5
Ab(Cys)-EGFR-
4
0.5
IV
1
t = 0
30
96

6
PEG5k (n = 1)
4
0.5
IV
1
t = 0
180
168

7
EGFR-Ab(Cys)-
4
0.5
IV
1
t = 0
5
24

8
PEG5k-
4
0.5
IV
1
t = 0
30
96

9
EGFR (n = 1)
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

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 mm³in 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 CO₂asphyxiation 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

Dose

siRNA

Vol-

Har-

Dose

ume

vest

(mg/

(mL/
# of
Time

Group
Test Article
N
kg)
ROA
kg)
Doses
(h)

1
EGFR-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR-

PEG5k (n = 1)

2
EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
96

EGFR-

PEG5k (n = 1)

3
EGFR-Ab(Cys)-
5
0.25
IV
5.0
1
96

EGFR-

PEG5k (n = 1)

4
EGFR2-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR-

PEG5k (n = 1)

5
EGFR2-Ab(Cys)-
5
0.5
IV
5.0
1
96

EGFR-

PEG5k (n = 1)

6
EGFR2-Ab(Cys)-
5
0.25
IV
5.0
1
96

EGFR-

PEG5k (n = 1)

7
EGFR-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
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 phosphoramidite 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-NH₂at 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=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank HCC827 tumors 100-300 mm³in 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 CO₂asphyxiation 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

Har-

Dose

Volume

vest

(mg/

(mL/
# of
Time

Group
Test Article
N
kg)
ROA
kg)
Doses
(h)

1
EGFR3-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR-

PEG5k (n = 1)

2
EGFR3-Ab(Cys)-
5
0.5
IV
5.0
1
96

EGFR-

PEG5k (n = 1)

3
EGFR3-Ab(Cys)-
5
0.25
IV
5.0
1
96

EGFR-

PEG5k (n = 1)

4
EGFR-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR-

PEG5k (n = 1)

5
EGFR-Ab(Cys)-
5
1
IV
5.0
1
96

scramble-

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 phosphoramidite 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-NH₂at 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=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank HCC827 tumors 100-300 mm³in 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 CO₂asphyxiation 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

Har-

Dose

Volume

vest

(mg/

(mL/
# of
Time

Group
Test Article
N
kg)
ROA
kg)
Doses
(h)

1
EGFR3-Ab(Cys)-N3′-
5
1
IV
5.0
1
96

EGFR-5′S-PEG5k

(n = 1)

2
EGFR3-Ab(Cys)-N3′-
5
0.5
IV
5.0
1
96

EGFR-5′S-PEG5k

(n = 1)

3
EGFR3-Ab(Cys)-N3′-
5
0.25
IV
5.0
1
96

EGFR-5′S-PEG5k

(n = 1)

4
EGFR-Ab(Cys)-N5′-
5
1
IV
5.0
1
96

EGFR-3′S-PEG5k

(n = 1)

5
EGFR-Ab(Cys)-N5′-
5
0.5
IV
5.0
1
96

EGFR-3′S-PEG5k

(n = 1)

6
EGFR-Ab(Cys)-N5′-
5
0.25
IV
5.0
1
96

EGFR-3′S-PEG5k

(n = 1)

7
EGFR-Ab(Cys)-N5′-
5
1
IV
5.0
1
96

scramble-3′S-

PEG5k (n = 1)

8
EGFR-Ab(Cys)-N5′-
5
0.5
IV
5.0
1
96

scramble-3′S-

PEG5k (n = 1)

9
EGFR-Ab(Cys)-N5′-
5
0.25
IV
5.0
1
96

scramble-3′S-

PEG5k (n = 1)

10
PBS Control
5
—
IV
5.0
1
96

Total # of Animals: 50
nu/nu mice with HCC827 tumors

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 phosphoramidite 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 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 phosphoramidite 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-NH₂at 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 mm³in 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 CO₂asphyxiation 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

Sur-
Ter-

siRNA

Dose

vival
minal
Harvest

Dose

Volume
# of
Bleed
Bleed
Time

Group
Test Article
N
(mg/kg)
ROA
(mL/kg)
Doses
(min)
(h)
(h)

EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
0.25
72
72

1
KRAS-PEG5k

(n = 1)

EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
3
96
96

2
KRAS-PEG5k

(n = 1)

3
EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
24
168
168

KRAS-PEG5k

(n = 1)

4
EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
0.25
72
72

EGFR-PEG5k

(n = 1)

5
EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
3
96
96

EGFR-PEG5k

(n = 1)

6
EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
24
168
168

EGFR-PEG5k

(n = 1)

7
PBS Control
5
—
IV
5.0
1
—
—
96

Total # of
35
nu/nu mice with

Animals:

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 phosphoramidite 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-NH₂at 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=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank Hep-3B2 1-7 tumors 100-300 mm³in 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 CO₂asphyxiation 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

Har-

Dose

Volume

vest

(mg/

(mL/
# of
Time

Group
Test Article
N
kg)
ROA
kg)
Doses
(h)

1
EGFR-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR-PEG5k (n = 1)

2
EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
96

EGFR-PEG5k (n = 1)

3
EGFR-Ab(Cys)-
5
0.25
IV
5.0
1
96

EGFR-PEG5k (n = 1)

4
EGFR-Ab(Cys)-
5
1
IV
5.0
1
96

scramble-

PEG5k (n = 1)

5
PBS Control
5
—
IV
5.0
1
96

Total # of Animals: 25
nu/nu mice with Hep3B tumors

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 phosphoramidite 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-NH₂at 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-NH₂and 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)-
4
2
IV
5.0
1
96

CTNNB1-PEG5k

(n = 1)

2
ASGR1-Ab(Lys)-
4
1
IV
5.0
1
96

CTNNB1-PEG5k

(n = 1)

3
ASGR1-Ab(Lys)-
4
0.5
IV
5.0
1
96

CTNNB1-PEG5k

(n = 1)

4
3GalNAc-
5
2
s.c.
5.0
1
96

CTNNB1

Control

5
PBS Control
5
—
IV
5.0
1
96

Total # of
22
WT mice (CD-1)

Animals:

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 phosphoramidite 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-NH₂at 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-NH₂and 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

Dose

Harvest

Dose

Volume
# of
Time

Group
Test Article
N
(mg/kg)
ROA
(mL/kg)
Doses
(h)

1
ASGR2-Ab(Lys)-
4
2
IV
5.0
1
96

KRAS-PEG5k

(n = 1)

2
ASGR2-Ab(Lys)-
4
1
IV
5.0
1
96

KRAS-PEG5k

(n = 1)

3
ASGR2-Ab(Lys)-
4
0.5
IV
5.0
1
96

KRAS-PEG5k

(n = 1)

4
3GalNAc-KRAS
5
2
s.c.
5.0
1
96

Control

5
PBS Control
5
—
IV
5.0
1
96

Total # of
22
WT mice (CD-1)

Animals:

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 phosphoramidite 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-NH₂at 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 phosphoramidite 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-NH₂at 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 CO₂asphyxiation 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

siRNA

Dose

Survival
Terminal

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
3
0.5
IV
5.0
1
30
96

3
(N = 1)
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
3
0.5
IV
5.0
1
30
96

6
(N = 1)
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).

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 phosphoramidite 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-NH₂at 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 mm³in 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 CO₂asphyxiation 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. 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 33

siRNA

Dose

Harvest

Dose

Volume
# of
Time

Group
Test Article
N
(mg/kg)
ROA
(mL/kg)
Doses
(h)

1
PSMA-Ab(Lys)-
5
2
IV
5.71
1
72

2
EGFR-PEG5k
5
1
IV
5.71
1
72

3
(n = 1)
5
0.5
IV
5.71
1
72

4
PSMA-Ab(Lys)-
5
4
IV
5.71
1
72

5
EGFR-PEG5k
5
2
IV
5.71
1
72

6
(n = 2)
5
1
IV
5.71
1
72

7
PSMA-Ab(Lys)-
5
2
IV
5.71
1
72

Scramble-PEG5k

(n = 1)

