Rna-sn-38 conjugate for the treatment of colorectal cancer and sequential lung metastasis

Abstract

Disclosed herein are compositions and methods for CMC production of RNA therapeutic complexes (nanostructures) that contain SN-38 (7-Ethyl-10-hydroxycamptothecin) and / or Irinotecan. In particular, disclosed herein is an RNA nanoparticle having at least three synthetic RNA nucleotides coupled to each other, wherein the at least three synthetic RNA oligonucleotides form a central ore domain and at least three double-stranded arms arranged around the core domain and extending away from the central core domain, wherein at least one of the three double-stranded arms is conjugated with Irinotecan and / or SN-38 with an esterbond that is cleavable by esterase in cancer tissue or cancer cells.

Description

TH Docket No.321502-2140 RNA-SN-38 CONJUGATE FOR THE TREATMENT OF COLORECTAL CANCER AND SEQUENTIAL LUNG METASTASIS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No.63 / 594,983, filed November 1, 2023, which is hereby incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with Government Support under Grant Nos. CA207946 and EB019036 awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0003] SN38, an active metabolite of irinotecan, stands as a pivotal antineoplastic drug in the realm of cancer therapy. Initially derived from the family of cytotoxic nucleoside analogues and nucleobases, SN38 has distinguished itself as a potent agent in the medical treatment of various cancer types. Its influence extends to both solid tumors and malignant blood disorders, showcasing its versatility in combating diverse forms of cancer. As an antimetabolite, SN38 competes with endogenous nucleosides, effectively targeting a multitude of intracellular sites to induce cytotoxic effects. However, its strong toxicity has led to the failure in the use of SN-39 as a pure chemical for the treatment of human cancers. Given its significance, efforts to refine the delivery mechanisms of SN38 remain a critical area of focus in maximizing its therapeutic potential. Currently, an antibody conjugated with SN-38 have been clinically approved for the treatment of non-small cell lung cancer, breast cancer, and pancreatic cancer. However, there is no SN-38 or other SN-38 conjugates have been approved clinically. SUMMARY OF THE INVENTION

[0004] The disclosed compositions and methods have revolutionized the production of RNA-SN-38 therapeutic complexes, presenting a breakthrough in the manufacturing of pharmaceutical agents. Specifically, this innovation extends to the streamlined creation of RNA complexes containing SN38, a potent antineoplastic drug, and irinotecan, a pivotal precursor compound. Through a two-step process in CMC production, this two chemotherapeutics are integrated into RNA strands via esterification reaction and click chemistry reaction.

[0005] In some embodiments, the RNA complex can be composed of at least 3, 4, 5, or 6 synthetic RNA oligonucleotides, wherein the synthetic RNA oligonucleotides self-assemble to form the RNA complex, wherein the RNA complex has a central core domain and at least 3, 4,TH Docket No.321502-2140 5, or 6 double-stranded arms arranged around the core domain and extending away from the central core domain.

[0006] In some embodiments, one, two, three or more of the at least three synthetic RNA oligonucleotides in the RNA complex contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more SN38, irinotecan, or a combination thereof.

[0007] In some embodiments, the RNA oligonucleotides are modified at the 5’ end with GalNAc (N-acetylgalactosamine), UAMC-1110 (SP-13786; (S)-N-(2-(2-Cyano-4,4- difluoropyrrolidin-1-yl)-2-oxoethyl)quinoline-4-carboxamide), FA (Folate), or DCL (N-[N- [(S)-1,3- dicarboxypropyl]carbamoyl]-(S)-lysine).

[0008] In some embodiments, at least 1, 2, 3, or 4 of the synthetic RNA oligonucleotides have an amino acid sequence that is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS:1-297. In some embodiments, synthetic RNA oligonucleotides containing nucleoside analogues are combined with un-modified RNA oligonucleotides to form the RNA nanostructure. For example, the at least 1, 2, 3, or 4 synthetic RNA oligonucleotides that have an amino acid sequence that is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS:1-297 can be combined with at least 1, 2, 3, 4, 5, or 6 synthetic RNA oligonucleotides that have an amino acid sequence that is about 80-100% identical to any one of SEQ ID NOS:298-394. Suitable combinations of these oligonucleotides to form RNA nanoparticles are described herein.

[0009] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF FIGURES

[0010] FIGs.1A to 1C show the sequence and stepwise assembly of M23WJ using 3WJ-a (SEQ ID NO:232), 3WJ-b (SEQ ID NO:233), and 3WJ-c (SEQ ID NO:234). FIG.1A shows the high-GC content sequence of M23WJ. The free energy of the thermodynamic ensemble is 60.54 kcal / mol. The frequency of the MFE structure in the ensemble is 57.16%. FIG.1B is a gel showing the stepwise assembly of M23WJ and size comparison of phi293WJ. FIG.1C shows stepwise assembly of 3WJ using 3WJ-a (SEQ ID NO:232), 3WJ-b (SEQ ID NO:233), and 3WJ-c (SEQ ID NO:234),

[0011] FIGs.2A to 2F show assembly and characterization of 4WJ-SN38-E RNA nanoparticles. FIG.2A is a schematic of functionalizing 4WJ with 24 copies of SN38 and oneTH Docket No.321502-2140 copy of EpCAM RNA aptamer. FIG.2B is a gel showing the stepwise assembly of 4WJ-SN38-E (M: monomer, D: dimer, T: trimer) and size comparison of 4WJ, 4WJ-SN38, and 4WJ-SN38-E. FIG.2C shows size distribution of 4WJ and 4WJ-SN38. FIG.2D shows zeta potential distribution of 4WJ and 4WJ-SN38. FIG.2E shows thermostability of 4WJ and 4WJ-SN38 demonstrated by annealing profile. FIG.2F shows thermostability of 4WJ-SN38 demonstrated by melting profile.

[0012] FIGs.3A to 3C show in vitro cell binding, internalization, and cancer suppression study of 4WJ-SN38-E RNA nanoparticles. FIG.3A contains confocal images of HT29 cells after incubation with PBS, 4WJ, and 4WJ-SN38-E, respectively. FIG.3B shows evaluation of cell viability by MTT assay in HT29 cells incubated with RNA nanoparticles and SN38 for 48, 72, and 96 hr, respectively. Statistics were calculated by two-tailed unpaired t-test presented as mean ± SEM. Significant results compared to the RNA nanoparticle at a concentration of 0.025 μM are marked with an asterisk (*p <^0.05, **p <^0.01, ***p^<^0.001). Significant results compared to the 4WJ at a concentration of 0.025 μM are marked with a hash (##p <^0.01 and ###p^<^0.001). FIG.3C shows in vitro apoptotic effects of RNA nanoparticles and SN38 by PI / Annexin V-FITC dual staining and FACS analysis (Q2 = Annexin V-FITC positive & PI positive, indicating late apoptotic and dead cells; Q3 = Annexin V-FITC positive & PI negative, indicating early apoptotic cells).

[0013] FIGs.4A to 4D show in vitro immune response and in vivo tumor inhibition of 4WJ-SN38-E RNA nanoparticles. FIGs.4A and 4B show evaluation of TNF-α (FIG.4A) and IL-6 (FIG.4B) production after incubating 4WJ-SN38-E with macrophage-like cells by ELISA. FIG. 4C shows intravenous treatment of nude mice bearing HT29 xenografts with 4WJ-SN38-E and control groups every three days for a total of 5 injections (indicated by arrows). Mice tumor size was monitored during the time course of treatments. FIG.4D is a comparison of tumor weight and size at the endpoint (n = 5 biologically independent animals). Statistics were calculated by two-tailed unpaired t-test presented as mean ± SEM, **p < 0.01, ***p < 0.001.

[0014] FIGs.5A to 5D show in vivo colorectal cancer lung metastasis model inhibition of 4WJ-SN38-E. FIG.5A shows Lago imaging to confirm metastasis establishment 5 days after IV injection. FIG.5B shows bioluminescence to compare metastasis in vivo between 4WJ and 4WJ-SN38-E groups. The mice in the PBS control group were so sick (see FIG.5D) and cannot survive till the whole body imaging. FIG.5C shows GFP imaging to compare metastasis ex vivo between PBS, 4WJ, and 4WJ-SN38-E groups. FIG.5D shows mice weight changes on day 5, 8, 11, 14, 17, 20, and 23.TH Docket No.321502-2140

[0015] FIG 6 is a CMC product for drug-contained RNA nanoparticles. Schematic of the procedures for the CMC product of drug-contained RNA nanoparticles.

[0016] FIGs.7A and 7B show preparation of SN38-N3. FIG.7A is a schema of Steglich Esterification mechanism. FIG.7B shows LC-MS data on the synthesized SN38-N3.

[0017] FIGs.8A to 8C shows improvement of water solubility of SN38 by conjugation to RNA strands. FIG.8A is a schematic of 6 copies of SN38 conjugated to one RNA strand via click reaction. FIG.8B shows gel electrophoresis and HPLC spectrum of RNA-6-ALK and RNA- 6-SN38 strands. FIG.8C is a solubility comparison of RNA-SN38 in water, SN38 in water, and SN38 in DMSO.

[0018] FIGs.9A to 9D shows combinational chemotherapy of GEM / PTX and GEM / SN38 achieved by 4WJ RNA nanoparticles. FIGs.9A and 9B is a dose-response matrix of 4WJ- GEM / PTX (FIG.4A) and HSA synergy map of 4WJ-GEM / PTX (HSA score: 15.293) (FIG.4B). FIGs.4C and 4D is a dose-response matrix of 4WJ-GEM / SN38 (FIG.4C) and HSA synergy map of 4WJ-GEM / SN38 (HSA score: 24.956) (FIG.4D).

[0019] FIG.10A is a schematic diagram of 3WJ nanoparticles using 3WJ-A (SEQ ID NO:160), 3WJ-B (SEQ ID NO:165), and 3WJ-C (SEQ ID NO:170).

[0020] FIG.10B is a schematic diagram of 3WJ-FA-siRNA nanoparticles using 3WJ-A (SEQ ID NO:160), 3WJ-B (SEQ ID NO:235), 3WJ-C (SEQ ID NO:170), and survivin anti-sense (SEQ ID NO:236). FIG.10C is a schematic diagram of 4WJ-FA-siRNA nanoparticles using 4WJ-A (SEQ ID NO:175), 4WJ-B (SEQ ID NO:180), 4WJ-C (SEQ ID NO:185), and 4WJ-D (SEQ ID NO:190).

[0021] FIGs.11A to 11C is a hemolysis data of 3WJ nanoparticles (FIG.11A) and 3WJ- FA-siRNA (FIG.11B) nanoparticles. BLOD = below limit of detection. NC is negative control. PC is positive control.

[0022] FIGs.12A to 12D is a platelet aggregation data of 3WJ nanoparticles alone (FIG. 12A) 3WJ-FA-siRNA (FIG.12B) nanoparticles alone, and 3WJ nanoparticles (FIG.12C) or 3WJ-FA-siRNA (FIG.12D) nanoparticles together with collagen. BLOD = below limit of detection. NC is negative control. PC is positive control.

[0023] FIGs.13A to 13C show plasma coagulation data on both 3WJ and 3WJ-FA- siRNA nanoparticles. FIG.13A shows prothrombin time of RNA nanoparticles. FIG.13B shows activated partial thromboplastin time of RNA nanoparticles. FIG.13C shows thrombin time of RNA nanoparticles. PT = Prothrombin time. APTT = Activated partial thromboplastin time. TT = Thrombin time. Coag.time = Coaggregation time. NC is negative control. PC is positive control.TH Docket No.321502-2140

[0024] FIGs.14A to 14B show immunogenicity profiles of RNA nanoparticles. FIG.14A shows complement activation data on 3WJ and 3WJ-FA-siRNA nanoparticles. BLOD = below limit of detection. NC is negative control. CVF = Cobra Venom Factor. FIG.14B shows interferons (IFN) induction data of 3WJ-FA-siRNA RNA nanoparticles.

[0025] FIG.15 shows in vivo organ level study of the safety profile of 4WJ-SN38. FIG. 14A shows organ weights study. FIG.15B shows H&E staining of major organs. *P < 0.05.

[0026] FIGs.16A and 16B shows in vivo toxicity biomarkers study on 4WJ-SN38. FIG. 16A shows a serum biochemistry study (FIG.16A) and Hematological study (FIG.16B) comparing PBS, SN38 free drug, and 4WJ-SN38 RNA nanoparticles. *P < 0.05. DETAILED DESCRIPTION

[0027] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0028] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0030] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtueTH Docket No.321502-2140 of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0031] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0032] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0033] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0034] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

[0035] 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.

[0036] As used herein, "self-assembly" refers to the ability of nucleic acids (and, in some instances, preformed nucleic acid nanostructures (e.g., crystals)) to anneal to each other, in a sequence- specific manner, in a predicted manner and without external control. In some aspects, nucleic acid nanostructure self-assembly methods include combining nucleic acids (e.g., single- stranded nucleic acids, or oligonucleotides) in a single vessel and allowing the nucleic acids to anneal to each other, based on sequence complementarity. In some aspects, this annealing process involves placing the nucleic acids at an elevated temperature and thenTH Docket No.321502-2140 reducing the temperature gradually in order to favor sequence- specific binding. Various nucleic acid nanostructures or self-assembly methods are known and described herein.

[0037] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0038] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0039] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0040] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0041] The term “prevent” refers to a treatment that forestalls or slows the onset of a disease or condition or reduced the severity of the disease or condition. Thus, if a treatment can treat a disease in a subject having symptoms of the disease, it can also prevent that disease in a subject who has yet to suffer some or all of the symptoms.

[0042] For purposes herein, the % sequence identity of a given nucleotides or amino acids sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given sequence C that has or comprises a certain % sequence identity to, with, or against a given sequence D) is calculated as follows:TH Docket No.321502-2140

[0043] 100 times the fraction W / Z,

[0044] where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program’s alignment of C and D, and where Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the % sequence identity of C to D will not equal the % sequence identity of D to C. RNA oligonucleotides

[0045] Disclosed herein are RNA oligonucleotides that can self-assemble into an RNA nanoparticle as described herein, wherein at least one of the RNA oligonucleotides in the RNA nanoparticle contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 SN38, Irinotecan, or a combination thereof.

[0046] In some embodiments, the RNA oligonucleotides are modified at the 5’ end with GalNAc (N-acetylgalactosamine), UAMC-1110 (SP-13786; (S)-N-(2-(2-Cyano-4,4- difluoropyrrolidin-1-yl)-2-oxoethyl)quinoline-4-carboxamide), FA (Folate), or DCL (N-[N- [(S)-1,3- dicarboxypropyl]carbamoyl]-(S)-lysine).

[0047] In some embodiments, the disclosed RNA oligonucleotide has one of the following sequences: 3WJ-A with SN38 and Irinotecan and derivatives

[0048] 5’- XXGYYAXGXGXAXGXGGG-3’; 5’-XXGYYAXGXGXAXGXGGGGGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGG C-3’ (SEQ ID NO:1); 5’-XXGYYAXGXGXAXGXGGGGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUU UCCC-3’ (SEQ ID NO:2); 5’-XXGYYXGXGXAXGXGGGGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:3); 5’-XXGYYAXGXGXAXGXGGGCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:4); 5’-GalNAc-XXGYYAXGXGXAXGXGGG-3’; 5’-FA-XXGYYAXGXGXAXGXGGG-3’; 5’- DCL-XXGYYAXGXGXAXGXGGG-3’; and 5’-UAMC1110-XXGYYAXGXGXAXGXGGG-3’, wherein X is SN38 and wherein Y is Irinotecan. 3WJ-B with SN38 and Irinotecan and derivatives

[0049] 5’-YYYAYAXAYXXXGXXGAXYY-3’; 5’-YYYAYAXAYXXXGXXGAXYYGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGG C-3’ (SEQ ID NO:5); 5’-YYYAYAXAYXXXGXXGAXYYGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACG UUUCCC-3’ (SEQ ID NO:6); 5’-YYYAYAXAYXXXGXXGAXYYGCGACUGGUUACCCGGUCG- 3’ (SEQ ID NO:7); 5’-YYYAYAXAYXXXGXXGAXYYCAGAACGUAUACUAUUCUG-3’ (SEQ IDTH Docket No.321502-2140 NO:8); 5’-GalNAc-YYYAYAXAYXXXGXXGAXYY-3’; 5’-FA-YYYAYAXAYXXXGXXGAXYY-3’; 5’- DCL-YYYAYAXAYXXXGXXGAXYY-3’; and 5’-UAMC1110-YYYAYAXAYXXXGXXGAXYY-3’, wherein X is SN38 and wherein Y is Irinotecan. 3WJ-C with SN38 and Irinotecan and derivatives

[0050] 5’-GGAXYAAXYAXGGYAA-3’ (SEQ ID NO:9); 5’-GGAXYAAXYAXGGYAAGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:10); 5’-GGAXYAAXYAXGGYAAGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUUUC CC-3’ (SEQ ID NO:11); 5’-GGAXYAAXYAXGGYAAGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:12); 5’-GGAXYAAXYAXGGYAACAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:13); 5’- GalNAc-GGAXYAAXYAXGGYAA-3’ (SEQ ID NO:9); 5’-FA-GGAXYAAXYAXGGYAA-3’ (SEQ ID NO:9); 5’-DCL-GGAXYAAXYAXGGYAA-3’ (SEQ ID NO:9); and 5’-UAMC1110- GGAXYAAXYAXGGYAA-3’ (SEQ ID NO:9), wherein X is SN38 and wherein Y is Irinotecan. 4WJ-A with SN38 and Irinotecan and derivatives

[0051] 5’-XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAA-3’ (SEQ ID NO:14); 5’-XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAAGCCUUAGUAACGUGCUUU GAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:15); 5’-XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAAGGGACCGAAAAAGACCUGA CUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:16); 5’-XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAAGCGACUGGUUACCCGGUC G-3’ (SEQ ID NO:17); 5’-XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAACAGAACGUAUACUAUUCUG -3’ (SEQ ID NO:18); 5’-GalNAc-XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAA- 3’ (SEQ ID NO:14); 5’-FA-XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAA-3’ (SEQ ID NO:14); 5’-DCL-XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAA-3’ (SEQ ID NO:14); 5’-UAMC1110-XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAA- 3’ (SEQ ID NO:14), wherein X is SN38 and wherein Y is Irinotecan. 4WJ-B with SN38 and Irinotecan and derivatives

[0052] 5’-XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAG-3’ (SEQ ID NO:19); 5’-XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAGGCCUUAGUAACGUGCUU UGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:20); 5’-XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAGGGGACCGAAAAAGACCUTH Docket No.321502-2140 GACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:21); 5’-XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAGGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:22); 5’-XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAGCAGAACGUAUACUAUUC UG-3’ (SEQ ID NO:23); 5’-GalNAc-XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAG-3’ (SEQ ID NO:19); 5’-FA-XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAG-3’ (SEQ ID NO:19); 5’-DCL-XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAG-3’ (SEQ ID NO:19); and 5’-UAMC1110-XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAG-3’ (SEQ ID NO:19), wherein X is SN38 and wherein Y is Irinotecan. 4WJ-C with SN38 and Irinotecan and derivatives