8
EGFR siRNA
5
2
IV
5.71
1
72

Alone

9
Vehicle
5
—
IV
5.71
1
72

Total # of
45
SCID SHO mice with LNCaP tumors

Animals:

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 phosphoramidite 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-NH₂at 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-NH₂and 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 phosphoramidite 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-NH₂at 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
4
3
IV
5.0
1
96

Ab(Cys)-HPRT-

PEG5k (n = 1)

2
Anti-B cell
4
1
IV
5.0
1
96

Ab(Cys)-HPRT-

PEG5k (n = 1)

3
Anti-B cell
4
0.3
IV
5.0
1
96

Ab(Cys)-HPRT-

PEG5k (n = 1)

4
Anti-B cell
4
3
IV
5.0
1
96

Ab(Lys)-HPRT-

PEG5k (n = 1)

5
Anti-B cell
4
1
IV
5.0
1
96

Ab(Lys)-HPRT-

PEG5k (n = 1)

6
Anti-B cell
4
0.3
IV
5.0
1
96

Ab(Lys)-HPRT-

PEG5k (n = 1)

7
Anti-B cell
4
3
IV
5.0
1
96

Ab(Cys)-

scramble-

PEG5k (n = 1)

8
Anti-B cell
4
1
IV
5.0
1
96

Ab(Cys)-

scramble-

PEG5k (n = 1)

9
Anti-B cell
4
0.3
IV
5.0
1
96

Ab(Cys)-

scramble-

PEG5k (n = 1)

10
PBS Control
4
—
IV
5.0
1
96

Total # of
77
WT mice (CD-1)

Animals:

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 phosphoramidite 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-NH₂at 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-NH₂and 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 phosphoramidite 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-NH₂at 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

Harvest

Dose

Volume
# of
Time

Group
Test Article
N
(mg/kg)
ROA
(mL/kg)
Doses
(h)

1
Anti-B cell
4
10
IV
5.1
1
96

Fab(Cys)-

HPRT-PEG5k

(n = 1)

2
Anti-B cell
4
3
IV
5.1
1
96

Fab(Cys)-

HPRT-PEG5k

(n = 1)

3
Anti-B cell
4
1
IV
5.1
1
96

Fab(Cys)-

HPRT-PEG5k

(n = 1)

4
Anti-B cell
4
10
IV
5.1
1
96

Fab(Cys)-

scramble-PEG5k

(n = 1)

5
Anti-B cell
4
3
IV
5.1
1
96

Fab(Cys)-

scramble-PEG5k

(n = 1)

6
Anti-B cell
4
1
IV
5.1
1
96

Fab(Cys)-

scramble-PEG5k

(n = 1)

7
PBS Control
5
—
IV
5.1
1
96

Total # of
29
WT mice (CD-1)

Animals:

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).

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 phosphoramidite 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-NH₂at 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-NH₂and 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

siRNA

Dose

Harvest

Dose

Volume
# of
Time

Group
Test Article
N
(mg/kg)
ROA
(mL/kg)
Doses
(h)

1
Anti-B cell
4
3
IV
5.0
1
96

Ab(Lys)-

CTNNB1-PEG5k

(n = 1)

2
Anti-B cell
4
1
IV
5.0
1
96

Ab(Lys)-

CTNNB1-PEG5k

(n = 1)

3
Anti-B cell
4
3
IV
5.0
1
96

Ab(Cys)-

CTNNB1-PEG5k

(n = 1)

4
Anti-B cell
4
1
IV
5.0
1
96

Ab(Cys)-

CTNNB1-PEG5k

(n = 1)

5
PBS Control
5
—
IV
5.0
1
96

Total # of
21
WT mice (CD-1)

Animals:

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.