[0053] 5’-YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGG-3’ (SEQ ID NO:20); 5-YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGGGCCUUAGUAACGUGCUUUGAU GUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:21); 5’-YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGGGGGACCGAAAAAGACCUGACU UCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:22); 5’-YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGGGCGACUGGUUACCCGGUCG- 3’ (SEQ ID NO:23); 5’-YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGGCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:24); 5’-GalNAc-YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGG-3’ (SEQ ID NO: 20); 5’-FA-YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGG-3’ (SEQ ID NO: 20); 5’-DCL-YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGG-3’ (SEQ ID NO: 20); and 5’-UAMC1110-YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGG-3’ (SEQ ID NO: 20), wherein X is SN38 and wherein Y is Irinotecan. 4WJ-D with SN38 and Irinotecan and derivatives

[0054] 5’-YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAA-3’ (SEQ ID NO:25); 5’-YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAAGCCUUAGUAACGUGCUU UGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:26); 5’-YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAAGGGACCGAAAAAGACCU GACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:27); 5’-YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAAGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:28);TH Docket No.321502-2140 5’-YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAACAGAACGUAUACUAUUC UG-3’ (SEQ ID NO:29); 5’-GalNAc--YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAA-3’ (SEQ ID NO:25); 5’-FA-YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAA-3’ (SEQ ID NO:25); 5’-DCL-YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAA-3’ (SEQ ID NO:25); and 5’-UAMC1110-YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAA-3’ (SEQ ID NO:25), wherein X is SN38 and wherein Y is Irinotecan. 6WJ-A with SN38 and Irinotecan and derivatives

[0055] 5’-GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYG-3’ (SEQ ID NO:30); 5’-GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYGGCCUUAGUAACGUGCU UUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:31); 5’-GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYGGGGACCGAAAAAGACC UGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:32); 5’-GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYGGCGACUGGUUACCCGG UCG-3’ (SEQ ID NO:33); 5’-GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYGCAGAACGUAUACUAUU CUG-3’ (SEQ ID NO:34); 5’-GalNAc-GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYG-3’ (SEQ ID NO:30); 5’-FA-GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYG-3’ (SEQ ID NO:30); 5’-DCL-GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYG-3’ (SEQ ID NO:30); and 5’-UAMC1110-GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYG- 3’ (SEQ ID NO:30), wherein X is SN38 and wherein Y is Irinotecan. 6WJ-B with SN38 and Irinotecan and derivatives

[0056] 5’-YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYY-3’ (SEQ ID NO:35); 5’-YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYYGCCUUAGUAACGUGCU UUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:36); 5’-YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYYGGGACCGAAAAAGACC UGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:37); 5’-YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYYGCGACUGGUUACCCGG UCG-3’ (SEQ ID NO:38); 5’-YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYYCAGAACGUAUACUAUU CUG-3’ (SEQ ID NO:39);TH Docket No.321502-2140 5’-GalNAc-YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYY-3’ (SEQ ID NO:35); 5’-FA-YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYY-3’ (SEQ ID NO:35); 5’-DCL-YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYY-3’ (SEQ ID NO:35); and 5’-UAMC1110-YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYY- 3’ (SEQ ID NO:35), wherein X is SN38 and wherein Y is Irinotecan. 6WJ-C with SN38 and Irinotecan and derivatives

[0057] 5’-GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYA-3’ (SEQ ID NO:40); 5’-GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYAGCCUUAGUAACGUGCU UUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:41); 5’-GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYAGGGACCGAAAAAGACC UGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:42); 5’-GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYAGCGACUGGUUACCCGG UCG-3’ (SEQ ID NO:43); 5’-GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYACAGAACGUAUACUAUUC UG-3’ (SEQ ID NO:44); 5’-GalNAc-GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYA-3’ (SEQ ID NO:40); 5’-FA-GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYA-3’ (SEQ ID NO:40); 5’-DCL-GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYA-3’ (SEQ ID NO:40); and 5’-UAMC1110-GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYA- 3’ (SEQ ID NO:40), wherein X is SN38 and wherein Y is Irinotecan. 6WJ-D with SN38 and Irinotecan and derivatives

[0058] 5’-XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXY-3’ (SEQ ID NO:45); 5’-XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXYGCCUUAGUAACGUGCUU UGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:46); 5’-XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXYGGGACCGAAAAAGACCU GACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:47); 5’-XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXYGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:48); 5’-XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXYCAGAACGUAUACUAUUC UG-3’ (SEQ ID NO:49); 5’-GalNAc-XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXY-3’ (SEQ ID NO:45); 5’-FA-XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXY-3’ (SEQ IDTH Docket No.321502-2140 NO:45); 5’-DCL-XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXY-3’ (SEQ ID NO:45); or 5’-UAMC1110-XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXY-3’ (SEQ ID NO:45), wherein X is SN38 and wherein Y is Irinotecan. 3WJ-A with only Irinotecan and derivatives

[0059] 5’-UUGYYAUGUGUAUGUGGG-3’ (SEQ ID NO:50); 5’-UUGYYAUGUGUAUGUGGGGGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAG GC-3’ (SEQ ID NO:51); 5’-UUGYYAUGUGUAUGUGGGGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGU UUCCC-3’ (SEQ ID NO:52); 5’-UUGYYAUGUGUAUGUGGGGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:53); 5’-UUGYYAUGUGUAUGUGGGCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:54); 5’-GalNAc-UUGYYAUGUGUAUGUGGG-3’ (SEQ ID NO:50); 5’-FA- UUGYYAUGUGUAUGUGGG-3’ (SEQ ID NO:50); 5’-DCL-UUGYYAUGUGUAUGUGGG-3’ (SEQ ID NO:50); and 5’-UAMC1110-UUGYYAUGUGUAUGUGGG-3’ (SEQ ID NO:50), wherein Y is Irinotecan. 3WJ-B with only Irinotecan and derivatives

[0060] 5’-YYYAYAUAYUUUGUUGAUYY-3’ (SEQ ID NO:55); 5’-YYYAYAUAYUUUGUUGAUYYGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAG GC-3’ (SEQ ID NO:56); 5’-YYYAYAUAYUUUGUUGAUYYGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACG UUUCCC-3’ (SEQ ID NO:57); 5’-YYYAYAUAYUUUGUUGAUYYGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:58); 5’-YYYAYAUAYUUUGUUGAUYYCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:59); 5’-GalNAc-YYYAYAUAYUUUGUUGAUYY-3’ (SEQ ID NO:55); 5’-FA-YYYAYAUAYUUUGUUGAUYY-3’ (SEQ ID NO:55); 5’-DCL-YYYAYAUAYUUUGUUGAUYY-3’ (SEQ ID NO:55); and 5’-UAMC1110-YYYAYAUAYUUUGUUGAUYY-3’ (SEQ ID NO:55) , wherein Y is Irinotecan. 3WJ-C with only Irinotecan and derivatives

[0061] 5’-GGAUYAAUYAUGGYAA-3’ (SEQ ID NO:60); 5’-GGAUYAAUYAUGGYAAGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:61); 5’-GGAUYAAUYAUGGYAAGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUUUC CC-3’ (SEQ ID NO:62); 5’-GGAUYAAUYAUGGYAAGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:63); 5’-GGAUYAAUYAUGGYAACAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:64); 5’-GalNAc-GGAUYAAUYAUGGYAA-3’ (SEQ ID NO: 60); 5’-FA-GGAUYAAUYAUGGYAA-3’TH Docket No.321502-2140 (SEQ ID NO: 60); 5’-DCL-GGAUYAAUYAUGGYAA-3’ (SEQ ID NO: 60); and 5’-UAMC1110-GGAUYAAUYAUGGYAA-3’ (SEQ ID NO: 60) , wherein Y is Irinotecan. 4WJ-A with only Irinotecan and derivatives

[0062] 5’-UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAA-3’ (SEQ ID NO:65); 5’-UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAAGCCUUAGUAACGUGCUUU GAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:66); 5’-UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAAGGGACCGAAAAAGACCUG ACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:67); 5’-UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAAGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:68); 5’-UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAACAGAACGUAUACUAUUCU G-3’ (SEQ ID NO:69); 5’-GalNAc-UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAA-3’ (SEQ ID NO: 65); 5’-FA-UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAA-3’ (SEQ ID NO: 65); 5’-DCL-UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAA-3’ (SEQ ID NO: 65); and 5’-UAMC1110-UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAA-3’ (SEQ ID NO: 65) , wherein Y is Irinotecan. 4WJ-B with only Irinotecan and derivatives

[0063] 5’-UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAG-3’ (SEQ ID NO:70); 5’-UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAGGCCUUAGUAACGUGCU UUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:71); 5’-UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAGGGGACCGAAAAAGACC UGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:72); 5’-UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAGGCGACUGGUUACCCGG UCG-3’ (SEQ ID NO:73); 5’-UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAGCAGAACGUAUACUAUU CUG-3’ (SEQ ID NO:74); 5’-GalNAc-UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAG-3’ (SEQ ID NO:70); 5’-FA-UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAG-3’ (SEQ ID NO:70); 5’-DCL-UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAG-3’ (SEQ ID NO:70); and 5’-UAMC1110-UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAG- 3’ (SEQ ID NO:70) , wherein Y is Irinotecan.TH Docket No.321502-2140 4WJ-C with only Irinotecan and derivatives

[0064] 5’-YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGG-3’ (SEQ ID NO:75); 5’-YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGGGCCUUAGUAACGUGCUUUGA UGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:76); 5’-YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGGGGGACCGAAAAAGACCUGAC UUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:77); 5’-YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGGGCGACUGGUUACCCGGUCG- 3’ (SEQ ID NO:78); 5’-YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGGCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:79); 5’-GalNAc-YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGG-3’ (SEQ ID NO:75); 5’-FA-YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGG-3’ (SEQ ID NO:75); 5’-DCL-YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGG-3’ (SEQ ID NO:75); and 5’-UAMC1110-YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGG-3’ (SEQ ID NO:75) , wherein Y is Irinotecan. 4WJ-D with only Irinotecan and derivatives

[0065] 5’-YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAA-3’ (SEQ ID NO:80); 5’-YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAAGCCUUAGUAACGUGCU UUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:81); 5’-YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAAGGGACCGAAAAAGACC UGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:82); 5’-YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAAGCGACUGGUUACCCGG UCG-3’ (SEQ ID NO:83); 5’-YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAACAGAACGUAUACUAUU CUG-3’ (SEQ ID NO:84); 5’-GalNAc-YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAA-3’ (SEQ ID NO:80); 5’-FA-YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAA-3’ (SEQ ID NO:80); 5’-DCL-YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAA-3’ (SEQ ID NO:80); 5’-UAMC1110-YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAA-3’ (SEQ ID NO:80) , wherein Y is Irinotecan. 6WJ-A with only Irinotecan and derivatives

[0066] 5’-GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYG-3’ (SEQ ID NO:85);TH Docket No.321502-2140 5’-GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYGGCCUUAGUAACGUGC UUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:86); 5’-GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYGGGGACCGAAAAAGAC CUGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:87); 5’-GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYGGCGACUGGUUACCCG GUCG-3’ (SEQ ID NO:88); 5’-GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYGCAGAACGUAUACUAU UCUG-3’ (SEQ ID NO:89); 5’-GalNAc-GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYG-3’ (SEQ ID NO:85); 5’-FA-GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYG-3’ (SEQ ID NO:85); 5’-DCL-GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYG-3’ (SEQ ID NO:85); and 5’-UAMC1110-GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYG-3’ (SEQ ID NO:85), wherein Y is Irinotecan. 6WJ-B with only Irinotecan and derivatives

[0067] 5’-YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYY-3’ (SEQ ID NO:90); 5’-YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYYGCCUUAGUAACGUGC UUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:91); 5’-YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYYGGGACCGAAAAAGAC CUGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:92); 5’-YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYYGCGACUGGUUACCCG GUCG-3’ (SEQ ID NO:93); 5’-YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYYCAGAACGUAUACUAUU CUG-3’ (SEQ ID NO:94); 5’-GalNAc-YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYY-3 (SEQ ID NO:90); 5’-FA-YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYY-3’ (SEQ ID NO:90); 5’-DCL-YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYY-3’ (SEQ ID NO:90); and 5’-UAMC1110-YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYY-3’ (SEQ ID NO:90) , wherein Y is Irinotecan. 6WJ-C with only Irinotecan and derivatives

[0068] 5’-GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYA-3’ (SEQ ID NO:95);TH Docket No.321502-2140 5’-GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYAGCCUUAGUAACGUGCU UUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:96); 5’-GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYAGGGACCGAAAAAGACC UGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:97); 5’-GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYAGCGACUGGUUACCCG GUCG-3’ (SEQ ID NO:98); 5’-GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYACAGAACGUAUACUAUU CUG-3’ (SEQ ID NO:99); 5’-GalNAc-GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYA-3’ (SEQ ID NO:95); 5’-FA-GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYA-3’ (SEQ ID NO:95); 5’-DCL-GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYA-3’ (SEQ ID NO:95); and 5’ UAMC1110 GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYA- 3’ (SEQ ID NO:95) , wherein Y is Irinotecan. 6WJ-D with only Irinotecan and derivatives

[0069] 5’-UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUY-3’ (SEQ ID NO:100); 5’-UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUYGCCUUAGUAACGUGCU UUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:101); 5’-UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUYGGGACCGAAAAAGACC UGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:102); 5’-UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUYGCGACUGGUUACCCGG UCG-3’ (SEQ ID NO:103); 5’-UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUYCAGAACGUAUACUAUU CUG-3’ (SEQ ID NO:104); 5’-GalNAc-UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUY-3’ (SEQ ID NO:100); 5’-FA-UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUY-3’ (SEQ ID NO:100); 5’-DCL-UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUY-3’ (SEQ ID NO:100); and 5’-UAMC1110-UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUY-3’ (SEQ ID NO:100), wherein Y is Irinotecan.TH Docket No.321502-2140

[0070] In some embodiments, the disclosed RNA oligonucleotide has one of the following sequences: 3WJ-A with SN38 only and derivatives

[0071] 5’-XXGCCAXGXGXAXGXGGG-3’ (SEQ ID NO:105); 5’-XXGCCAXGXGXAXGXGGGGGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGG C-3’ (SEQ ID NO:106); 5’-XXGCCAXGXGXAXGXGGGGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGU UUCCC-3’ (SEQ ID NO:107); 5’-XXGCCAXGXGXAXGXGGGGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:108); 5’-XXGCCAXGXGXAXGXGGGCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:109); 5’-GalNAc-XXGCCAXGXGXAXGXGGG-3’ (SEQ ID NO:105); 5’-FA-XXGCCAXGXGXAXGXGGG-3’ (SEQ ID NO:105); 5’-DCL-XXGCCAXGXGXAXGXGGG- 3’ (SEQ ID NO:105); and 5’-UAMC1110-XXGCCAXGXGXAXGXGGG-3’ (SEQ ID NO:105), wherein X is SN38. 3WJ-B with SN38 only and derivatives

[0072] 5’-CCCACAXACXXXGXXGAXCC-3’ (SEQ ID NO:110); 5’-CCCACAXACXXXGXXGAXCCGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAG GC-3’ (SEQ ID NO:111); 5’-CCCACAXACXXXGXXGAXCCGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACG UUUCCC-3’ (SEQ ID NO:112); 5’-CCCACAXACXXXGXXGAXCCGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:113); 5’-CCCACAXACXXXGXXGAXCCCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:114); 5’-GalNAc-CCCACAXACXXXGXXGAXCC-3’ (SEQ ID NO:110); 5’-FA-CCCACAXACXXXGXXGAXCC-3’ (SEQ ID NO:110); 5’-DCL-CCCACAXACXXXGXXGAXCC-3’ (SEQ ID NO:110); and 5’-UAMC1110-CCCACAXACXXXGXXGAXCC-3’ (SEQ ID NO:110) , wherein X is SN38. 3WJ-C with SN38 only and derivatives

[0073] 5’-GGAXCAAXCAXGGCAA-3’ (SEQ ID NO:115); 5’-GGAXCAAXCAXGGCAAGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:116); 5’-GGAXCAAXCAXGGCAAGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUUUC CC-3’ (SEQ ID NO:117); 5’-GGAXCAAXCAXGGCAAGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:118); 5’-GGAXCAAXCAXGGCAACAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:119); 5’-GalNAc-GGAXCAAXCAXGGCAA-3’ (SEQ ID NO:115); 5’-FA-GGAXCAAXCAXGGCAA-3’TH Docket No.321502-2140 (SEQ ID NO:115); 5’-DCL-GGAXCAAXCAXGGCAA-3’ (SEQ ID NO:115); 5’-UAMC1110- GGAXCAAXCAXGGCAA-3’ (SEQ ID NO:115) , wherein X is SN38. 4WJ-A with SN38 only and derivatives

[0074] 5’-XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAA-3’ (SEQ ID NO:120); 5’-XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAAGCCUUAGUAACGUGCUUU GAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:121); 5’-XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAAGGGACCGAAAAAGACCUG ACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:122); 5’-XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAAGCGACUGGUUACCCGGUC G-3’ (SEQ ID NO:123); 5’-XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAACAGAACGUAUACUAUUCU G-3’ (SEQ ID NO:124); 5’-GalNAc-XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAA-3’ (SEQ ID NO:120); 5’-FA-XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAA-3’ (SEQ ID NO:120); 5’-DCL-XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAA-3’ (SEQ ID NO:120); and 5’-UAMC1110-XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAA-3’ (SEQ ID NO:120) , wherein X is SN38. 4WJ-B with SN38 only and derivatives

[0075] 5’-XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAG-3’ (SEQ ID NO:125); 5’-XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAGGCCUUAGUAACGUGCU UUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:126); 5’-XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAGGGGACCGAAAAAGACC UGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:127); 5’-XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAGGCGACUGGUUACCCGG UCG-3’ (SEQ ID NO:128); 5’-XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAGCAGAACGUAUACUAUUC UG-3’ (SEQ ID NO:129); 5’-GalNAc-XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAG-3’ (SEQ ID NO:125); 5’-FA-XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAG-3’ (SEQ ID NO:125); 5’-DCL-XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAG-3’ (SEQ ID NO:125); andTH Docket No.321502-2140 5’-UAMC1110-XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAG-3’ (SEQ ID NO:125) , wherein X is SN38. 4WJ-C with SN38 only and derivatives

[0076] 5’-CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGG-3’ (SEQ ID NO:130); 5’-CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGGGCCUUAGUAACGUGCUUUG AUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:131); 5’-CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGGGGGACCGAAAAAGACCUGA CUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:132); 5’-CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGGGCGACUGGUUACCCGGUCG -3’ (SEQ ID NO:133); 5’-CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGGCAGAACGUAUACUAUUCUG- 3’ (SEQ ID NO:134); 5’-GalNAc-CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGG-3’ (SEQ ID NO:130); 5’-FA-CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGG-3’ (SEQ ID NO:130); 5’-DCL-CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGG-3’ (SEQ ID NO:130); and 5’-UAMC1110-CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGG-3’ (SEQ ID NO:130) , wherein X is SN38. 4WJ-D with SN38 only and derivatives

[0077] 5’-CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAA-3’ (SEQ ID NO:135); 5’-CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAAGCCUUAGUAACGUGCU UUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:136); 5’-CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAAGGGACCGAAAAAGACC UGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:137); 5’-CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAAGCGACUGGUUACCCGG UCG-3’ (SEQ ID NO:138); 5’-CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAACAGAACGUAUACUAUUC UG-3’ (SEQ ID NO:139); 5’-GalNAc-CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAA-3’ (SEQ ID NO:135); 5’-FA-CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAA-3’ (SEQ ID NO:135); 5’-DCL-CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAA-3’ (SEQ ID NO:135); and 5’-UAMC1110-CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAA-3’ (SEQ ID NO:135) , wherein X is SN38.TH Docket No.321502-2140 6WJ-A with SN38 only and derivatives

[0078] 5’-GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCG-3’ (SEQ ID NO:140); 5’-GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCGGCCUUAGUAACGUGC UUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:141); 5’-GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCGGGGACCGAAAAAGACC UGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:142); 5’-GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCGGCGACUGGUUACCCG GUCG-3’ (SEQ ID NO:143); 5’-GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCGCAGAACGUAUACUAUU CUG-3’ (SEQ ID NO:144); 5’-GalNAc-GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCG-3’ (SEQ ID NO:140); 5’-FA-GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCG-3’ (SEQ ID NO:140); 5’-DCL-GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCG-3’ (SEQ ID NO:140); 5’-UAMC1110-GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCG- 3’ (SEQ ID NO:140), wherein X is SN38. 6WJ-B with SN38 only and derivatives

[0079] 5’-CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCC-3’ (SEQ ID NO:145); 5’-CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCCGCCUUAGUAACGUGC UUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:146); 5’-CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCCGGGACCGAAAAAGACC UGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:147); 5’-CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCCGCGACUGGUUACCCG GUCG-3’ (SEQ ID NO:148); 5’-CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCCCAGAACGUAUACUAUU CUG-3’ (SEQ ID NO:149); 5’-GalNAc-CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCC-3’ (SEQ ID NO:145); 5’-FA-CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCC-3’ (SEQ ID NO:145); 5’-DCL-CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCC-3’ (SEQ ID NO:145); and 5’-UAMC1110-CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCC-3’ (SEQ ID NO:145), wherein X is SN38.TH Docket No.321502-2140 6WJ-C with SN38 only and derivatives

[0080] 5’-GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACA-3’ (SEQ ID NO:150); 5’-GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACAGCCUUAGUAACGUGC UUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:151); 5’-GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACAGGGACCGAAAAAGACC UGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:152); 5’-GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACAGCGACUGGUUACCCG GUCG-3’ (SEQ ID NO:153); 5’-GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACACAGAACGUAUACUAUU CUG-3’ (SEQ ID NO:154); 5’-GalNAc-GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACA-3’ (SEQ ID NO:150); 5’-FA-GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACA-3’ (SEQ ID NO:150); 5’-DCL-GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACA-3’ (SEQ ID NO:150); and 5’-UAMC1110-GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACA-3’ (SEQ ID NO:150), wherein X is SN38. 6WJ-D with SN38 only and derivatives

[0081] 5’-XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXC-3’ (SEQ ID NO:155); 5’-XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXCGCCUUAGUAACGUGCU UUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:156); 5’-XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXCGGGACCGAAAAAGACC UGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:157); 5’-XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXCGCGACUGGUUACCCG GUCG-3’ (SEQ ID NO:158); 5’-XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXCCAGAACGUAUACUAUU CUG-3’ (SEQ ID NO:159); 5’-GalNAc-XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXC-3’ (SEQ ID NO:155); 5’-FA-XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXC-3’ (SEQ ID NO:155); 5’-DCL-XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXC-3’ (SEQ ID NO:155); or 5’-UAMC1110-XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXC-3’ (SEQ ID NO:155), wherein X is SN38.TH Docket No.321502-2140

[0082] In some embodiments, the disclosed RNA oligonucleotide has one of the following unmodified sequences: 3WJ-A

[0083] 5’-UUGCCAUGUGUAUGUGGG-3’ (SEQ ID NO:160); 5’-UUGCCAUGUGUAUGUGGGGGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAG GC-3’ (SEQ ID NO:161); 5’-UUGCCAUGUGUAUGUGGGGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGU UUCCC-3’ (SEQ ID NO:162); 5’-UUGCCUGUGUAUGUGGGGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:163); 5’-UUGCCAUGUGUAUGUGGGCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:164); 5’-GalNAc UUGCCAUGUGUAUGUGGG-3’ (SEQ ID NO:160); 5’-FA UUGCCAUGUGUAUGUGGG-3’ (SEQ ID NO:160); 5’-DCL UUGCCAUGUGUAUGUGGG-3’ (SEQ ID NO:160); and 5’-UAMC1110 UUGCCAUGUGUAUGUGGG-3’ (SEQ ID NO:160); 3WJ-B

[0084] 5’-CCCACAUACUUUGUUGAUCC-3’ (SEQ ID NO:165); 5’-CCCACAUACUUUGUUGAUCCGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAG GC-3’ (SEQ ID NO:166); 5’-CCCACAUACUUUGUUGAUCCGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUAC GUUUCCC-3’ (SEQ ID NO:167); 5’-CCCACAUACUUUGUUGAUCCGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:168); 5’-CCCACAUACUUUGUUGAUCCCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:169); 5’-GalNAc CCCACAUACUUUGUUGAUCC-3’ (SEQ ID NO:165); 5’-FA-CCCACAUACUUUGUUGAUCC-3’ (SEQ ID NO:165); 5’-DCL-CCCACAUACUUUGUUGAUCC-3’ (SEQ ID NO:165); and 5’-UAMC1110-CCCACAUACUUUGUUGAUCC-3’ (SEQ ID NO:165). 3WJ-C

[0085] 5’-GGAUCAAUCAUGGCAA-3’ (SEQ ID NO:170); 5’-GGAUCAAUCAUGGCAAGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:171); 5’-GGAUCAAUCAUGGCAAGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUUU CCC-3’ (SEQ ID NO:172); 5’-GGAUCAAUCAUGGCAAGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:173); 5’-GGAUCAAUCAUGGCAACAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:174); 5’-GalNAc-GGAUCAAUCAUGGCAA-3’ (SEQ ID NO:170); 5’-FA-GGAUCAAUCAUGGCAA-3’ (SEQ ID NO:170); 5’-DCL-GGAUCAAUCAUGGCAA-3’ (SEQ ID NO:170); and 5’-UAMC1110-GGAUCAAUCAUGGCAA-3’ (SEQ ID NO:170);TH Docket No.321502-2140 4WJ-A

[0086] 5’-UUAGGUAAAGCCACCUGCAGGUGCUACCGAUGUAAUUCAA-3’ (SEQ ID NO:175); 5’-UUAGGUAAAGCCACCUGCAGGUGCUACCGAUGUAAUUCAAGCCUUAGUAACGUGCUU UGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:176); 5’-UUAGGUAAAGCCACCUGCAGGUGCUACCGAUGUAAUUCAAGGGACCGAAAAAGACCU GACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:177); 5’-UUAGGUAAAGCCACCUGCAGGUGCUACCGAUGUAAUUCAAGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:178); 5’-UUAGGUAAAGCCACCUGCAGGUGCUACCGAUGUAAUUCAACAGAACGUAUACUAUUC UG-3’ (SEQ ID NO:179); 5’-GalNAc-UUAGGUAAAGCCACCUGCAGGUGCUACCGAUGUAAUUCAA-3’ (SEQ ID NO:175); 5’-FA-UUAGGUAAAGCCACCUGCAGGUGCUACCGAUGUAAUUCAA-3’ (SEQ ID NO:175); 5’-DCL-UUAGGUAAAGCCACCUGCAGGUGCUACCGAUGUAAUUCAA-3’ (SEQ ID NO:175); and 5’-UAMC1110-UUAGGUAAAGCCACCUGCAGGUGCUACCGAUGUAAUUCAA-3’ (SEQ ID NO:175); 4WJ-B

[0087] 5’-UUGAAUUACAUCGGUAGCACGGGCUGUGCGAGGCUGAACAG-3’ (SEQ ID NO:180); 5’-UUGAAUUACAUCGGUAGCACGGGCUGUGCGAGGCUGAACAGGCCUUAGUAACGUGC UUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:181); 5’-UUGAAUUACAUCGGUAGCACGGGCUGUGCGAGGCUGAACAGGGGACCGAAAAAGACC UGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:182); 5’-UUGAAUUACAUCGGUAGCACGGGCUGUGCGAGGCUGAACAGGCGACUGGUUACCCG GUCG-3’ (SEQ ID NO:183); 5’-UUGAAUUACAUCGGUAGCACGGGCUGUGCGAGGCUGAACAGCAGAACGUAUACUAUU CUG-3’ (SEQ ID NO:184); 5’-GalNAc-UUGAAUUACAUCGGUAGCACGGGCUGUGCGAGGCUGAACAG-3’ (SEQ ID NO:180); 5’-FA-UUGAAUUACAUCGGUAGCACGGGCUGUGCGAGGCUGAACAG-3’ (SEQ ID NO:180); 5’-DCL-UUGAAUUACAUCGGUAGCACGGGCUGUGCGAGGCUGAACAG 3’ (SEQ ID NO:180); and 5’-UAMC1110-UUGAAUUACAUCGGUAGCACGGGCUGUGCGAGGCUGAACAG-3’ (SEQ ID NO:180).TH Docket No.321502-2140 4WJ-C

[0088] 5’-CUGUUCAGCCUCGCACAGCCAGCACGCACCUGAAUAGG-3’ (SEQ ID NO:185); 5’-CUGUUCAGCCUCGCACAGCCAGCACGCACCUGAAUAGGGCCUUAGUAACGUGCUUUG AUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:186); 5’-CUGUUCAGCCUCGCACAGCCAGCACGCACCUGAAUAGGGGGACCGAAAAAGACCUGA CUUCUAUACUAAGUCUACGUUUCCC 3’ (SEQ ID NO:187); 5’-CUGUUCAGCCUCGCACAGCCAGCACGCACCUGAAUAGGGCGACUGGUUACCCGGUC G-3’ (SEQ ID NO:188); 5’-CUGUUCAGCCUCGCACAGCCAGCACGCACCUGAAUAGGCAGAACGUAUACUAUUCUG- 3’ (SEQ ID NO:189); 5’-GalNAc-CUGUUCAGCCUCGCACAGCCAGCACGCACCUGAAUAGG-3’ (SEQ ID NO:185); 5’-FA-CUGUUCAGCCUCGCACAGCCAGCACGCACCUGAAUAGG 3’ (SEQ ID NO:185); 5’-DCL-CUGUUCAGCCUCGCACAGCCAGCACGCACCUGAAUAGG-3’ (SEQ ID NO:185); and 5’-UAMC1110 CUGUUCAGCCUCGCACAGCCAGCACGCACCUGAAUAGG-3’ (SEQ ID NO:185). 4WJ-D

[0089] 5’-CCUAUUCAGGUGCGUGCUGGGCUGCAGGUGGCUUUACCUAA-3’ (SEQ ID NO:190); 5’-CCUAUUCAGGUGCGUGCUGGGCUGCAGGUGGCUUUACCUAAGCCUUAGUAACGUGC UUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:191); 5’-CCUAUUCAGGUGCGUGCUGGGCUGCAGGUGGCUUUACCUAAGGGACCGAAAAAGAC CUGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:192); 5’-CCUAUUCAGGUGCGUGCUGGGCUGCAGGUGGCUUUACCUAAGCGACUGGUUACCCG GUCG-3’ (SEQ ID NO:193); 5’-CCUAUUCAGGUGCGUGCUGGGCUGCAGGUGGCUUUACCUAACAGAACGUAUACUAUU CUG-3’ (SEQ ID NO:194); 5’-GalNAc-CCUAUUCAGGUGCGUGCUGGGCUGCAGGUGGCUUUACCUAA-3’ (SEQ ID NO:190); 5’-FA-CCUAUUCAGGUGCGUGCUGGGCUGCAGGUGGCUUUACCUAA-3’ (SEQ ID NO:190); 5’-DCL-CCUAUUCAGGUGCGUGCUGGGCUGCAGGUGGCUUUACCUAA-3’ (SEQ ID NO:190); and 5’-UAMC1110-CCUAUUCAGGUGCGUGCUGGGCUGCAGGUGGCUUUACCUAA-3’ (SEQ ID NO:190).TH Docket No.321502-2140 6WJ-A

[0090] 5’-GAGUAUAUGUUAGGCCUGGGUGAGUCCUUGCGUCUUCUACCG-3’ (SEQ ID NO:195); 5’-GAGUAUAUGUUAGGCCUGGGUGAGUCCUUGCGUCUUCUACCGGCCUUAGUAACGUG CUUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:196); 5’-GAGUAUAUGUUAGGCCUGGGUGAGUCCUUGCGUCUUCUACCGGGGACCGAAAAAGA CCUGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:197); 5’-GAGUAUAUGUUAGGCCUGGGUGAGUCCUUGCGUCUUCUACCGGCGACUGGUUACCC GGUCG 3’ (SEQ ID NO:198); 5’-GAGUAUAUGUUAGGCCUGGGUGAGUCCUUGCGUCUUCUACCGCAGAACGUAUACUAU UCUG-3’ (SEQ ID NO:199); 5’-GalNAc-GAGUAUAUGUUAGGCCUGGGUGAGUCCUUGCGUCUUCUACCG-3’ (SEQ ID NO:195); 5’-FA-GAGUAUAUGUUAGGCCUGGGUGAGUCCUUGCGUCUUCUACCG-3’ (SEQ ID NO:195); 5’-DCL GAGUAUAUGUUAGGCCUGGGUGAGUCCUUGCGUCUUCUACCG-3’ (SEQ ID NO:195); and ID6WJ-B

[0091] 5’-CGGUAGAAGACGCAAGGACUUGCUAGUUGUGGUACUGUUCCC-3’ (SEQ ID NO:200); 5’-CGGUAGAAGACGCAAGGACUUGCUAGUUGUGGUACUGUUCCCGCCUUAGUAACGUG CUUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:201); 5’-CGGUAGAAGACGCAAGGACUUGCUAGUUGUGGUACUGUUCCCGGGACCGAAAAAGAC CUGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:202); 5’-CGGUAGAAGACGCAAGGACUUGCUAGUUGUGGUACUGUUCCCGCGACUGGUUACCC GGUCG-3’ (SEQ ID NO:203); 5’-CGGUAGAAGACGCAAGGACUUGCUAGUUGUGGUACUGUUCCCCAGAACGUAUACUAU UCUG-3’ (SEQ ID NO:204); 5’-GalNAc-CGGUAGAAGACGCAAGGACUUGCUAGUUGUGGUACUGUUCCC-3’ (SEQ ID NO:200); 5’-FA-CGGUAGAAGACGCAAGGACUUGCUAGUUGUGGUACUGUUCCC-3’ (SEQ ID NO:200); 5’-DCL-CGGUAGAAGACGCAAGGACUUGCUAGUUGUGGUACUGUUCCC-3’ (SEQ ID NO:200); and 5’-UAMC1110-CGGUAGAAGACGCAAGGACUUGCUAGUUGUGGUACUGUUCCC 3’ (SEQ ID NO:200).TH Docket No.321502-2140 6WJ-C

[0092] 5’-GGGAACAGUACCACAACUAGUGUCCCGGGAUAGGGACAUACA-3’ (SEQ ID NO:205); 5-GGGAACAGUACCACAACUAGUGUCCCGGGAUAGGGACAUACAGCCUUAGUAACGUGC UUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:206); 5’-GGGAACAGUACCACAACUAGUGUCCCGGGAUAGGGACAUACAGGGACCGAAAAAGAC CUGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:207); 5’-GGGAACAGUACCACAACUAGUGUCCCGGGAUAGGGACAUACAGCGACUGGUUACCCG GUCG-3’ (SEQ ID NO:208); 5’-GGGAACAGUACCACAACUAGUGUCCCGGGAUAGGGACAUACACAGAACGUAUACUAU UCUG-3’ (SEQ ID NO:209); 5’-GalNAc-GGGAACAGUACCACAACUAGUGUCCCGGGAUAGGGACAUACA-3’ (SEQ ID NO:205); 5’-FA-GGGAACAGUACCACAACUAGUGUCCCGGGAUAGGGACAUACA-3’ (SEQ ID NO:205); 5’-DCL-GGGAACAGUACCACAACUAGUGUCCCGGGAUAGGGACAUACA-3’ (SEQ ID NO:205); and 5’-UAMC1110-GGGAACAGUACCACAACUAGUGUCCCGGGAUAGGGACAUACA 3’ (SEQ ID NO:205). 6WJ-D

[0093] 5’-UGUAUGUCCCUAUCCCGGGAUGCCCAGGCCUAACAUAUACUC-3’ (SEQ ID NO:210); 5’-UGUAUGUCCCUAUCCCGGGAUGCCCAGGCCUAACAUAUACUCGCCUUAGUAACGUGC UUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:211); 5’-UGUAUGUCCCUAUCCCGGGAUGCCCAGGCCUAACAUAUACUCGGGACCGAAAAAGAC CUGACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:212); 5’-UGUAUGUCCCUAUCCCGGGAUGCCCAGGCCUAACAUAUACUCGCGACUGGUUACCCG GUCG-3’ (SEQ ID NO:213); 5’-UGUAUGUCCCUAUCCCGGGAUGCCCAGGCCUAACAUAUACUCCAGAACGUAUACUAU UCUG-3’ (SEQ ID NO:214); 5’-GalNAc-UGUAUGUCCCUAUCCCGGGAUGCCCAGGCCUAACAUAUACUC 3’ (SEQ ID NO:210); 5’-FA-UGUAUGUCCCUAUCCCGGGAUGCCCAGGCCUAACAUAUACUC 3’ (SEQ ID NO:210); 5’-DCL-UGUAUGUCCCUAUCCCGGGAUGCCCAGGCCUAACAUAUACUC 3’ (SEQ ID NO:210); and 5’-UAMC1110-UGUAUGUCCCUAUCCCGGGAUGCCCAGGCCUAACAUAUACUC 3’ (SEQ ID NO:210).TH Docket No.321502-2140 RNA Nanostructures

[0094] Described herein are RNA nanostructures that can be composed of one or more synthetic RNA oligonucleotides that are designed (or configured) to self-assemble into the RNA nanostructures. When assembled, the RNA nanostructures can be composed of double- stranded arms (DAs) that can be arranged around a core domain.