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 phosphoramidite 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-NH₂at 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=5) of female SCID SHO mice bearing subcutaneous flank LNCaP tumors 100-350 mm³in 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 CO₂asphyxiation 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)-
5
1
IV
5.0
1
96

AR-PEG5k (n = 1)

2
ANT4044(Lys)-
5
0.5
IV
5.0
1
96

AR-PEG5k (n = 1)

3
ANT4044(Lys)-
5
0.25
IV
5.0
1
96

AR-PE5k (n = 1)

4
ANT4044(Lys)-
5
1
IV
5.0
1
96

scramble-

PEG5k (n = 1)

5
PBS Control
5
—
IV
5.0
1
96

Total # of
30
castrated SCID SHO mice with LNCaP

Animals:

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

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 18 G 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 10⁶cells 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 phosphoramidite 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-NH₂at 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 phosphoramidite 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-NH₂at 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 CO₂asphyxiation 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

siRNA
Dose

Survival
Terminal

Dose
Volume
# of
Bleed
Bleed

Gr
Test Article
N
(mg/kg)
(mL/kg)
Doses
(h)
(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 phosphoramidite 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-NH₂at 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=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank HCC827 tumors 100-300 mm³in 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 CO₂asphyxiation 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

Harvest

Dose

Volume
# of
Time

Gr
Test Article
N
(mg/kg)
ROA
(mL/kg)
Doses
(h)

1
EGFR-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR-PEG5k

(n = 1)

2
EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
96

EGFR-PEG5k

(n = 1)

3
EGFR-Ab(Cys)-
5
0.25
IV
5.0
1
96

EGFR-PEG5k

(n = 1)

4
EGFR-Ab(Cys)-
5
1
IV
5.0
1
96

ECL-EGFR-

PEG5k (n = 1)

5
EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
96

ECL-EGFR-

PEG5k (n = 1)

6
EGFR-Ab(Cys)-
5
0.25
IV
5.0
1
96

ECL-EGFR-

PEG5k (n = 1)

7
EGFR-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR-SS-

PEG5k (n = 1)

8
EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
96

EGFR-SS-

PEG5k (n = 1)

9
EGFR-Ab(Cys)-
5
0.25
IV
5.0
1
96

EGFR-SS-

PEG5k (n = 1)

10
EGFR-Ab(Cys)-
5
1
IV
5.0
1
96

ECL-EGFR-SS-

PEG5k (n = 1)

11
EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
96

ECL-EGFR-SS-

PEG5k (n = 1)

12
EGFR-Ab(Cys)-
5
0.25
IV
5.0
1
96

ECL-EGFR-SS-

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

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 phosphoramidite 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-NH₂at 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 1-7 (n=5) of female SCID SHO mice bearing subcutaneous flank LNCaP tumors 100-350 mm³in 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 CO₂asphyxiation 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)-
5
1
IV
5.0
1
96

SS-EGFR-PEG5k

(n = 1)

2
PSMA-Ab(Lys)-
5
0.5
IV
5.0
1
96

SS-EGFR-PEG5k

(n = 1)

3
PSMA-Ab(Cys)-
5
1
IV
5.0
1
96

ECL-EGFR-

PEG5k (n = 1)

4
PSMA-Ab(Cys)-
5
0.5
IV
5.0
1
96

ECL-EGFR-

PEG5k (n = 1)

5
PSMA-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR-PEG5k

(n = 1) FRESH

6
PSMA-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR-PEG5k

(n = 1)

FROZEN

7
PSMA-Ab(Cys)-
5
1
IV
5.0
1
96

svcramble-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 phosphoramidite 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-NH₂at 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=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank HCC827 tumors 100-300 mm³in 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 CO₂asphyxiation 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

siRNA

Dose

Harvest

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-PEG5k

(n = 1)

2
EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
96

EGFR-PEG5k

(n = 1)

3
EGFR-Ab(Cys)-
5
1
IV
5.0
1
96

SS-EGFR-

PEG5k (n = 1)

4
EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
96

SS-EGFR-

PEG5k (n = 1)

6
EGFR-Ab(Cys)-
5
1
IV
5.0
1
96

scramble-

PEG5k (n = 1)

7
EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
96

scramble-

PEG5k (n = 1)

8
PBS Control
5
—
IV
5.0
1
96

Total # of
80
nu/nu mice with HCC827 tumors

Animals:

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 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 phosphoramidite 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-NH₂at 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 CO₂asphyxiation 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)-SMPT-
4
0.5
IV
5.0
1
5
24

8
KRAS-PEG5k (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
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 phosphoramidite 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-NH₂at 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 CO₂asphyxiation 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

Two different passenger strands were made containing two conjugation handles (C6-NH₂and C6-SH) in two different orientations (55′-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-NH₂and C6-SH were connected through the phosphodiester, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank HCC827 tumors 100-300 mm³in 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 CO₂asphyxiation 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.