[0095] The modular RNA nanostructures can be composed of 3, 4, 5, 6, 7, 8, 9 or more synthetic single-stranded RNA oligonucleotides that can self-assemble into the RNA nanostructures via hybridization. Each synthetic single-stranded RNA oligonucleotide can be about 16 to about 120 bases in length. The exact sequence of each synthetic single-stranded RNA oligonucleotide in each modular RNA nanostructures can be designed such that they achieve specific physical characteristics when assembled with 2 or more other synthetic single- stranded RNA oligonucleotides. The RNA nanostructures can be composed of 3, 4, 5, 6, 7, 8, 9, or more synthetic RNA oligonucleotides. The synthetic RNA oligonucleotides can be designed such that they form highly ordered 2-D and / or 3-D structures upon self-assembly. The RNA nanostructures can have 3, 4, 5, 6, 7, 8, 9, or more double-stranded arms (DAs) that stem off of a core domain. The DAs can be symmetrically or asymmetrically arranged around the core domain.

[0096] The core domain can have 0-4 symmetric or asymmetric bulge nucleotides separating individual DAs, which can allow for optimization of thermodynamic stability, steric constraints, and / or structural arrangements of individual loops. Changes in duplex sequence (the DAs) and number unpaired core nucleotides can both affect thermodynamic stability of the RNA nanostructures. Thus the physical properties and functional characteristics can be optimized by altering the sequence of the synthetic RNA oligonucleotides that form the RNA nanostructures.

[0097] The melting temperature (Tm) of the RNA nanostructure can be about 65 °C or more. In some aspects, the melting temperature of the RNA nanostructure can be greater than 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 °C. In some aspects, the Tm of the RNA nanostructure can be 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 °C.

[0098] The synthetic RNA oligonucleotides can be single-stranded. Each individual synthetic RNA oligonucleotide can be composed of 16-120 nucleotides. The nucleotides can be native ribonucleotide or can be modified. In some aspects, the synthetic RNA oligonucleotide(s) can be 2’ modified. The 2’ or other modification can be a 2’Fluoro-, 2’O-methyl-, LNA- or anyTH Docket No.321502-2140 other backbone, sugar, or base modified ribonucleotide or any combination of native, backbone, sugar, and base modified ribonucleotides. Modifications are futher discussed elsewhere herein. Each synthetic RNA oligonucleotide can be composed of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 nucleotides or any range therein. Each synthetic RNA oligonucleotide can be designed and configured such that it can self assemble with 2, 3, 4, 5, 6, 7, 8, or more other synthetic RNA oligonucleotides into a RNA nanostructure as described herein.

[0099] In addition to the SN38 or Irinotecan conjugated nucleotides described herein, the remaining nucleotides can be unmodified or modified nucleotides. The modifications can be 5’-terminal modifications and / or 3’-terminal modifications and / or 2’-internal sugar modifications and / or base-internal modifications. Typical 5’ terminal modifications include amino, carboxy, phosphate, thiol, maleimide, alkyne, cholesterol, aldehyde, carbon spacers, Peg-spacer, doubler, trebler, photocleavable amino, photocleavable spacer, fluorophores (e.g. Cyanine 3, 3.5, 5, 5.5, 7, Fluorescein, etc.), biotin, desthiobiotin, digoxigenin, quenchers (dabcyl, dabsyl, BlackHole, BBQ650, etc.) or other 5’ modifications known to an experienced user of the art. Typical 3’ terminal modifications include amino, carboxy, phosphate, thiol, alkyne, cholesterol, carbon spacers, Peg-spacer, fluorophores (e.g. Cyanine 3, 3.5, 5, 5.5, 7, Fluorescein, etc.), biotin, desthiobiotin, digoxigenin, quenchers (dabcyl, dabsyl, BlackHole, BBQ650, etc.) or other 3’ modifications known to an experienced user of the art. Typical internal modifications include amino-dA, amino-dC, amino-dT, carboxy-dT, 2’O-propargyl, 2’amino, 2’fluoro, 2’methoxy, 5- ethynyl-dU, C8-alkyne-dC, C8-alkyne-dT, carbon spacers, Peg-spacer, fluorophores (e.g. Cyanine 3, 3.5, 5, 5.5, 7, Fluorescein, etc.), biotin, desthiobiotin, digoxigenin, quenchers (dabcyl, dabsyl, BlackHole, BBQ650, etc.) or other 5’ modifications known to an experienced user of the art. The modification can be an alkyne group attached to a nucleotide. The modification can be a functional group attached to a nucleotide. One or more of the terminal (e.g. the 5’ and / or 3’ end) nucleotides can be modified in a synthetic RNA oligonucleotide.

[0100] The RNA nanostructure, when measured along its longest or largest dimension, can have a size of up to a micrometer. The size of the RNA nanostructure, when measured along its longest or largest dimension, can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 , 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,TH Docket No.321502-2140 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800 or about 900 nm. In some aspects, the size of the RNA nanostructure, when measured along its longest or largest dimension, can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 , 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 nm or any range of values therein. In some aspects, the size of the RNA nanostructure, when measured along its longest or largest dimension, can be about 1-30, 1-40, 1-50, 10-50, 10-40, 10-30, 30-50, 30-40, or 40-50 nm. In some aspects, the RNA nanostructure can be substantially spherical. In other aspects, the RNA nanostructure can be trigonal planar, trigonal pyramidal, T-shaped, tetrahedral, square planar, seesaw, trigonal bipyramidal, square pyramidal, pentagonal planar, octahedral, trigonal prismatic, pentagonal pyramidal, pentagonal bipyramidal, square antiprismatic, tricapped trigonal prismatic, capped square antiprismatic, arrowhead shaped, arrow tail shaped, X-shaped, or a distorted version of any of these shapes.

[0101] A principle for designing self-assembled nucleic acid nanostructures is that sequence complementarity in nucleic acid strands is encoded such that, by pairing up complementary segments, the nucleic acid strands self-organize into a predefined nanostructure under appropriate physical conditions. From this basic principle (see, e.g., Seeman N.C. J. Theor. Biol.99: 237, 1982, incorporated by reference herein), researchers have created diverse synthetic nucleic acid nanostructures (see, e.g., Seeman N.C. Nature 421: 427, 2003; Shih W.M. et al. Curr. Opin. Struct. Biol.20: 276, 2010, each of which is incorporated by reference herein). Examples of nucleic acid (e.g., DNA) nanostructures, and methods of producing such structures, that may be used in accordance with the present disclosure are known and include, without limitation, lattices (see, e.g., Winfree E. et al. Nature 394: 539, 1998; Yan H. et al. Science 301: 1882, 2003; Yan H. et al. Proc. Natl. Acad. ofSci. USA 100; 8103, 2003; Liu D. et al. J. Am. Chem. Soc.126: 2324, 2004; Rothemund P.W.K. et al. PLoS Biology 2: 2041, 2004, each of which is incorporated by reference herein), ribbons (see, e.g., Park S.H. et al. Nano Lett.5: 729, 2005; Yin P. et al. Science 321: 824, 2008, each of which is incorporated by reference herein), tubes (see, e.g., Yan H. Science, 2003; P. Yin, 2008, each of which is incorporated by reference herein), finite two-dimensional and three dimensional objects with defined shapes (see, e.g., Chen J. et al. Nature 350: 631, 1991; Rothemund P. W. K., Nature, 2006; He Y. et al. Nature 452: 198, 2008; Ke Y. et al. Nano. Lett.9: 2445, 2009; Douglas S. M. et al. Nature 459: 414, 2009; Dietz H. et al. Science 325: 725, 2009; Andersen E.TH Docket No.321502-2140 S. et al. Nature 459: 73, 2009; Liedl T. et al. Nature Nanotech.5: 520, 2010; Han D. et al. Science 332: 342, 2011, each of which is incorporated by reference herein), and macroscopic crystals (see, e.g., Meng J. P. et al. Nature 461: 74, 2009, incorporated by reference herein). The synthetic RNA oligonucleotides can be single- stranded nucleic acids, double- stranded nucleic acids, or a combination of single- stranded and double- stranded nucleic acids.

[0102] The RNA nanostructure components (synthetic RNA oligonucleotides and modular RNA motifs can be designed using a computer aided design methodology described herein. Although the computer design is demonstrated using specific RNA nanostructures, it will be appreciated that the principles taught therein will be able to be extrapolated to any desired RNA nanostrcutre by the skilled artisan.

[0103] Each synthetic RNA oligonucleotide can be designed such that when it is combined with 2 or more additional synthetic RNA oligonucleotides that base pairing occurs to produce a highy ordered 2-D and 3-D structure having 3 or more DAs surrounding a core domain that may or may not contain base-pairing between strands and can include 0-4 nucleotides that form symmetric or asymmetric bulges bweteen the DAs. Each synthetic RNA oligonucleotide can be designed to include 16-120 nucleotides. Each synthetic RNA oligonucleotide can be designed to include 1 or more modified nucleotides.

[0104] The rational design of the synthetic RNA oligonucleotides and / or RNA nanostructures described herein can be carried out in a computing environment. The computing environment can include one or more computing devices that can include at least one processor circuit, for example, that can have a processor and a memory. Various applications and / or other functionality may be executed in the compouting environment according to various aspects of the disclosure. Also, various data can be stored in one or more data stores that can be accessible to the computing environement.

[0105] The components executed on the computing environment, for example, can include a rational RNA design system and other applications, services, processes, systems, engines, or functionalities not discussed in detail herein. The rational RNA design system can be executed to facilitate the design of synthetic RNA oligonucleotides and / or RNA nanostructures as described herein. The rational RNA design system can also perform various back end functions that can be associated with the design of RNA oligonucleotides, such as those synthetic RNA oligonucleotides and / or RNA nanostructures described herein.

[0106] The synthetic RNA oligonucleotides can be synthesized using standard molecular biologic and biochemical techniques. In other words, the various nucleic acids thatTH Docket No.321502-2140 can form the RNA nanoparticles can be de novo synthesized as desired. Such synthesis techniques will be known to the skilled artisan.

[0107] Pharmaceutical Formulations

[0108] Also provided herein are pharmaceutical formulations that can include an amount of an RNA nanostructure described herein and a pharmaceutical carrier appropriate for administration to an individual in need thereof. The individual in need thereof can have or can be suspected of a cancer or other disease or disorder in need of treatment or prevention. In some embodiments, the subject in need thereof is in need of a diagnostic procedure, such as an imaging procedure. The pharmaceutical formulations can include an amount of an RNA nanostructure described herein that can be effective to treat or prevent a cancer.

[0109] Formulations can be administered via any suitable administration route. For example, the formulations (and / or compositions) can be administered to the subject in need thereof orally, intravenously, ocularly, intraocularly, intramuscularly, intravaginally, intraperitoneally, rectally, parenterally, topically, intranasally, or subcutaneously. Other suitable routes are described herein. In some embodiments, the RNA nanostructure contains an effective amount of a cargo molecule.

[0110] The RNA nanostructure can be formulated for parenteral delivery, such as injection or infusion, in the form of a solution or suspension. The formulation can be administered via any route, such as, the blood stream or directly to the organ or tissue to be treated.

[0111] Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

[0112] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and / or by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.TH Docket No.321502-2140

[0113] Solutions and dispersions of the RNA nanostructures as described herein can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, and combinations thereof.

[0114] Suitable surfactants can be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Suitable anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)- sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Suitable cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Suitable nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-β-alanine, sodium N- lauryl-β-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

[0115] The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation can also contain an antioxidant to prevent degradation of the RNA nanostructures.

[0116] The formulation can be buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

[0117] Water-soluble polymers can be used in the formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol. Sterile injectable solutions can be prepared by incorporating the RNA nanostructures in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Dispersions can be prepared by incorporating the various sterilized RNA nanostructures into a sterile vehicle which contains theTH Docket No.321502-2140 basic dispersion medium and the required other ingredients from those listed above. Sterile powders for the preparation of sterile injectable solutions can be prepared by vacuum-drying and freeze-drying techniques, which yields a powder of the RNA nanostructures plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art.

[0118] Pharmaceutical formulations for parenteral administration can be in the form of a sterile aqueous solution or suspension of particles formed from one or more RNA nanostructures. Acceptable solvents include, for example, water, Ringer's solution, phosphate buffered saline (PBS), and isotonic sodium chloride solution. The formulation can also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1,3-butanediol.

[0119] In some instances, the formulation can be distributed or packaged in a liquid form. In other aspects, formulations for parenteral administration can be packed as a solid, obtained, for example by lyophilization of a suitable liquid formulation. The solid can be reconstituted with an appropriate carrier or diluent prior to administration.

[0120] Solutions, suspensions, or emulsions for parenteral administration can be buffered with an effective amount of buffer necessary to maintain a pH suitable for ocular administration. Suitable buffers include, but are not limited to, acetate, borate, carbonate, citrate, and phosphate buffers.

[0121] Solutions, suspensions, or emulsions for parenteral administration can also contain one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents include, but are not limited to, glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes.

[0122] Solutions, suspensions, or emulsions for parenteral administration can also contain one or more preservatives to prevent bacterial contamination of the ophthalmic preparations. Suitable preservatives include, but are not limited to, polyhexamethylenebiguanidine (PHMB), benzalkonium chloride (BAK), stabilized oxychloro complexes (otherwise known as Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixtures thereof.

[0123] Solutions, suspensions, or emulsions for use of nanotechnology including nanoformulations for parenteral administration can also contain one or more excipients, such as dispersing agents, wetting agents, and suspending agents.TH Docket No.321502-2140

[0124] The RNA nanostructures as described herein can be formulated for topical administration. Suitable dosage forms for topical administration include creams, ointments, salves, sprays, gels, lotions, emulsions, liquids, and transdermal patches. The formulation can be formulated for transmucosal, transepithelial, transendothelial, or transdermal administration. The topical formulations can contain one or more chemical penetration enhancers, membrane permeability agents, membrane transport agents, emollients, surfactants, stabilizers, and combination thereof.

[0125] In some aspects, the RNA nanostructures can be administered as a liquid formulation, such as a solution or suspension, a semi-solid formulation, such as a lotion or ointment, or a solid formulation. In some aspects, the RNA nanostructures can be formulated as liquids, including solutions and suspensions, such as eye drops or as a semi-solid formulation, such as ointment or lotion for topical application to the skin, to the mucosa, such as the eye, to the vagina, or to the rectum.

[0126] The formulation can contain one or more excipients, such as emollients, surfactants, emulsifiers, penetration enhancers, and the like.

[0127] Suitable emollients include, without limitation, almond oil, castor oil, ceratonia extract, cetostearoyl alcohol, cetyl alcohol, cetyl esters wax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycol palmitostearate, glycerin, glycerin monostearate, glyceryl monooleate, isopropyl myristate, isopropyl palmitate, lanolin, lecithin, light mineral oil, medium- chain triglycerides, mineral oil and lanolin alcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil, starch, stearyl alcohol, sunflower oil, xylitol and combinations thereof. In some aspects, the emollients can be ethylhexylstearate and ethylhexyl palmitate.

[0128] Suitable surfactants include, but are not limited to, emulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate, poloxamer, povidone and combinations thereof. In some aspects, the surfactant can be stearyl alcohol.

[0129] Suitable emulsifiers include, but are not limited to, acacia, metallic soaps, certain animal and vegetable oils, and various polar compounds, anionic emulsifying wax, calcium stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolin alcohols, lecithin, medium-chain triglycerides, methylcellulose, mineral oil and lanolin alcohols, monobasic sodium phosphate, monoethanolamine, nonionic emulsifying wax, oleic acid, poloxamer, poloxamers,TH Docket No.321502-2140 polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, propylene glycol alginate, self-emulsifying glyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulfate, sorbitan esters, stearic acid, sunflower oil, tragacanth, triethanolamine, xanthan gum and combinations thereof. In some aspects, the emulsifier can be glycerol stearate.

[0130] Suitable classes of penetration enhancers include, but are not limited to, fatty alcohols, fatty acid esters, fatty acids, fatty alcohol ethers, amino acids, phospholipids, lecithins, cholate salts, enzymes, amines and amides, complexing agents (liposomes, cyclodextrins, modified celluloses, and diimides), macrocyclics, such as macrocylic lactones, ketones, and anhydrides and cyclic ureas, surfactants, N-methyl pyrrolidones and derivatives thereof, DMSO and related compounds, ionic compounds, azone and related compounds, and solvents, such as alcohols, ketones, amides, polyols (e.g., glycols).

[0131] Suitable emulsions include, but are not limited to, oil-in-water and water-in-oil emulsions. Either or both phases of the emulsions can include a surfactant, an emulsifying agent, and / or a liquid non-volatile non-aqueous material. In some aspects, the surfactant can be a non-ionic surfactant. In other aspects, the emulsifying agent is an emulsifying wax. In further aspects, the liquid non-volatile non-aqueous material is a glycol. In some aspects, the glycol is propylene glycol. The oil phase can contain other suitable oily pharmaceutically acceptable excipients. Suitable oily pharmaceutically acceptable excipients include, but are not limited to, hydroxylated castor oil or sesame oil can be used in the oil phase as surfactants or emulsifiers.

[0132] Lotions containing RNA nanostructures as described herein are also provided. In some aspects, the lotion can be in the form of an emulsion having a viscosity of between 100 and 1000 centistokes. The fluidity of lotions can permit rapid and uniform application over a wide surface area. Lotions can be formulated to dry on the skin leaving a thin coat of their medicinal components on the skin’s surface.

[0133] Creams containing an RNA nanostructure as described herein are also provided. The cream can contain emulsifying agents and / or other stabilizing agents. In some aspects, the cream is in the form of a cream having a viscosity of greater than 1000 centistokes, typically in the range of 20,000-50,000 centistokes. Creams, as compared to ointments, can be easier to spread and easier to remove.

[0134] One difference between a cream and a lotion is the viscosity, which is dependent on the amount / use of various oils and the percentage of water used to prepare the formulations. Creams can be thicker than lotions, can have various uses, and can have more varied oils / butters, depending upon the desired effect upon the skin. In some aspects of a creamTH Docket No.321502-2140 formulation, the water-base percentage can be about 60% to about 75% and the oil-base can be about 20% to about 30% of the total, with the other percentages being the emulsifier agent, preservatives and additives for a total of 100%.