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 phosphoramidite 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-NH₂and 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-NH₂and 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

Har-

siRNA

Dose

vest

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

N5′-HPRT-3′S-

PEG5k (n = 1)

2
Anti-B cell Ab(Cys)-
4
1
IV
5.0
1
96

N5′-HPRT-3′S-

PEG5k (n = 1)

3
Anti-B cell Ab(Cys)-
4
0.3
IV
5.0
1
96

N5′-HPRT-3′S-

PEG5k (n = 1)

4
Anti-B cell Ab(Cys)-
4
3
IV
5.0
1
96

N3′-HPRT-5′S-

PEG5k (n = 1)

5
Anti-B cell Ab(Cys)-
4
1
IV
5.0
1
96

N3′-HPRT-5′S-

PEG5k (n = 1)

6
Anti-B cell Ab(Cys)-
4
0.3
IV
5.0
1
96

N3′-HPRT-5′S-

PEG5k (n = 1)

7
Anti-B cell Ab(Lys)-
4
2
IV
5.0
1
96

S3′-HPRT-5′N-

PEG5k (n = 1)

8
Anti-B cell Ab(Lys)-
4
0.75
IV
5.0
1
96

S3′-HPRT-5′N-

PEG5k (n = 1)

9
Anti-B cell Ab(Lys)-
4
0.25
IV
5.0
1
96

S3′-HPRT-5′N-

PEG5k (n = 1)

10
Anti-B cell Ab(Lys)-
4
2
IV
5.0
1
96

S5′-HPRT-3′N-

PEG5k (n = 1)

11
Anti-B cell Ab(Lys)-
4
0.75
IV
5.0
1
96

S5′-HPRT-3′N-

PEG5k (n = 1)

12
Anti-B cell Ab(Lys)-
4
0.25
IV
5.0
1
96

S5′-HPRT-3′N-

PEG5k (n = 1)

13
PBS Control
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

Two different passenger strands were made containing two conjugation handles (C6-NH₂and C6-SH) in two different orientations (55′-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-NH₂and 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

TABLE 47

Har-

siRNA

Dose

vest

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

N5′-CTNNB1-3′

S-PEG5k (n = 1)

2
Anti-B cell Ab(Cys)-
4
1
IV
5.0
1
96

N5′-CTNNB1-3′

S-PEG5k (n = 1)

3
Anti-B cell Ab(Cys)-
4
0.3
IV
5.0
1
96

N5′-CTNNB1-3′

S-PEG5k (n = 1)

4
Anti-B cell Ab(Lys)-
4
3
IV
5.0
1
96

S5′-CTNNB1-

3′N-PEG5k (n = 1)

5
Anti-B cell Ab(Lys)-
4
1
IV
5.0
1
96

S5′-CTNNB1-

3′N-PEG5k (n = 1)

6
Anti-B cell Ab(Lys)-
4
0.3
IV
5.0
1
96

S5′-CTNNB1-

3′N-PEG5k (n = 1)

7
Anti-B cell Ab(Cys)-
4
3
IV
5.0
1
96

N3′-CTNNB1-

5′S-PEG5k (n = 1)

8
Anti-B cell Ab(Cys)-
4
1
IV
5.0
1
96

N3′-CTNNB1-

5′S-PEG5k (n = 1)

9
Anti-B cell Ab(Cys)-
4
0.3
IV
5.0
1
96

N3′-CTNNB1-

5′S-PEG5k (n = 1)

10
Anti-B cell Ab(Lys)-
4
3
IV
5.0
1
96

S3′-CTNNB1-

5′N-PEG5k (n = 1)

11
Anti-B cell Ab(Lys)-
4
1
IV
5.0
1
96

S3′-CTNNB1-

5′N-PEG5k (n = 1)

12
Anti-B cell Ab(Lys)-
4
0.3
IV
5.0
1
96

S3′-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.

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 phosphoramidite 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-NH₂at 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 CO₂asphyxiation 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)-
4
IV
5.0
1
5
24

2
EGFR-
4
IV
5.0
1
30
96

3
PEG5k (n = 1)
4
IV
5.0
1
180
168

4
EGFR-Ab(Cys)-
4
IV
5.0
1
5
24

5
ECL-EGFR-
4
IV
5.0
1
30
96

6
PEG5k (n = 1)
4
IV
5.0
1
180
168

7
EGFR-Ab(Cys)-
4
IV
5.0
1
5
24

8
EGFR-SS-
4
IV
5.0
1
30
96

9
PEG5k (n = 1)
4
IV
5.0
1
180
168

10
EGFR-Ab(Cys)-
4
IV
5.0
1
5
24

11
ECL-EGFR-
4
IV
5.0
1
30
96

12
SS-PEG5k (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 phosphoramidite 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-NH₂at 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 1-7 (n=5) of female SCID SHO mice bearing subcutaneous flank LNCaP tumors 100-350 mm³in 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 CO₂asphyxiation 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

siRNA

Dose

Harvest

Test

Dose

Volume
# of
Time

Group
Article
N
(mg/kg)
ROA
(mL/kg)
Doses
(h)

1
PSMA-
5
1
IV
5.0
1
96

Ab(Cys)-

EGFR-

SS-PEG5k

(n = 1)

2
PSMA-
5
0.5
IV
5.0
1
96

Ab(Cys)-

EGFR-

SS-PEG5k

(n = 1)

3
PSMA-
5
1
IV
5.0
1
96

Ab(Cys)-

EGFR-

ECL-

PEG5k

(n = 1)

4
PSMA-
5
0.5
IV
5.0
1
96

Ab(Cys)-

EGFR-

ECL-

PEG5k

(n = 1)

5
PSAM-
5
1
IV
5.0
1
96

Ab(Cys)-

EGFR-

PEG5k

(n = 1)

6
PSAM-
5
0.5
IV
5.0
1
96

Ab(Cys)-

EGFR-

PEG5k

(n = 1)

7
PSMA-
5
1
IV
5.0
1
96

Ab(Cys)-

scramble-

PEG5k

(n = 1)

8
PBS
5
—
IV
5.0
1
96

Control

Total # of
40
SCID SHO mice with LNCaP tumors

Animals:

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 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 phosphoramidite 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-NH₂at 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 1-15 (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank HCC827 tumors 100-300 mm³in 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 CO₂asphyxiation 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

siRNA

Dose

Har-

Dose

Volume

vest

(mg/

(mL/
# of
Time

Group
Test Article
N
kg)
ROA
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 phosphoramidite 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-NH₂at 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 CO₂asphyxiation 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

siRNA

Dose

Sur-
Ter-

Dose

Volume

vival
minal

(mg/

(mL/
# of
Bleed
Bleed

Group
Test Article
N
kg)
ROA
kg)
Doses
(min)
(h)

1
EGFR-
4
0.5
IV
5.0
1
5
24

2
Ab(Cys)-
4
0.5
IV
5.0
1
30
96

3
EGFR-
4
0.5
IV
5.0
1
180
168

PEG2k

(n = 1)

4
EGFR-
4
0.5
IV
5.0
1
5
24

5
Ab(Cys)-
4
0.5
IV
5.0
1
30
96

6
EGFR-
4
0.5
IV
5.0
1
180
168

dPEG₄₈

(n = 1)

7
EGFR-
4
0.5
IV
5.0
1
5
24

8
Ab(Cys)-
4
0.5
IV
5.0
1
30
96

9
EGFR-
4
0.5
IV
5.0
1
180
168

dPEG₂₄

(n = 1)