[0135] Ointments containing an RNA nanostructure as described herein and a suitable ointment base are also provided. Suitable ointment bases include hydrocarbon bases (e.g., petrolatum, white petrolatum, yellow ointment, and mineral oil); absorption bases (hydrophilic petrolatum, anhydrous lanolin, lanolin, and cold cream); water-removable bases (e.g., hydrophilic ointment), and water-soluble bases (e.g., polyethylene glycol ointments). Pastes typically differ from ointments in that they contain a larger percentage of solids. Pastes are typically more absorptive and less greasy that ointments prepared with the same components.

[0136] Also described herein are gels containing an RNA nanostructure as described herein, a gelling agent, and a liquid vehicle. Suitable gelling agents include, but are not limited to, modified celluloses, such as hydroxypropyl cellulose and hydroxyethyl cellulose; carbopol homopolymers and copolymers; thermoreversible gels and combinations thereof. Suitable solvents in the liquid vehicle include, but are not limited to, diglycol monoethyl ether; alkylene glycols, such as propylene glycol; dimethyl isosorbide; alcohols, such as isopropyl alcohol and ethanol. The solvents can be selected for their ability to dissolve the drug. Other additives, which can improve the skin feel and / or emolliency of the formulation, can also be incorporated. Such additives include, but are not limited to, isopropyl myristate, ethyl acetate, C12-C15alkyl benzoates, mineral oil, squalane, cyclomethicone, capric / caprylic triglycerides, and combinations thereof.

[0137] Also described herein are foams that can include an RNA nanostructure as described herein. Foams can be an emulsion in combination with a gaseous propellant. The gaseous propellant can include hydrofluoroalkanes (HFAs). Suitable propellants include HFAs such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoropropane (HFA 227), but mixtures and admixtures of these and other HFAs that are currently approved or can become approved for medical use are suitable. The propellants can be devoid of hydrocarbon propellant gases, which can produce flammable or explosive vapors during spraying. Furthermore, the foams can contain no volatile alcohols, which can produce flammable or explosive vapors during use.

[0138] Buffers can be used to control pH of a composition. The buffers can buffer the composition from a pH of about 4 to a pH of about 7.5, from a pH of about 4 to a pH of about 7, or from a pH of about 5 to a pH of about 7. In some aspects, the buffer can be triethanolamine.TH Docket No.321502-2140

[0139] Preservatives can be included to prevent the growth of fungi and microorganisms. Suitable preservatives include, but are not limited to, benzoic acid, butylparaben, ethyl paraben, methyl paraben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, and thimerosal.

[0140] In certain aspects, the formulations can be provided via continuous delivery of one or more formulations to a patient in need thereof. For topical applications, repeated application can be done or a patch can be used to provide continuous administration of the noscapine analogs over an extended period of time.

[0141] The RNA nanostructures as described herein can be prepared in enteral formulations, such as for oral administration. Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can be prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art.

[0142] Formulations containing RNA nanostructures as described herein can be prepared using pharmaceutically acceptable carriers. As generally used herein “carrier” includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof. Polymers used in the dosage form include, but are not limited to, suitable hydrophobic or hydrophilic polymers and suitable pH dependent or independent polymers. Suitable hydrophobic and hydrophilic polymers include, but are not limited to, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxy methylcellulose, polyethylene glycol, ethylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetate, and ion exchange resins. “Carrier” also includes all components of the coating composition which can include plasticizers, pigments, colorants, stabilizing agents, and glidants.

[0143] Formulations containing an RNA nanostructure as described herein can be prepared using one or more pharmaceutically acceptable excipients, including diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.

[0144] Delayed release dosage formulations containing an RNA nanostructure as described herein can be prepared as described in standard references such as “Pharmaceutical dosage form tablets”, eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), “Remington – The science and practice of pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD,TH Docket No.321502-2140 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules. These references provide information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.

[0145] The formulations containing an RNA nanostructure as described herein can be coated with a suitable coating material, for example, to delay release once the particles have passed through the acidic environment of the stomach. Suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0146] Coatings can be formed with a different ratio of water soluble polymer, water insoluble polymers and / or pH dependent polymers, with or without water insoluble / water soluble non polymeric excipient, to produce the desired release profile. The coating can be performed on a dosage form (matrix or simple) which includes, but is not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particle compositions, “ingredient as is” formulated as, but not limited to, suspension form or as a sprinkle dosage form.

[0147] Additionally, the coating material can contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants. Optional pharmaceutically acceptable excipients include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants.

[0148] Diluents, also referred to as "fillers," can be used to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar. The usual diluents include inert powdered substances such as starches, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours andTH Docket No.321502-2140 similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful.

[0149] Binders can impart cohesive qualities to a solid dosage formulation, and thus can ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid and polyvinylpyrrolidone. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders.

[0150] Lubricants can be included to facilitate tablet manufacture. Suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil. A lubricant can be included in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant can be chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.

[0151] Disintegrants can be used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as cross-linked PVP (Polyplasdone® XL from GAF Chemical Corp).

[0152] Stabilizers can be used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA).

[0153] In some aspects, an amount of one or more additional active agents are included in the pharmaceutical formulation containing an RNA nanostructure. Suitable additional active agents include, but are not limited to, DNA, RNA, modified ribonucleotides, amino acids,TH Docket No.321502-2140 peptides, polypeptides, antibodies, aptamers, ribozymes, guide sequences for ribozymes that inhibit translation or transcription of essential tumor proteins and genes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti- inflammatories, anti-histamines, anti-infectives, and chemotherapeutics (anti-cancer drugs). Other suitable additional active agents include, sensitizers (such as radiosensitizers). The RNA nanostructure can be used as a monotherapy or in combination with other active agents for treatment or prevention of a disease or disorder.

[0154] Suitable hormones include, but are not limited to, amino-acid derived hormones (e.g. melatonin and thyroxine), small peptide hormones and protein hormones (e.g. thyrotropin- releasing hormone, vasopressin, insulin, growth hormone, luteinizing hormone, follicle- stimulating hormone, and thyroid-stimulating hormone), eiconsanoids (e.g. arachidonic acid, lipoxins, and prostaglandins), and steroid hormones (e.g. estradiol, testosterone, tetrahydro testosteron cortisol).

[0155] Suitable immunomodulators include, but are not limited to, prednisone, azathioprine, 6-MP, cyclosporine, tacrolimus, methotrexate, interleukins (e.g. IL-2, IL-7, and IL- 12), cytokines (e.g. interferons (e.g. IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, and IFN-γ), granulocyte colony-stimulating factor, and imiquimod), chemokines (e.g. CCL3, CCL26 and CXCL7), cytosine phosphate-guanosine, oligodeoxynucleotides, glucans, antibodies, and aptamers).

[0156] Suitable analgesics include, but are not limited to, paracetamol / acetaminophen, non-steroidal anti-inflammants (e.g. ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g. rofecoxib, celecoxib, and etoricoxib), opioids (e.g. morphine, codeine, oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine), tramadol, norepinephrine, flupiretine, nefopam, orphenadrine, pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone, ketobemidone, piritramide, and aspirin and related salicylates (e.g. choline salicylate, magnesium salicylae, and sodium salicaylate).

[0157] Suitable antispasmodics include, but are not limited to, mebeverine, papverine, cyclobenzaprine, carisoprodol, orphenadrine, tizanidine, metaxalone, methodcarbamol, chlorzoxazone, baclofen, dantrolene, baclofen, tizanidine, and dantrolene.

[0158] Suitable anti-inflammatories include, but are not limited to, prednisone, non- steroidal anti-inflammants (e.g. ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g. rofecoxib, celecoxib, and etoricoxib), and immune selective anti-inflammatory derivatives (e.g. submandibular gland peptide-T and its derivatives).

[0159] Suitable anti-histamines include, but are not limited to, H1-receptor antagonists (e.g. acrivastine, azelastine, bilastine, brompheniramine, buclizine, bromodiphenhydramine,TH Docket No.321502-2140 carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, clemastine, cyproheptadine, desloratadine, dexbromapheniramine, dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, doxylamine, ebasine, embramine, fexofenadine, hydroxyzine, levocetirzine, loratadine, meclozine, mirtazapine, olopatadine, orphenadrine, phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine, tripelennamine, and triprolidine), H2-receptor antagonists (e.g. cimetidine, famotidine, lafutidine, nizatidine, rafitidine, and roxatidine), tritoqualine, catechin, cromoglicate, nedocromil, and β2- adrenergic agonists.

[0160] Suitable anti-infectives include, but are not limited to, amebicides (e.g. nitazoxanide, paromomycin, metronidazole, tnidazole, chloroquine, and iodoquinol), aminoglycosides (e.g. paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin), anthelmintics (e.g. pyrantel, mebendazole, ivermectin, praziquantel, abendazole, miltefosine, thiabendazole, oxamniquine), antifungals (e.g. azole antifungals (e.g. itraconazole, fluconazole, posaconazole, ketoconazole, clotrimazole, miconazole, and voriconazole), echinocandins (e.g. caspofungin, anidulafungin, and micafungin), griseofulvin, terbinafine, flucytosine, and polyenes (e.g. nystatin, and amphotericin b), antimalarial agents (e.g. pyrimethamine / sulfadoxine, artemether / lumefantrine, atovaquone / proquanil, quinine, hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine, and halofantrine), antituberculosis agents (e.g. aminosalicylates (e.g. aminosalicylic acid), isoniazid / rifampin, isoniazid / pyrazinamide / rifampin, bedaquiline, isoniazid, ethanmbutol, rifampin, rifabutin, rifapentine, capreomycin, and cycloserine), antivirals (e.g. amantadine, rimantadine, abacavir / lamivudine, emtricitabine / tenofovir, cobicistat / elvitegravir / emtricitabine / tenofovir, efavirenz / emtricitabine / tenofovir, avacavir / lamivudine / zidovudine, lamivudine / zidovudine, emtricitabine / tenofovir, emtricitabine / opinavir / ritonavir / tenofovir, interferon alfa-2v / ribavirin, peginterferon alfa-2b, maraviroc, raltegravir, dolutegravir, enfuvirtide, foscarnet, fomivirsen, oseltamivir, zanamivir, nevirapine, efavirenz, etravirine, rilpiviirine, delaviridine, nevirapine, entecavir, lamivudine, adefovir, sofosbuvir, didanosine, tenofovir, avacivr, zidovudine, stavudine, emtricitabine, xalcitabine, telbivudine, simeprevir, boceprevir, telaprevir, lopinavir / ritonavir, fosamprenvir, dranuavir, ritonavir, tipranavir, atazanavir, nelfinavir, amprenavir, indinavir, sawuinavir, ribavirin, valcyclovir, acyclovir, famciclovir, ganciclovir, and valganciclovir), carbapenems (e.g. doripenem, meropenem, ertapenem, and cilastatin / imipenem), cephalosporins (e.g. cefadroxil, cephradine, cefazolin, cephalexin, cefepime, ceflaroline, loracarbef, cefotetan, cefuroxime, cefprozil, loracarbef, cefoxitin, cefaclor, ceftibuten, ceftriaxone, cefotaxime, cefpodoxime, cefdinir, cefixime, cefditoren, cefizoxime, andTH Docket No.321502-2140 ceftazidime), glycopeptide antibiotics (e.g. vancomycin, dalbavancin, oritavancin, and telvancin), glycylcyclines (e.g. tigecycline), leprostatics (e.g. clofazimine and thalidomide), lincomycin and derivatives thereof (e.g. clindamycin and lincomycin ), macrolides and derivatives thereof (e.g. telithromycin, fidaxomicin, erthromycin, azithromycin, clarithromycin, dirithromycin, and troleandomycin), linezolid, sulfamethoxazole / trimethoprim, rifaximin, chloramphenicol, fosfomycin, metronidazole, aztreonam, bacitracin, beta lactam antibiotics (benzathine penicillin (benzatihine and benzylpenicillin), phenoxymethylpenicillin, cloxacillin, flucoxacillin, methicillin, temocillin, mecillinam, azlocillin, mezlocillin, piperacillin, amoxicillin, ampicillin, bacampicillin, carbenicillin, piperacillin, ticarcillin, amoxicillin / clavulanate, ampicillin / sulbactam, piperacillin / tazobactam, clavulanate / ticarcillin, penicillin, procaine penicillin, oxacillin, dicloxacillin, nafcillin, cefazolin, cephalexin, cephalosporin C, cephalothin, cefaclor, cefamandole, cefuroxime, cefotetan, cefoxitin, cefiximine, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone, cefepime, cefpirome, ceftaroline, biapenem, doripenem, ertapenem, faropenem, imipenem, meropenem, panipenem, razupenem, tebipenem, thienamycin, azrewonam, tigemonam, nocardicin A, taboxinine, and beta-lactam), quinolones (e.g. lomefloxacin, norfloxacin, ofloxacin, qatifloxacin, moxifloxacin, ciprofloxacin, levofloxacin, gemifloxacin, moxifloxacin, cinoxacin, nalidixic acid, enoxacin, grepafloxacin, gatifloxacin, trovafloxacin, and sparfloxacin), sulfonamides (e.g. sulfamethoxazole / trimethoprim, sulfasalazine, and sulfasoxazole), tetracyclines (e.g. doxycycline, demeclocycline, minocycline, doxycycline / salicyclic acid, doxycycline / omega-3 polyunsaturated fatty acids, and tetracycline), and urinary anti-infectives (e.g. nitrofurantoin, methenamine, fosfomycin, cinoxacin, nalidixic acid, trimethoprim, and methylene blue).

[0161] Suitable chemotherapeutics include, but are not limited to, paclitaxel, brentuximab vedotin, doxorubicin, 5-FU (fluorouracil), everolimus, pemetrexed, melphalan, pamidronate, anastrozole, exemestane, nelarabine, ofatumumab, bevacizumab, belinostat, tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide, lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin, ramucirumab, cytarabine, cytoxan, cyclophosphamide, decitabine, dexamethasone, docetaxel, hydroxyurea, decarbazine, leuprolide, epirubicin, oxaliplatin, asparaginase, estramustine, cetuximab, vismodegib, aspargainase erwinia chyrsanthemi, amifostine, etoposide, flutamide, toremifene, fulvestrant, letrozole, degarelix, pralatrexate, methotrexate, floxuridine, obinutuzumab, gemcitabine, afatinib, imatinib mesylatem, carmustine, eribulin, trastuzumab, altretamine, topotecan, ponatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferon alfa-2a, gefitinib, romidepsin, ixabepilone, ruxolitinib, cabazitaxel, ado-trastuzumab emtansine, carfilzomib,TH Docket No.321502-2140 chlorambucil, sargramostim, cladribine, mitotane, vincristine, procarbazine, megestrol, trametinib, mesna, strontium-89 chloride, mechlorethamine, mitomycin, busulfan, gemtuzumab ozogamicin, vinorelbine, filgrastim, pegfilgrastim, sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone, pegaspargase, denileukin diftitox, alitretinoin, carboplatin, pertuzumab, cisplatin, pomalidomide, prednisone, aldesleukin, mercaptopurine, zoledronic acid, lenalidomide, rituximab, octretide, dasatinib, regorafenib, histrelin, sunitinib, siltuximab, omacetaxine, thioguanine (tioguanine), dabrafenib, erlotinib, bexarotene, temozolomide, thiotepa, thalidomide, BCG, temsirolimus, bendamustine hydrochloride, triptorelin, aresnic trioxide, lapatinib, valrubicin, panitumumab, vinblastine, bortezomib, tretinoin, azacitidine, pazopanib, teniposide, leucovorin, crizotinib, capecitabine, enzalutamide, ipilimumab, goserelin, vorinostat, idelalisib, ceritinib, abiraterone, epothilone, tafluposide, azathioprine, doxifluridine, vindesine, all-trans retinoic acid, and other anti-cancer agents listed elsewhere herein.

[0162] Methods of Using the RNA Nanostructures and Formulations Thereof

[0163] The RNA nanostructure as provided herein can be administered to a subject in need thereof, cell, or population thereof. The subject in need thereof can have a cancer or any other disease or disorder that would benefit from a nucleoside analogue. The amount delivered can be an effective amount of an RNA nanostructure provided herein. The subject in need thereof can be symptomatic or asymptomatic. In some aspects, the RNA nanostructures provided herein can be co-administered with another active agent. It will be appreciated that co- administered can refer to an additional compound that is included in the formulation or provided in a dosage form separate from the RNA nanostructure or formulation thereof. The effective amount of the RNA nanostructure or formulation thereof, such as those described herein, can range from about 0.1 mg / kg to about 500 mg / kg. In some aspects, the effective amount ranges from about 0.1 mg / kg to 10 mg / kg. In additional aspects, the effective amount ranges from about 0.1 mg / kg to 100 mg / kg. If further aspects, the effective amount ranges from about 0.1 mg to about 1000 mg. In some aspects, the effective amount can be about 500 mg to about 1000 mg.

[0164] Administration of the RNA nanostructures and formulations thereof can be systemic or localized. The compounds and formulations described herein can be administered to the subject in need thereof one or more times per day. In an aspect, the compound(s) and / or formulation(s) thereof can be administered once daily. In some aspects, the compound(s) and / or formulation(s) thereof can be administered given once daily. In another aspect, the compound(s) and / or formulation(s) thereof can be administered twice daily. In some aspects, when administered, an effective amount of the compounds and / or formulations are administeredTH Docket No.321502-2140 to the subject in need thereof. The compound(s) and / or formulation(s) thereof can be administered one or more times per week. In some aspects the compound(s) and / or formulation(s) thereof can be administered 1 day per week. In other aspects, the compound(s) and / or formulation(s) thereof can be administered 2 to 7 days per week.

[0165] In some aspects, the RNA nanostructure(s) and / or formulation(s) thereof, can be administered in a dosage form. The amount or effective amount of the compound(s) and / or formulation(s) thereof can be divided into multiple dosage forms. For example, the effective amount can be split into two dosage forms and the first dosage form can be administered, for example, in the morning, and the second dosage form can be administered in the evening. Although the effective amount is given over two doses, in one day, the subject receives the effective amount. In some aspects the effective amount is about 0.1 to about 1000 mg per day. The effective amount in a dosage form can range from about 0.1 mg / kg to about 1000 mg / kg. The dosage form can be formulated for oral, vaginal, intravenous, transdermal, subcutaneous, intraperitoneal, or intramuscular administration. Preparation of dosage forms for various administration routes are described elsewhere herein.

[0166] The modular RNA motifs described herein and the RNA nanostructures described herein can be used in the preparation of a medicament for treatmet of a disease or a cancer.

[0167] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0168] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. EXAMPLES Example 1: RNA 4WJ for Spontaneous Cancer-Targeting, Effective Tumor- Regression, Metastasis Suppression, Fast Renal Excretion and Undetectable Toxicity Abstract

[0169] The field of RNA therapeutics has been emerging as the third milestone in pharmaceutical drug development. RNA nanoparticles have displayed motile and deformable properties to allow for high tumor accumulation with undetectable healthy organ accumulation. Therefore, RNA nanoparticles have potential to serve as potent drug delivery vehicles withTH Docket No.321502-2140 strong anti-cancer responses. Herein, we report the physicochemical basis for the rational design of a branched RNA four-way junction (4WJ) nanoparticle that result in advantageous high-thermostability and -drug payload for cancer therapy, including metastatic tumors in the lung. The 4WJ nanostructure displayed versatility through functionalization with four different anti-cancer chemical drugs, including SN38, fluorouridine, gemcitabine, and paclitaxel for the treatment of three cancer models including breast cancer patient-derived xenograft (PDX), colorectal cancer xenograft, and orthotopic lung metastases of colon cancer. The resulting 4WJ RNA drug complex spontaneously targeted cancers effectively for cancer inhibition with and without ligands. The 4WJ displayed fast renal excretion, rapid body clearance, and little organ accumulation with undetectable toxicity and immunogenicity. The safety parameters were documented by organ histology, blood biochemistry, and pathological analysis. The highly efficient cancer inhibition, undetectable drug toxicity, and favorable CMC production of RNA nanoparticles documents a candidate with high potential for translation in cancer therapy. Introduction

[0170] It was predicted that RNA therapeutics would be the third milestone in drug pharmaceutical drug development [1, 2]. Now the spring of RNA therapeutics is coming. Nanoparticles have been developed as drug delivery platforms with the goal to improve the therapeutic outcome of small molecule drugs by increasing solubility and stability, improving bioavailability, extending circulation time, and reducing side effects [3]. Ribonucleic acid (RNA) is a novel biomaterial used to construct novel nanoparticles to deliver drugs for disease treatment. RNA nanoparticles with various two- and three-dimensional (2D and 3D) structures have been constructed using bottom-up assembly, rolling circle transcription, and RNA origami approaches [4-9]. RNA nanoparticles have many advantages, such as net negative charge for repulsion to nontargeted cells, multivalence for multiple conjugates, programmability, one-pot self-assembly from short oligos, and favorable biodistribution profile, that make them suitable as drug delivery platforms [10, 11].

[0171] RNA nanoparticles are highly programmable in terms of size, shape, stability, and stoichiometry [12-15]. Short RNA strands can be synthesized through solid-phase synthesis with various of modifications with defined quantity and position. Multi-functional RNA nanoparticles can be easily produced by mixing functionalization RNA strands at a defined molar ratio with high assembly efficiency [7, 16]. This allows for the chemistry, manufacturing, and controls (CMC) production of RNA nanoparticles, which makes them feasible for clinical applications. Furthermore, RNA nanoparticles have been functionalized with small molecule drugs, including as camptothecin (CPT), and therapeutic oligos, such as anti-miRNA21 orTH Docket No.321502-2140 siRNAs, which demonstrated significant cancer inhibition effects in various tumor-bearing animal models [17-22]. Click reactions have been used to conjugate anti-cancer drugs and targeting ligands onto RNA strands [17, 23, 24]. Current methods to incorporate small molecules into RNA nanoparticles include non-covalent intercalation, such as doxorubicin (DOX), and covalent conjugation, such as paclitaxel (PTX)

[0025] . Additionally, a new approach of incorporating an anti-cancer nucleoside analog with a defined quantity and position during RNA synthesis is presented in this study. Previous studies have demonstrated that RNA nanoparticles loaded with different anti-cancer drugs showed tumor inhibition effects in various tumor models due to the EPR effect, deformative property, and active targeting [17, 25-27].

[0172] RNA molecules possess a dynamic property that is critical for their biological functions, such as catalysis in ribozymes. RNA nanoparticles also have deformative properties, which enables their prolonged tumor accumulation and fast renal excretion [5, 11, 28]. This is favorable for the tumor inhibition effect and low toxicity. In addition to the programmability and deformative property, RNA nanoparticles are highly hydrophilic, which can be utilized to solubilize hydrophobic drugs [17, 25]. Many anti-cancer drugs suffer from low water solubility leading to high toxicities. The hydrophilic property of RNA solubilizes the conjugated drugs in an aqueous solution, thus eliminating the toxic formulations [17, 25]. Furthermore, RNA nanoparticles have demonstrated a great safety profile with low toxicity, including immune and organ toxicity in various animal trials [5, 11, 29].

[0173] In this study, after extensive investigation, the RNA 4WJ was identified as the optimal nanoparticle to carry small molecule drugs for cancer therapy. To carry a drug for combinational therapy requires an RNA nanoparticle to have a high melting temperature (Tm), allowing for a high drug-loading capacity without interfering with the stability of the nanoparticle. Meanwhile, the high efficiency in assembly overcomes challenges in CMC production.

[0174] Constructed RNA nanoparticles harboring various anti-cancer drugs, including SN38, floxuridine (FUDR), and gemcitabine (GEM), were evaluated as drug delivery platforms against various cancer models including breast cancer patient-derived xenograft (PDX), colorectal cancer (CRC) xenograft, and orthotopic lung metastases of colon cancer. The drug payload, therapeutic effect, and pathology parameters were evaluated. Functionalized RNA nanoparticles demonstrated tumor inhibition effect in all tested tumor models demonstrating the versatility of RNA nanoparticles while no observable toxicity in biochemical and histological analysis were identified.TH Docket No.321502-2140 Results Rational design of 4WJ for high drug loading and high stability

[0175] The three-way junction (3WJ) of phi29 motor pRNA has been found to have a high thermostability and has been extensively investigated as a vehicle to carry and deliver therapeutics for cancer therapy [2, 5, 30-32]. However, the conjugation of eight paclitaxel molecules to the 3WJ led to the destabilization of the 3WJ / drug complex, resulting in the reduction of a Tmto 30°C. This suggests that the pRNA 3WJ is not a suitable vehicle for the delivery of chemical drugs with a high payload

[0025] . To optimize the drug delivery system for the chemotherapeutics, different viral pRNA 3WJ pseudoknot from literature, including the 3WJ of phi29, M2, B103, SF5 and Ga1 [33-37] were compared for chemical drug conjugation and tested for the stability of the resulting 3WJ-drug complexes.

[0176] The previously reported 4WJ, derived from phi29-3WJ, can carry 24 chemical drugs with a stable Tmof 70-80°C

[0025] , while the original phi293WJ can only carry 8-PTX with a Tmof around 30 degrees. The higher payload of 24 chemical drugs will enhance therapeutic efficacies. It is hoped that a higher drug payload might be achieved by increasing the length of the 3WJ sequence. However, when the size of the RNA nanoparticles increases, healthy organ accumulation occurs due to macrophage trapping [14, 29]. The Immunol response also increases [38, 39]; the kidney excretion is reduced due to the kidney filtration size limit; biodistribution will not be favorable; and nanoparticle production becomes complicated with numerous component strands.

[0177] In RNA structure and folding investigation, it is commonly believed that increasing the ratio of GC content will increase the Tmand thermostability. This hypothesis was tested. While each of the pRNA 3WJs from phages phi29, M2, SF5, GA1, and B103 show very similar secondary structures, while their primary sequences are very different [33-36]. The M2 3WJ has been reported as the most stable 3WJ with the highest Tm. Therefore, the M2 was mutated through GC base pair substitution in the quest for a more stable 3WJ (Fig.1A). However, it was found that increasing GC content led to the failure of RNA 3WJ assembly. The data showed that after increasing GC content to 3WJ, smearing and misfolding appeared. Each oligo subunit self-forms into a stronger secondary structure due to the random GC base pairing within the subunit (Fig.1B). The high GC content of each RNA strand also lead to the formation of Quadruplexs and resulted in random GC interactions among the three strands. The self- folding of the high-GC content strands and the presence of multiple complementary sequences leads to the failure in RNA nanoparticle assembly, leading to misfolding and the failure in producing homogenous RNA nanoparticles (Fig.1B).TH Docket No.321502-2140

[0178] The mechanism of 3WJ assembly from three component strands has been reported (Fig.1C)

[0040] . It has been found that each 3WJ oligo offers eight or nine nucleotides (50% of the 16 nucleotides) to interact with each other. First, the b-strand interacts with 50% of the c-strand due to the lowest ΔG of the dimerization in comparison to that in a-c or a-b interaction. The generated b-c dimer creates a fork structure to recruit the a-strand, resulting in the formation of the 3WJ

[0040] . Similar mechanism if 3WJ formation were applied to the design of 4WJ. The 4WJ possesses four helixes that, each contain a core domain that controls the structure formation and a payload domain that is used for functionalization. The folding of the 4WJ motif might start with the generation of a helix structure between two strands, which creates two fork structures for a third strand and a fourth strand to bind with it. This principle has been applied to the design of the 4WJ with high Tmand high drug loading capacity via an alternation and screening process. CMC (Chemical, Manufacturing, and Control) production of drug-contained RNA nanoparticles

[0179] RNA nanoparticles are able to be constructed under CMC production due to defined and highly programmable in terms of size, shape, stability, stoichiometry, and modification [5, 11, 16, 41]. The procedures for the CMC product of drug-contained RNA nanoparticles are shown in Fig.6. Single RNA oligo strands were generated using solid-phase synthesis that is highly controlled and produced specific products. These oligos were then used to construct RNA nanoparticles through the one-pot self-assembly [25, 42, 43].4WJ has high thermostability, which was demonstrated by its high melting and annealing temperature (Fig. 2D-2E)

[0025] , thus allowing the 4WJ to carry anti-cancer drugs with high drug-loading capacity

[0025] . RNA strands were functionalized with a defined number of various anti-cancer drugs, including small molecule drugs such as SN38, PTX, GEM, and FUDR. RNA strands with alkyne modification were synthesized and then conjugated with azide modified prodrugs such as PTX- N3and SN38-N3(Fig.7). Additionally, nucleoside drugs, like GEM and FUDR, were directly incorporated into the strands during the solid-phase syncretization using nucleoside phosphoramidites. Construction of functionalized thermostable 4WJ RNA nanoparticles with SN38 and EpCAMapt

[0180] SN38 is a highly potent small molecule drug but has unfavorable pharmacokinetics due to its rapid metabolization and excretion, which limits the amount of drug reaching the disease site

[0044] . Various nanoparticles have been designed as the delivery platform for SN38 to extend its circulation and increase its tumor accumulation

[0045] . In addition,TH Docket No.321502-2140 RNA nanoparticles have unique deformative properties, which allow them to penetrate through leaky blood vessels at the tumor site with better tumor accumulation compared with other types of nanoparticles [5, 11, 28]. Thus, a highly thermostable 4WJ RNA nanoparticle, composing four 42-nt RNA strands, A, B, C, and D, was designed as the drug delivery platform for SN38 (Fig. 2A). Each RNA strand was conjugated with 6 copies of SN38 at a three-nucleotide interval to achieve the maximal drug loading without causing steric hindrance during conjugation and aggregation after conjugation (Fig.8).4WJ RNA nanoparticles were self-assembled by mixing and annealing four composing RNA strands at the equal molar ratio in one-step. Both 4WJ and 4WJ-SN38 RNA nanoparticles were assembled with high efficiency demonstrated by gel electrophoresis, which indicates the drug conjugation did not interfere with the assembly (Fig. 2B). With the attachment of SN38, the size of 4WJ increased from 10.13 nm to 12.19 nm which demonstrates no aggregation of conjugated SN38 in water (Fig.2C). In addition, the drug conjugation did not affect the negative charge of 4WJ RNA nanoparticles (Fig.2D).4WJ RNA nanoparticles were designed with high thermostability through the incorporation of 2’F-C and U as well the high GC content to overcome impacts of drug conjugation. The annealing curve showed that both 4WJ and 4WJ-SN38 exhibited high thermostability, with the annealing temperature (Ta) above 80 °C, which is much higher than physiological condition (37°C) (Fig. 2E). The TGGE gel further demonstrated the high thermostability of 4WJ-SN38 with the Tmhigher than 80°C (Fig.2F).

[0181] Additionally, RNA aptamers are single-stranded RNA with a defined folding structure that are capable of binding to target molecules with high affinity and selectivity

[0046] . RNA 4WJ nanoparticles were constructed with epithelial cell adhesion molecule (EpCAM) RNA aptamers (EpCAMapt) to instill specific tumor targeting

[0021] (Fig.2A). EpCAM has been found to be overexpressed in numerous cancers, including colorectal cancer

[0047] , and provides a good biomarker and cell surface receptor for targeting of RNA nanoparticles. Therefore, a well published and proven EpCAMaptwas placed onto the a helical branch of the 4WJ through sequence through extending B strand during synthesis without additional conjugation and purification steps

[0048] . Resulting RNA nanoparticles demonstrated homogenous products by PAGE analysis (Fig.2B) and the inclusion of the EpCAMaptshould provide selective binding to tumor expressed EpCAM for receptor mediated endocytosis. Improvement of poor drug water-solubility by covalent conjugation to RNA strand: a case study on SN38

[0182] Many chemotherapeutics suffer from poor water solubility and are highly hydrophobic thus limiting their pharmaceutical formulations, including SN38

[0053] . To solve thisTH Docket No.321502-2140 solubility issue, we designed RNA-SN38 conjugates taking advantage of the highly hydrophilic property of RNA. The multivalency of RNA enables the high drug loading capacity and precise control over the amount of modification. Multiple copies of hydrophobic SN38 were conjugated to hydrophilic RNA strands with defined stoichiometry as described above (Fig.7 and 8). The conjugation resulted in a significant improvement of its water solubility as demonstrated by UV / Vis spectrometry and visual observation. Dissolving RNA-6-SN38 conjugate in DEPC water yield clear solutions at increasing concentration from 12.5 μM to 800 μM (4.9 – 313.9 µg / mL) (Fig.8C). The absorbance of free SN38 in water remains at low level within the measured concentration range, which suggests the extremely poor water solubility of SN38. In contrast, the absorbance of RNA-SN38 in water showed linear increase with concentration which indicates that RNA-SN38 was completely dissolved water (Fig.8C). The solubility of RNA-SN38 in water is comparable to the solubility of free SN38 in DMSO, a common solvent for SN38, as indicated by the similar proportional increase of absorbance as a function of concentration. And the comparison of the clear solution of RNA-6-SN38 versus the cloudy suspension of SN38 in water at high concentration (313 µg / mL, 800 μM) further proved the improvement of water solubility after conjugation (Fig.8C). Collectively, the water solubility of SN38 was increased by at least 60-fold after conjugation to RNA, compared to the reported solubility of free SN38 (5 µg / mL, 12.7 μM) and its prodrug, irinotecan, (107µg / mL, 182.4 μM) in water. In vitro cell binding and internalization of SN38 RNA nanoparticles mediated by the EpCAM aptamer

[0183] Specificity is one of the key considerations in the drug delivery field. Targeted delivery can not only improve therapeutic outcome but also reduce the accumulation in normal organ and tissue, thus lowering side effects. EpCAM is expressed at low levels in normal epithelial tissues but highly expressed in 70-90% of carcinomas such as colorectal cancer cells

[0047] . To evaluate the specific targeting efficiency, AFDye 647 was attached to RNA nanoparticles as a fluorescent indicator. HT29 cells were incubated with 4WJ RNA nanoparticles which were then imaged by confocal microscope.4WJ RNA nanoparticle itself showed no observable binding to HT29 cells due to its negative charge repulsion from the cell membrane. However, with the incorporation of EpCAMapt, both 4WJ-EpCAM and 4WJ-SN38- EpCAM (4WJ-SN38-E) showed strong overlap of red signals from RNA nanoparticle and green signals from cytosol (Fig.3A). These results together demonstrated that EpCAMaptdisplaying RNA nanoparticles specifically bind to EpCAM-overexpressed tumor cells and are further internalized into the cells efficiently by receptor-mediated endocytosis. The enhancedTH Docket No.321502-2140 internalization profile provides a foundation for 4WJ-SN38-EpCAM to be applied in CRC targeting and therapy. In vitro cytotoxicity and immunogenicity of SN38 RNA nanoparticles

[0184] To determine the cytotoxicity of RNA-SN38 nanoparticles, dose-dependent MTT assay with different incubation time was performed. The cell viability results show that 4WJ RNA nanoparticle has no observable toxicity within the tested concentrations (0.025 – 1 μM) and time points (48, 72, 96 hr), which indicates its safety to be used as a delivery platform (Fig.3B). However, 4WJ-SN38-EpCAM RNA nanoparticle inhibited HT29 cell growth in a concentration dependent manner which demonstrated that SN38 was released from the RNA nanoparticles and retained its pharmacological activity to inhibit tumor cell growth. Interestingly, the cytotoxicity of 4WJ-SN38-EpCAM was relatively lower at 48 hr but became the same at 96 hr in comparison to free SN38, which suggests that SN38 is gradually released from RNA nanoparticles. Another explanation is that 4WJ-SN38-EpCAM internalization may take a longer time compared to the free SN38. To further confirm the inhibitive effect, PI and FITC Annexin V double staining analysis was performed. Consistent with the MTT assay, 4WJ RNA nanoparticles induced no observable apoptotic effect while 4WJ-SN38-EpCAM induced 31.6% of cells which is similar to the 38.0% induced by SN38 (Fig.3C).

[0185] Special formulations such as Cremephor EL / Ethanol have been used to dissolve hydrophobic drugs such as paclitaxel for cancer treatment

[0054] . However, these formulations could induce undesirable immune responses. Immunogenicity is also a concern for the use of nanoparticles fabricated with biomaterials. RNA nanoparticles are highly biocompatible and has no or low immune response with careful sequence design [10, 38]. To evaluate the immunogenicity of 4WJ RNA nanoparticles, production of pro-inflammatory cytokines, including TNF-α (tumor necrosis factor α) and IL-6 (interleukin-6) was investigated in vitro. TNF-α is cytokine involved in systemic inflammation and one of the cytokines that make up the acute phase reaction

[0055] . IL-6 is an interleukin that acts as both a pro-inflammatory cytokine and an anti-inflammatory myokine, which is secreted to stimulate immune response during infection

[0056] . The ELISA results showed that both bare 4WJ and functionalized 4WJ-SN38-EpCAM at the therapeutic concentration (100 nM) induced negligible TNF-α nor IL-6 production compared to LPS (lipopolysaccharide) positive control while incubating with mouse macrophage like RAW 264.7 cells

[0057] (Fig.4A and 4B). The low induction of cytokine suggests that RNA is a biocompatible and relative safe biomaterial.TH Docket No.321502-2140 In vivo colorectal tumor inhibition by 4WJ-SN38-EpCAMapt

[0186] Given the demonstration of specific tumor cell binding, high apoptotic effect, and negligible immune response, we performed the tumor suppression study in CRC tumor xenograft model. The therapeutic effect of 4WJ-SN38-EpCAM nanoparticles was validated by a tumor inhibition study in an HT29 xenografic model in nude mice. After tumors grew to approximately 50 mm3, mice bearing HT29 xenograft were randomly divided into four groups of PBS, SN38, 4WJ-SN38, and 4WJ-SN38-EpCAM. Samples were administrated via intravenous injection (IV) at the dose of 2 mg / kg (SN38 / body weight) on day 0, 3, 6, 9, and 12 for a total of five injections. Both the tumor growth curve and the tumor weight comparison demonstrated 4WJ-SN38-EpCAM has significant tumor suppression in comparison to PBS group (Fig.4C). Both RNA-SN38 nanoparticles did not cause any fatality or obvious weight changes during experiments, which suggests a favorable safety profile of the RNA-SN38 nanoparticles. Due to the recognition between EpCAM aptamer and its overexpressed receptor on HT29 cells, 4WJ- SN38-EpCAM outperformed 4WJ-SN38 in tumor suppression by about 20.37% more reduction in tumor weight (Fig.4D). In vivo colorectal cancer lung metastasis inhibition by 4WJ-SN38-EpCAM

[0187] The therapeutic effect of 4WJ-SN38-EpCAM nanoparticles was further validated by a tumor inhibition study in a colorectal cancer lung metastasis model. Five days after IV injection of lung metastatic trained HT29 cell lines with GFP protein expression, Lago imaging was conducted to verify the existence of lung metastasis (Fig.5A).