10
EGFR-
4
0.5
IV
5.0
1
5
24

11
Ab(Cys)-
4
0.5
IV
5.0
1
30
96

12
EGFR-
4
0.5
IV
5.0
1
180
168

dPEG₁₂

(n = 1)

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 phosphoramidite 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-NH₂at 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 CO₂asphyxiation 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 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)-
4
0.5
IV
5.0
1
5
24

2
EGFR-PEG10k
4
0.5
IV
5.0
1
30
96

3
(n-1)
4
0.5
IV
5.0
1
180
168

4
EGFR-Ab(Cys)-
4
0.5
IV
5.0
1
5
24

5
EGFR-(dPEG24)₃
4
0.5
IV
5.0
1
30
96

6
(n = 1)
4
0.5
IV
5.0
1
180
168

7
EGFR-Ab(Cys)-
4
0.5
IV
5.0
1
5
24

8
EGFR-(dPEG12)3
4
0.5
IV
5.0
1
30
96

9
(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
EGFR-(dPEG4)₃
4
0.5
IV
5.0
1
30
96

12
(n = 1)
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 phosphoramidite 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-NH₂at 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 1-12 (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flank HCC827 tumors 100-300 mm³in 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 CO₂asphyxiation 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

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-PEG10k

(n = 1)

2
EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
96

EGFR-PEG10k

(n = 1)

3
EGFR-Ab(Cys)-
5
0.25
IV
5.0
1
96

EGFR-PEG10k

(n = 1)

4
EGFR-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR-(dPEG24)3

(n = 1)

5
EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
96

EGFR-(dPEG24)3

(n = 1)

6
EGFR-Ab(Cys)-
5
0.25
IV
5.0
1
96

EGFR-(dPEG24)3

(n = 1)

7
EGFR-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR-(dPEG12)3

(n = 1)

8
EGFR-Ab(Cys)-
5
0.5
IV
5.0
1
96

EGFR-(dPEG12)3

(n = 1)

9
EGFR-Ab(Cys)-
5
0.25
IV
5.0
1
96

EGFR-(dPEG12)3

(n = 1)

10
EGFR-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR-(dPEG4)3

(n = 1)

11
EGFR-Ab(Cys)-
5
1
IV
5.0
1
96

EGFR-PEG5k

(n = 1)

12
EGFR-Ab(Cys)-
5
1
IV
5.0
1
96

scramble-PEG5k

(n = 1)

13
PBS Control
5
—
IV
5.0
1
96

Total # of
65
nu/nu mice with HCC827 tumors

Animals:

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 PEGSK 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 PEGSK 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 phosphoramidite 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-NH₂at 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-NH₂and 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 phosphoramidite 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-NH₂at 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

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 phosphoramidite 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-NH₂at 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

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

Sur-
Ter-

siRNA

Dose

vival
minal

Dose

Volume
# of
Bleed
Bleed

Gr
Test Article
N
(mg/kg)
ROA
(mL/kg)
Doses
(h)
(h)

1
EGFR-
4
0.5
IV
5.0
1
0.25
24

Ab(Cys)-N5′-

Cy5.KRAS-

3′S-PEG5k

(n = 1)

2
EGFR-
4
0.5
IV
5.0
1
1
48

Ab(Cys)-N5′-

Cy5.KRAS-

3′S-PEG5k

(n = 1)

3
EGFR-
4
0.5
IV
5.0
1
4
72

Ab(Cys)-N5′-

Cy5.KRAS-

3′S-PEG5k

(n = 1)

4
EGFR-
4
0.5
IV
5.0
1
0.25
24

Ab(Cys)-N5′-

Cy5.KRAS-

3′S--NEM

(n = 1)

5
EGFR-
4
0.5
IV
5.0
1
1
48

Ab(Cys)-N5′-

Cy5.KRAS-

3′S--NEM

(n = 1)

6
EGFR-
4
0.5
IV
5.0
1
4
72

Ab(Cys)-N5′-

Cy5.KRAS-

3′S--NEM

(n = 1)

7
EGFR-
4
0.5
IV
5.0
1
0.25
24

Ab(Lys)-S3′-

Cy5.KRAS-

5′N-PEG5k

(n = 1)