[0188] Compared to PBS group, 4WJ nanoparticle control, 4WJ-SN38-EpCAM shows significant inhibition on the colorectal cancer lung metastasis both in vivo and ex vivo as verified by the decreased GFP protein expression. (Fig.5B and 5C) Meanwhile, 4WJ-SN38-EpCAM treatment did not cause any fatality, and the mouse weight shown no significant change in 4WJ- SN38-EpCAM treatment group compared to the decreased weight among all other three groups. (Fig.5D) All data show enhanced therapeutic efficacy and a limited toxicity profile of 4WJ-SN38-EpCAM treatment against the CRC lung metastasis. Discussion

[0189] Since its inception in 1998

[0066] , the field of RNA nanotechnology has quickly grown into a viable drug delivery vehicle that offers many benefits such as improved pharmacological parameters, avoidance of healthy organs for low toxicities, rapid renal clearance, and high spontaneous tumor accumulation [5, 11]. RNA nanoparticles have been found to be motile and deformative to allow enhanced passage through tumor vasculature and rapid renal excretion to avoid interactions with healthy organs

[0028] . Additionally, RNATH Docket No.321502-2140 nanoparticles have shown to specifically bind and target various cancers through the inclusion of chemical or RNA targeting ligands [17-21, 25-27, 67-69]. Their multivalent nature allows for the easy modification with several functional groups, and high thermostability allows for stable conjugation of ligands or therapeutic groups [11, 27, 42, 70]. Here we demonstrate the versatility of 4WJ nanoparticles to harbor numerous copies of small molecule drugs and nucleoside therapies, including paclitaxel, SN38, GEM, and FUDR. The 4WJ RNA nanoparticle is easily adaptable to accept different drugs and combinations of drugs while remaining stable. The conjugation of these therapeutics results in several benefits including solubilization of hydrophobic therapeutics (Fig.8), specific delivery to tumors, avoidance of immune responses (Fig.4), controlled release in tumor environments, while remaining safe and presenting no toxicities (Accompanying manuscript)

[0025] .

[0190] In another recent research, we have shown that our RNA nanoparticles do not show detectable hemolysis phenomenon, platelet aggregation, complement activation, plasma coagulation, interferons induction, or cytokine secretion induction in vitro following Nanotechnology Characterization Laboratory protocol. At the same time, RNA nanoparticles carrying chemotherapeutics do not show any organ toxicity, serum biochemistry markers, or hematological markers changes. However, free chemotherapeutics especially free SN38 at the same drug concentration have shown obvious liver toxicity in vivo via increased Glutamic Oxaloacetic Transaminase and Glutamate Pyruvate Transaminase concentration. Meanwhile, White blood cell count, Platelet count, and Hemoglobin concentration are significantly reduced in the free SN38 treatment group (Accompanying manuscript), which further shows the advantage in the safety aspect of using RNA nanoparticles to carry chemotherapeutics.

[0191] The novelty of the presented RNA nanoparticle platform is its ability to easily adapt to the treatment of various cancers. Here we demonstrated the 4WJ RNA nanoparticle is able to target and significantly inhibit triple negative breast cancer and colorectal cancer. Most notably the 4WJ nanoparticle is able to target and nearly completely treat CRC metastases to the lungs in mice. These results are accomplished by simply changing drug conjugates on the nanoparticle to match clinically relevant therapies to each tumor subtype. In all, RNA nanotechnology is a very capable platform for the treatment of many cancers and is poised for translation into the clinic. Materials and Methods Synthesis of RNA strands

[0192] All RNA strands were synthesized by ASM-800ET DNA / RNA synthesizer (Biosset) using RNA A, RNA G, 2’-Fluoro C and 2’-Fluoro U phosphoramidites (ChemGenes).TH Docket No.321502-2140 5’-Hexynyl and 2’-O-propargyl phosphoramidites (Glen Research, ChemGenes) were incorporated into RNA-ALK strands for PTX or SN38 conjugation. RNA-FUDR and RNA-GEM strands were synthesized using 5-Fluoro-deoxy uridine CED phosphoramidite (ChemGenes) and N4-Benzoyl-2’-deoxy-5-O-DMT-2’,2’-difluorocytidine 3’-CE phosphoramidite (BOC Sciences), respectively. The sequences of all RNA strands are listed in SI table (Table 1). Table 1. Sequences of RNA nanoparticles. RNA strand Sequence (5’-3’) A C A U 5 G d C F UTH Docket No.321502-2140 19 4WJ-D-14-GEM UGUAUGUdFdCdFdCdFdCUAUdFdCdFdCdFdCGGGAUGdFdCdF (42-nt) dCdFdCAGGdFdCdFdCUAAdFdCAUAUAdFdCUdFdC (SEQ ID NO:231) reperformed using copper(I)-catalyzed alkyne-azide cycloaddition (“Click chemistry”), as previously described

[0025] . RNA-6-ALK strand dissolved in diethyl pyrocarbonate-treated water (DEPC-H2O) were thoroughly mixed with PTX-N3or SN38-N3dissolved in 3:1 (v / v) dimethyl sulfoxide / tert-butanol (DMSO / t-BuOH). Freshly prepared “click solution” (a mixture of CuBr / TBTA at a 1:2 molar ratio in DMSO / t-BuOH) was then added with the final molar ratio of RNA / SN38 / Cu+at 1:15:20.

[0194] After incubation at room temperature overnight, RNA-PTX or RNA-SN38 was initially purified by ethanol precipitation with 1 / 10 volumes of 0.3 M sodium acetate and 2.5 volumes of 100% ethanol followed by resuspension in DEPC-H2O. RNA was subsequently purified by Ion-Pair Reverse Phase high performance liquid chromatography (HPLC) using PLRP-S 4.6 ൈ 250 mm 300 Å column (Agilent Technologies). Fully conjugated RNA-PTX or RNA-SN38 was separated from unreacted and not fully reacted RNA-ALK strands under gradient elution with 0.1% Triethylammonium acetate (TEAA) in H2O as solvent A and 0.1% TEAA in 75% Acetonitrile (AcN) as solvent B. Fractions were collected and analyzed by PAGE and pure product were combined, dried, and resuspended in DEPC-H2O.

[0195] Synthesized RNA strands were characterized by 16% 8 M urea PAGE in TBE buffer (89 mM Tris base, 200 mM boric acid and 2 mM EDTA) at 200 V for 1 hr. After staining by ethidium bromide (EtBr, Sigma-Aldrich) and washing, gel was visualized and analyzed by a Li- Cor Odyssey Fc imaging system. Preparation of RNA nanoparticle

[0196] To assemble 4WJ RNA nanoparticle, four single RNA strands (A, B, C, and D) were mixed at equal molar ratio in TES buffer (50 mM Tris pH = 8.0, 50 mM NaCl, 1 mM EDTA). The mixture solution was subjected to a one-hour annealing program that starts with denaturing at 95°C for 5 min followed by slow cooling to 4°C over 1 hr in Mastercycler (Eppendorf).4WJ- PTX or -SN38 RNA nanoparticle was generated using the same procedures with four RNA-PTX or -SN38 strands.4WJ-FUDR and 4WJ-GEM RNA nanoparticles were assembled using four RNA-FUDR or -GEM strands, respectively. After annealing, RNA nanoparticles were checked by 10% native PAGE in TBE buffer at 120 V for 1 hr to check the assembly efficiency. AfterTH Docket No.321502-2140 staining by ethidium bromide (EtBr, Sigma-Aldrich) and washing, gel was visualized and analyzed by a Li-Cor Odyssey Fc imaging system. Size measurement by DLS

[0197] The hydrodynamic diameter and zeta potential of the RNA nanoparticles with the concentration of 10 ^M in TES buffer were measured using Zetasizer Nano-ZS (Malvern Instrument) at room temperature. Size distributions were plotted with data points using GraphPad Prism. Measurement of melting profile by thermal gradient gel electrophoresis (TGGE)

[0198] The melting profile of RNA nanoparticles in TES buffer was measured by TGGE in TBE buffer. RNA nanoparticles with a final concentration of 1 μM were subjected to 10% native PAGE gel 100 V for 60 min with a perpendicular temperature gradient (Left to right: 39.6 – 79.6 °C). After staining by EtBr and washing, gels were imaged by a Li-Cor Odyssey Fc imaging system and quantified by ImageJ. Cell culture

[0199] HT29 cells and HT29 G-L LungM3 cells were cultured in MyCoy’s 5A medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) at 37°C in humidified air containing 5% CO2. Mouse 4T1 cells were culture in Gibco Roswell Park Memorial Institute (RPMI) 1640 (Thermo Fisher Scientific) medium with 10% FBS at 37°C in humidified air containing 5% CO2. Mouse macrophage-like RAW 264.7 cells were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) at 37°C in humidified air containing 5% CO2. In vitro cell binding assay using confocal microscopy

[0200] HT29 cells were grown on glass slides in 24-well plate and cultured at 37°C overnight. RNA nanoparticles labeled with AFDye 647 were incubated with 2 × 105HT29 cells with a final concentration of 200 nM at 37°C for 2 hr. The cells were washed twice with PBS and fixed by 4% paraformaldehyde (PFA). After washing with PBS, the fixed cells were stained by Alexa Fluor 488 Phalloidin (Life Technologies) for cytoskeleton and Fluoroshield Mounting Medium with DAPI (Abcam) for nucleus, respectively. The cells were then imaged and analyzed by FluoView FV3000-Filter Confocal Microscope System (Olympus Corp). In vitro cytotoxicity by MTT assay

[0201] For 4WJ-SN38 RNA nanoparticles, the cytotoxicity was evaluated by CellTiter 96®Non-Radioactive Cell Proliferation Assay (Promega). HT29 cells were plated into 96-well plate and cultured at 37°C overnight. The cells were then incubated with samples with different concentration in 100 ^l cell medium containing 10% FBS at 37°C for different time point (48, 72,TH Docket No.321502-2140 96 hr). After the addition of 15 ^l MTT dye solution and incubation at 37°C for 4 hr, 100 μl stop solution was added to each well and incubated at room temperature overnight. The absorbance at 570 nm was measured using the Synergy 4 microplate reader (BioTek).

[0202] For the cytotoxicity study of 4WJ-FUDR and 4WJ-GEM RNA nanoparticles, HT29 or 4T1 cells were plated in 96-well plate and grow overnight, respectively. Cells were incubated with samples for 48 h. CellTiter 96®Non-Radioactive Cell Proliferation Assay (Promega) was performed was performed according to manual, by adding MTT dye and incubating at 37°C for 4 hr, adding stop solution and incubating at room temperature overnight, and measuring absorbance at 570 nm by Synergy 4 microplate reader (BioTek). In vitro apoptosis assay

[0203] The cell apoptosis was studied by FITC Annexin V Apoptosis Detection Kit (BD Pharmingen). HT29 cells were plated into 24-well plate and cultured at 37°C overnight. The cells were then incubated with samples in 400 μl cell medium containing 10% FBS at 37°C for 24 hr. After trypsinization, HT29 cells were washed with PBS and re-suspended in 100 μl 1× Annexin V-FITC binding buffer. After the addition of 2 μl Annexin V-FITC and 2 μl propidium iodide (PI) and incubation at room temperature for 15 min, the cell samples were transferred to flow tubes which contained 150 μl binding buffer for fluorescence-activated cell sorting (FACS) analysis by LSR II Flow Cytometer (Becton Dickinson), and the data were analyzed by FlowJo 7.6.1 software. In vitro cytokine induction assay

[0204] RAW 264.7 cells were plated into 24-well plates and cultured at 37°C overnight. RNA nanoparticles and lipopolysaccharide (LPS, 5.5 μg / mL, equal amount as 100 nM 4WJ RNA nanoparticles) were each incubated with macrophage cells in 200 μl Opti-MEM cell medium (Thermo Fisher Scientific) at 37°C for 16 hr. The supernatants of cell medium were collected and frozen at −80°C for further analysis. The TNF-α and IL-6 in diluted supernatants were measured by Mouse ELISA MAX Deluxe sets (BioLegend) following manufacturer’s protocols. Animal model preparation

[0205] Tumor inhibition study in colorectal cancer model. All animal procedures were performed in accordance with Subcommittee on Research Animal Care of The Ohio State University guidelines approved by the Institutional Review Board. The protocol for this animal experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of The Ohio State University. Colorectal cancer cell line HT29 (2 × 106cells) were transplanted into nude mice (4 - 6 weeks old) (Charles River Laboratories). Mice with established tumor nodules were randomly divided into four groups (n = 5, biologically independent animals). Samples wereTH Docket No.321502-2140 intravenously administrated through IV injection with a total of 5 doses (2 mg / kg, SN38, 4WJ- SN38, 4WJ-SN38-E / body weight) every 3 days for 15 days. Tumor volume was monitored everyday by caliper, calculated as (length × width2) / 2, and mouse weight was monitored every day. On day 15, mice were sacrificed, and tumors extracted and weighted.

[0206] Tumor inhibition study in lung metastasis model. All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Kentucky and were conducted in accordance with guidelines issued by the National Institutes of Health for the care of laboratory animals. HT29 cells were trained to be lung tropic through initial IV injection. Lung metastases were harvested and re-injected into mice; the in vivo selection process which is repeated three times to develop the HT29 G-L LungM3 cell line. HT29 G-L LungM3 cell line expresses GFP signal. [71, 72] NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ male and female mice were injected intravenously with 1 ൈ 106HT29 LungM3 cells. Mice were randomly divided into PBS, 4WJ, and 4WJ-SN38-EpCAM groups (n = 5, biologically independent animals).4WJ-SN38- EpCAM samples were administrated via IV injection at the dose of 2 mg / kg (SN38 / body weight) on day 5, 8, 11, 14, 17, 20, and 23 for a total of seven injections.4WJ samples were administrated at the same RNA concentration and frequency as the 4WJ-SN38-EpCAM group. On day 24, Lago imaging was conducted using Lago SII (Spectral Instruments Imaging, Tucson, AZ) to measure the GFP signal from the lung metastasis in vivo. Mice were then euthanized, and lungs were harvested for ex vivo Lago imaging. Mice weights of different groups were measured at day 5, 8, 11, 14, 17, 20, and 23.

[0207] Tumor inhibition study in breast cancer PDX model. All animals were kept in the National Defense Animal Center according to the protocol approved by the Association for Laboratory Animal Care Evaluation and Certification (LAC-101-0064). Triple negative breast cancer (TNBC) tissue from patient were transplanted into NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ female mice to generate PDX model. Mice with established tumor nodules were randomly divided into six groups (n = 5, biologically independent animals). Samples were intravenously administrated through IV injection with a total of 5 doses (2 mg / kg, GEM, 4WJ-GEM, FUDR, 4WJ-FUDR per body wright) every 2 days for 9 days. Tumor volume was monitored every week by caliper, calculated as (length × width2) / 2, and mouse weight was monitored every week. On day 32, mice were sacrificed, and tumors extracted and weighted. Immunohistochemistry (IHC) staining

[0208] The protein expression in PDX tumor tissues was detected using IHC. Paraffin- embedded PDX tumor tissues were cut into 8-μm sections and preincubated in 3% H2O2and 0.3% Triton X-100 before microwaving for antigen retrieval. For detection of Ki-67 proteinTH Docket No.321502-2140 immunostaining, sections were microwaved in Tris buffer (pH 6) for 30 min. The antigenicity of the tumor cells in sections was blocked in 5% horse serum (Chemicon, Temecula, CA) for 30 min and incubated with a diluted (1:200) Ki-67 (GTX16667, GeneTex, California, USA)-specific antibody for 2 hr at room temperature. Staining was developed using the streptavidin-biotin- peroxidase method and an LSAB 2 kit purchased from Dako (Carpinteria, CA). Briefly, sections were washed in phosphate-buffered saline (PBS) and incubated with a biotinylated anti-rabbit secondary antibody. The samples were rewashed in the same buffer and incubated with a streptavidin-biotin-peroxidase complex. Staining was completed after incubation with substrate- chromogen solution. The incubation duration in solution with 3,3’-diaminobenzidine was determined using low-power microscopic inspection. Slides were washed, dehydrated, and coverslipped using a mixture of di-styrene, plasticizer, and xylene (DPX mounting medium) (44581, Sigma-Aldrich, St. Louis, MO, USA). Adjacent sections on the same slides were counterstained with hematoxylin for general histological orientation. Statistics

[0209] Each experiment was repeated at least three times with triplication for each sample tested. The results were presented as mean േ SEM, unless otherwise indicated. Statistical mean differences were evaluated using unpaired t-test with GraphPad software, and p < 0.05 was considered statistically significant. References

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[0072] P. Rychahou, Y. Bae, D. Reichel, Y.Y. Zaytseva, E.Y. Lee, D. Napier, H.L. Weiss, N. Roller, H. Frohman, A.-T. Le, B. Mark Evers, Colorectal cancer lung metastasis treatment with polymer-drug nanoparticles, Journal of controlled release : official journal of the Controlled Release Society 275 (2018) 85-91. Example 2: In vitro and in vivo Evaluation of the Pathology and Safety Aspects of RNA Nanoparticles Abstract

[0282] RNA has developed into the third milestone in pharmaceutical drug development following chemical and protein therapeutics. RNA itself can serve either as therapeutics,TH Docket No.321502-2140 carriers, regulators, or substrates in drug development. Due to RNA’s motile, dynamic, and deformable properties, RNA nanoparticles have demonstrated spontaneous targeting and accumulation in cancer vasculature and fast excretion through the kidney glomerulus to urine to prevent possible interactions with healthy organs. Furthermore, the negatively charged phosphate backbone of RNA results in general repulsion from negatively charged lipid cell membranes for further avoidance of vital organs. Thus, RNA nanoparticles can first and efficiently target tumor tissue while clearing from the body quickly. These favorable parameters have led to the expectation that RNA has low or little toxicity. RNA nanoparticles have been well characterized for their anti-cancer efficacy; however, little detail on RNA nanoparticle pathology and safety is known. Here we report the in vitro and in vivo assessment of the pathology and safety aspects of different RNA nanoparticles including Three-way Junction (3WJ) and Four- way Junction (4WJ) with or without conjugation to chemical drugs. Both animal models and patient serum were investigated. In vitro studies include hemolysis, platelet aggregation, complement activation, plasma coagulation, and Interferon induction. In vivo studies include Hematoxylin and Eosin (H&E) staining, hematological and biochemical analysis as the serum profiling, and animal organ weight study. No significant toxicity, side-effect or immune responses were detected during the extensive safety evaluations of RNA nanoparticles. These results further complement previous cancer inhibition studies and demonstrate RNA nanoparticles as an effective and safe drug delivery vehicle for future clinical translations. Introduction

[0283] Only 1.5% of the human genome codes for proteins, while the remaining 98.5% codes for small or long non-coding RNAs proving its biological importance. RNA can serve as either a therapeutic that is delivered or as a substrate targeted by drugs. In recent years, RNA has gained focus in pharmaceutic and vaccine field, most notably through the FDA approval of RNA-based therapeutics1-3and authorization of mRNA COVID-19 vaccines4-7. These approvals confirm the safety and stability of RNA in clinical applications8-11. RNA therapeutics have now emerged as a prominent field and is becoming the third milestone in pharmaceutical drug development12-19.