8
EGFR-
4
0.5
IV
5.0
1
1
48

Ab(Lys)-S3′-

Cy5.KRAS-

5′N-PEG5k

(n = 1)

9
EGFR-
4
0.5
IV
5.0
1
4
72

Ab(Lys)-S3′-

Cy5.KRAS-

5′N-PEG5k

(n = 1)

10
EGFR-
4
0.5
IV
5.0
1
0.25
24

Ab(Lys)-S3′-

Cy5.KRAS-

5′NH₂

(n = 1)

11
EGFR-
4
0.5
IV
5.0
1
1
48

Ab(Lys)-S3′-

Cy5.KRAS-

5′NH₂

(n = 1)

12
EGFR-
4
0.5
IV
5.0
1
4
72

Ab(Lys)-S3′-

Cy5.KRAS-

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 CO₂asphyxiation 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

Cross-reference for

Dose

# of
synthesis and

Group
Test Article
N
(mg/kg)
ROA
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 CO₂asphyxiation 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 mm³in 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 CO₂asphyxiation 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

Cross-

siRNA
mol

Har-
reference

Dose
EEP/

vest
to synthesis

Test

(mg/
mol

# of
Time
and char-

Group
Article
N
kg)
Ratio
ROA
Doses
(h)
acterization

1
chol-
3
5
—
IV
1
24
General

2
KRAS
3
5
—
IV
1
72
experimental

3

3
5
—
IV
1
144
(Example 2)

4
chol-
3
5
1
IV
1
24
General

5
KRAS +
3
5
1
IV
1
72
experimental

6
chol-
3
5
1
IV
1
144
(Example 2)

Melittin

7
chol-
3
5
3
IV
1
24
General

8
KRAS +
3
5
3
IV
1
72
experimental

9
chol-
3
5
3
IV
1
144
(Example 2)

Melittin

10
chol-
3
5
10
IV
1
24
General

11
KRAS +
3
5
10
IV
1
72
experimental

12
chol-
3
5
10
IV
1
144
(Example 2)

Melittin

13
chol-
3
5
1
IV
1
24
General

14
KRAS +
3
5
1
IV
1
72
experimental

15
chol-
3
5
1
IV
1
144
(Example 2)

INF7

16
chol-
3
5
3
IV
1
24
General

17
KRAS +
3
5
3
IV
1
72
experimental

18
chol-
3
5
3
IV
1
144
(Example 2)

INF7

19
chol-
3
5
10
IV
1
24
General

20
KRAS +
3
5
10
IV
1
72
experimental

21
chol-
3
5
10
IV
1
144
(Example 2)

INF7

22
Vehicle
4
—
—
IV
1
24
General

23
(PBS)
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 mm³in 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 CO₂asphyxiation 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/

Cross-

siRNA
siRNA

Harvest
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
5
5
1
IV
3
96
experimental

(1:1)

(Example 2)

5
chol-KRAS +
5
5
3
IV
3
24
General

6
chol-INF7
5
5
3
IV
3
96
experimental

(3:1)

(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 mm³in 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 CO₂asphyxiation 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:

Cross-

siRNA
EGFR-
siRNA

Harvest
reference to

Dose
Ab Ratio
Ratio

# of
Time
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

PEG5k-EGFR +

3 and 6

EGFR-Ab-

melittin

3
EGFR-Ab-

KRAS-PEG5k +
5
0.5
1
1:1
IV
1
96
Example

EGFR-Ab-

3 and 6

melittin

4
EGFR
5
—
—
—
IV
1
96
General

antibody

experimental

Alone

(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 CO₂asphyxiation 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-
11
5
IV
9
Weekly
24
General

CTNNB1

experimental

(Example 2)

8
Chol-
11
5
SC
9
Weekly
24
General

CTNNB1

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.

	Number	Date	Country
Parent	PCT/US2017/025608	Mar 2017	US
Child	16128440		US

NUCLEIC ACID-POLYPEPTIDE COMPOSITIONS AND USES THEREOF

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