[0284] RNA nanotechnology, similar to the system of Lego13, is to deal with the self- assembling of nanometer-scale structures composed mainly of RNA20-29. RNA nanoparticles can serve as therapeutics or drug delivery vehicles to cancers with strong inhibition15, 30-38. RNA nanoparticle-based drug delivery systems exhibit several advantages, including efficient and spontaneous accumulation in tumors, improved pharmacokinetics, and the undetectable gross toxicity and low immune responses25, 31, 39-44. These features also facilitate the repeatedTH Docket No.321502-2140 treatment of chronic diseases. It was found that RNA nanoparticles can be stretched or released over multiple rounds of repeats much like rubber45and this stretching is important for the beneficial in vivo features. Furthermore, RNA naturally exhibits elasticity, deformation, and motility, making it highly promising for spontaneous targeting toward cancer vasculature and fast excretion through the kidney glomerulus to urine13, 45. RNA nanoparticles exhibit rubber-like properties45-47, making them highly attractive as potential cancer therapeutics.

[0285] Our team has created multifunctional, ultra-stable RNA complexes with uniform nano-scale size, controllable structure, and precise stoichiometry, including Phi293WJ31and 4WJ10. These branched RNA nanoparticles offer multivalency and can be conjugation to chemical drugs for controlled delivery to the tumor microenvironment10, 31, 33, 48. These nanoparticles have proven great potential as cancer therapeutics by demonstrating specific tumor accumulation, delivery of therapeutics resulting in apoptosis, and initial safety with no gross toxicity. However, further data is necessary to overcome existing challenges and maximize RNA nanoparticle’s clinical benefits. It will be beneficial to evaluate in-depth the pathology, side effects, and toxicity of these RNA nanoparticles.

[0286] In this study, we aim to further prove the capability of RNA nanoparticles for cancer treatments by providing detailed pathology and safety parameters.3WJ and 4WJ nanoparticles with and without chemotherapeutic conjugates (Fig.10) were evaluated in vitro and in vivo, including hemolysis study, Platelet aggregation study, complement activation study, plasma coagulation study, interferons induction study, animal organ weight, H&E staining, serum biochemistry, and hematological study. Extensive evaluations revealed that these 3WJ and 4WJ RNA nanoparticles with and without drug conjugation did not have detectable toxicity, side effects, or immune responses. In general, the RNA nanoparticles provided safe profiles to serve as a viable drug delivery vehicle. In combination with the strong tumor inhibition of previous studies, there is a strong rationale to more towards the translation of RNA nanoparticles. Materials and Methods Design and preparation of RNA Nanoparticles

[0287] Multifunctional RNA nanoparticles were designed and constructed via bottom-up self-assembly approaches. The 3WJ nanoparticle is composed of three RNA fragments harboring 3WJ-A, 3WJ-B, and 3WJ-C strands. The three-way junction-Folic acid-Survivin siRNA RNA nanoparticles (3WJ-FA-siRNA) contained four RNA fragments harboring 3WJ-A-Folate, 3WJ-B-Survivin Sense, 3WJ-C, and Survivin anti-sense. The four-way junction SN38 conjugated RNA nanoparticles (4WJ-SN38) contained four RNA fragments respectablyTH Docket No.321502-2140 including 1) 4WJ-A-6 SN38, 2) 4WJ-B-6 SN38, 3) 4WJ-C-6 SN38, and 4) 4WJ-D-6 SN38. RNA sequences can be found in Supplementary information.

[0288] RNA strands were synthesized chemically using solid-phase synthesis.2′-Fluoro (2′-F) modified cytosine (2′F-C) and uracil (2′F-U) nucleotides were incorporated into all RNA strands except Survivin anti-sense stand for improving thermostability and nuclease-resistance of the RNA nanoparticles. SN38 was conjugated to RNA strands via click chemistry between Drug-Azido and RNA-6 alkyne using methods previously published10, 37.

[0289] After synthesis, RNA component strands were purified by ion-pair reversed- phase HPLC to remove salts, incomplete synthesis strands, or unconjugated chemicals. All RNA nanoparticles were prepared via thermocycler annealing via rapid heating to 95 degrees to denature all secondary structures and following by slow cooling down to 4 degrees. Analysis Hemolytic Properties of Nanoparticles

[0290] Hemolysis tests were performed in vitro using the NCL protocol ITA-149. First, fresh human blood was treated with lithium heparin to prevent clotting and was diluted using PBS into 10 mg / ml of total hemoglobin. Then the diluted sample was combined with the experimental materials and incubated at 37°C for 3h. After incubation, the cell-free liquid was processed, and the hemoglobin that was not bound to plasma was assessed by converting the hemoglobin and its metabolites into cyanmethemoglobin (CMH) with Drabkin’s reagent for measurement. The quantification of CMH was then carried out against a hemoglobin standardby measuring sample absorbance at 540 nm. The rate of Hemolysis was determined as% ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ൌ ^^^^^^^^^^ ^^ ௧^^ ௧^^௧ ^^^^^^்^ுௗ ൈ 100, where TBHd refers to total bloodhemoglobin

[0291] There are alternative versions of the hemolysis test accessible. These procedures do not involve converting hemoglobin into its enduring CMH state. Instead, they gauge the level of hemolysis by examining oxyhemoglobin at one of its main absorption points, specifically at the wavelength of 415, 541, or 577nm50. Analysis of Platelet Aggregation

[0292] The Platelet aggregation tests were conducted in vitro according to the NCL protocol ITA-2.251. Whole human blood was collected in sodium citrate tubes and then centrifuged to obtain Platelet rich plasma (PRP) and plasma poor plasma (PPP). The PRP was mixed with test samples and ChronoLum reagent and incubated at 37 °C for 6 mins in a Platelet aggregation profiler to measure whether RNA nanoparticle may induce Platelet aggregation. Additionally, The PRP was mixed with test samples, ChronoLum reagent, and Collagen and incubated at 37 °C for 6 mins in a Platelet aggregation profiler to measure whether RNATH Docket No.321502-2140 nanoparticle may block the Platelet aggregation caused by collagen. Collagen (100 μg / ml) mixed PRP was used as a positive control, PPP alone was used as a negative control, and non- treated PRP runs were internal test controls. A positive response was determined by a Platelet aggregation >20%. The ChronoLum reagent was employed for tracking ATP release from Platelets, providing an extra measure to verify that alterations in light transmission were indeed a result of Platelet aggregation. The ChronoLog aggregometer was used to determine the sample turbidity. However, a definition of PRP and PPP based on Platelet quantification would be more helpful to obtain a more precise result.

[0293] The aggregation percentage was determined using the following formula:

[0294] % ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ൌ^^^ ^^^^^^^^^ ^^^^௧^௩^ൈ 100. RNA nanoparticlesto measure complement activation using the NCL protocol ITA-5.252. Plasma was extracted from recently drawn human blood and mixed with test samples, and Cobra Venom Factor (CVF) was used as a positive control. After a 30-minute incubation at 37 °C, the samples were examined using a commercial enzyme immunoassay kit to detect the iC3b component of complement. The RNA nanoparticles were assessed at four RNA concentrations (0.14, 0.72, 3.6, and 35.6 µg / mL) while Doxil, a commercially relevant nanoparticle, was used as a positive control. Plasma Coagulation Test

[0296] The NCL ITA-1253method was used to perform this assay. Initially, human blood was collected in tubes containing sodium citrate. After centrifuging the blood at 2500 g for 10 minutes, plasma was collected. The samples were then incubated with plasma in a microcentrifuge tube for 30 minutes at 37 °C and subsequently centrifuged at 17000 g for 5 minutes. The STA-R Evolution coagulometer (Diagnostica Stago) was used to measure activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time after exposure. Non-treated plasma, PBS, and abnormal control plasma were utilized as untreated, negative control, and positive control respectively.

[0297] The standard coagulation times for the PT assay are 13.4 seconds or less, APTT is 34.1 seconds or less, and Thrombin assay is 21 seconds or less. Abnormal control plasma should have coagulation times above these limits. Interferon and Cytokine Induction Assay

[0298] The NCL method ITA-1054, ITA-2555, and ITA-2756were used to conduct this assay. In short, human PBMC were suspended in RPMI 1640 medium containing 10% FBS. The cells were then exposed to samples and incubated at 37 °C for 24 hours. Positive andTH Docket No.321502-2140 negative controls were lipopolysaccharides (LPS) at 20 ng / ml and PBS, respectively. After exposure, the supernatants were collected and underwent centrifugation at 12,000 RPM for 15 minutes. ELISA kits were used to measure interferon type I (IFN α, β, and ω), type II (IFN γ), and type III (IFN λ). Treatment plan of different RNA nanoparticles

[0299] All animals were kept in the National Defense Animal Center according to the protocol approved by the Association for Laboratory Animal Care Evaluation and Certification (LAC-101-0064). A549 lung cancer cells were utilized to establish xenograft models in male non-obese diabetic-severe combined immunodeficiency-IL2R gamma null (NSG) mice. The cells were transplanted into the dorsal region of 4-5 weeks-old mice. Once the tumor size reached 100 mm3, the mice bearing A549 xenografts were randomly allocated into three groups: PBS, SN38 free drug, and 4WJ-SN38, with each group consisting of five mice. To assess the efficacy of the treatments, samples were administered via intravenously (IV) through tail-vein injections. The dose was set at 2 mg / kg, and five injections were given on days 1, 3, 5, 7, and 9. The dosage used for the 4WJ-24 SN38 RNA nanoparticles in vivo safety experiments is kept the same as described in the in vivo treatment experiments (Xin Li and Peixuan Guo, unpublished results). The negative control group received PBS injections. Tumor size was monitored every two days throughout the treatment period, and the volume was calculated using the formula (length × width2) / 2. A two-tailed unpaired t-test was used as statistical analysis to compare the different treatment groups. The results were reported as mean ± SEM, with significance levels denoted as *p^<^0.05. Organ weight measurement

[0300] After the in vivo delivery of RNA nanoparticles, all animals, including tumor- bearing mice and healthy mice, were euthanized. The major organs, namely the liver, spleen, kidney, heart, and lung, were excised and harvested from the sacrificed mice. The organs were weighed directly using an electronic balance. The organ weight data were expressed as mean ± SEM. Statistical analysis of the organ weights was performed using a two-tailed t-test. Significance levels were indicated as *p < 0.05. H&E staining

[0301] After weighing major organs from the sacrificed mice, organs were subjected to fixation in 4% paraformaldehyde, embedding, and sectioning processes to generate formalin- fixed, paraffin-embedded (FFPE) sections. H&E staining was performed on the FFPE sections. Hematoxylin dye was used to impart a blue color to the cell nuclei, while eosin dye wasTH Docket No.321502-2140 employed to stain the cell cytoplasm and extracellular matrix pink. The H&E-stained sections were directly captured using a light microscope at a 10× magnification. Analysis of serum biochemistry in treated NSG mice

[0302] After in vivo delivery of RNA nanoparticles, cardiac blood was collected from each NSG mouse, including both the treated and healthy control groups, using cardiac puncture. The collected blood was then divided into two parts. One part was utilized for whole blood hematological analysis, while the other was centrifuged at 1200 g for 10 minutes to obtain serum for biochemical analysis. Hematological and biochemical analyses were performed at TMU Hospital following standard protocols. The hematological analysis was conducted using the Beckman Coulter DxH 800 automated hematology analyzer. The biochemical analysis used the Roche Cobas c702 analyzer. Statistical analysis of the data was performed using a two- tailed unpaired t-test. The results were presented as mean ± SEM. Significance levels were denoted as *p < 0.05. Analysis of hematology of treated NSG mice

[0303] All animals, including tumor-bearing mice, were included in the study. The data obtained were presented as mean ± SEM values. Cardiac blood samples were collected from each NSG mouse at the endpoint of the treatment for hematological analysis. Statistical analysis of the organ weight data was performed using a two-tailed t-test. Significance levels were denoted as *p < 0.05. Statistics

[0304] The experiment was conducted multiple times, ensuring a minimum of three repetitions for each sample tested. The results were reported as the mean value accompanied by the standard error of the mean (SEM), unless stated otherwise. Statistical analysis of mean differences was performed using GraphPad software, employing the unpaired t-test. A significance level of p < 0.05 was used to determine statistical significance. Results Design and quality check of RNA Nanoparticles

[0305] Two 3WJ RNA nanoparticles were designed based on our previous published results (Fig.10A-10B)31. The first nanoparticle was a 3WJ scaffold with pyramidines modified with fluorine at the 2′ location (Fig.10A). The second nanoparticle is a 2’F 3WJ scaffold with the 3WJ-A strand 5′ end modified with Folate Acid (FA), the 3WJ-B strand 5′ end extended with the sense strand of survivin (BRCCA1) siRNA, the 3WJ-C strand, and the survivin (BRCCA1) anti- sense strand (Fig.1B). No bacterial contamination was detected in either 3WJ or 3WJ-FA- siRNA nanoparticle samples, and endotoxin levels were below the Endotoxin Limit (EL) for bothTH Docket No.321502-2140 samples. The selection of nanoparticle and RNA nanoparticle concentration used in the in vitro experiments is a follow-up of our past pharmacological characterization research9. In that research, the RNA nanoparticles did not induce interferon response in vitro at a concentration of 4ug / ml. A further investigation on the hemolysis, Platelet aggregation, plasma coagulation, complement activation, and Interferon (IFN) induction profile of the RNA nanoparticles is in reference to the concentrations as reported before9.

[0306] We also previously designed a 4WJ RNA nanoparticle to carry different chemotherapeutics10. For 4WJ-24 SN38 RNA nanoparticles (Fig.10C), 4WJ A, 4WJ B, 4WJ C, and 4WJ D RNA strands were modified with A) 2′F modified pyrimidines to result in nuclease- resistance and B) 2′-O-Propargyl Amidites to replace 6 bases on each 4WJ-A, B, C, and D strands for the click chemistry. SN38-N3was then conjugated to bases with 2′-O-Propargyl group via the click reaction (Fig.10C).4WJ-A-SN38, 4WJ-B-SN38, 4WJ-C-SN38, and 4WJ-D- SN38 are purified via RP-HPLC.

[0307] Following synthesis of component strands of both the 3WJ and 4WJ, the RNA nanoparticles were assembled through controlled thermocycling. Each of the three RNA nanoparticles assembled in one-pot single step with high efficiency. Polyacrylamide native gels confirmed RNA nanoparticle step-wise formation (data not shown) as previously published with similar nanoparticles10 31. As a result, stable RNA nanoparticles were created that with and without chemotherapeutics or siRNA to act as drug delivery vehicles. Our prior testing of these nanoplatforms have demonstrated significant inhibition in tumor growth in triple negative breast cancer10, prostate cancer41, colorectal cancer57, and more. Hemolysis study on RNA nanoparticles

[0308] Assaying interactions of RNA nanoparticles with blood components, specifically with Red blood cells, is of importance as RNA nanoparticles are best delivered intravenously and have the ability to continually interact with blood components until reaching the tumor microenvironment. Nanoparticles interacting with blood have resulted in significant toxicities and negative implications for clinical translation58, 59. However, the information about the interaction of RNA nanoparticles with Red blood cells is minimal. Therefore, a hemolysis study on the RNA nanoparticles was carried out to determine whether RNA nanoparticles can cause Red blood cell destruction. The hemolysis study is the starting point to evaluate the interaction between RNA nanoparticles and Red blood cells. Compared to the positive control group triggered by Triton X-100, hemolysis of 3WJ nanoparticles was below the limit of detection (BLOD) in a concentration ranging from 0.14 µg / ml to 3.6 µg / ml siRNA equivalent, same as the negative control PBS group (Fig.11A). Similar to 3WJ nanoparticles, 3WJ-FA-siRNA RNA nanoparticlesTH Docket No.321502-2140 show the same result without hemolysis phenomenon after administration (Fig.11B), and Folic Acid or siRNA clearly did not trigger hemolysis. This result clearly indicates the safety when administrating our RNA nanoparticles, and RNA nanoparticles alone or RNA nanoparticles with targeting and therapeutic groups clearly did not affect the integrity of human Red blood cells at any of the tested concentrations. Platelet aggregation and coagulation study on RNA nanoparticles

[0309] Nanoparticles become in contact with the Platelet within the blood after administration and may cause Platelet activation and aggregation60, 61. Serious bleeding or thrombosis phenomenon caused by interfering with normal Platelet function may risk patients’ lives. RNA nanoparticles have been used in vivo without causing blood aggregation, but their overall safety profile on the Platelet activation and aggregation is liminted. As a result, understanding the interaction between Platelet and RNA nanoparticles is critical for translational medicine. Compared to positive control group triggered by Collagen, both 3WJ and 3WJ-FA- siRNA RNA nanoparticles did not induce Platelet aggregation itself (Fig.12A and Fig.12B) and did not inhibit collagen induced aggregation (Fig.12C and Fig.12D) at any of the tested concentrations. This result clearly indicates the safety when administrating our RNA nanoparticles, and RNA nanoparticles alone or RNA nanoparticles with targeting and therapeutic groups clearly did not have blood aggregation concerns. Plasma coagulation study on RNA nanoparticles

[0310] Apart from Red blood cells and Platelets, nanoparticles may interact with many other protein components within the plasma62. The published information on plasma coagulation profile of RNA nanoparticles is limited. A blockage of important coagulation factors within plasma could be dangerous as it might increase the coagulation time. As a result, plasma coagulation test could be a valid tool to understand the overall safety profile after RNA nanoparticles being administered into the body. Compared to the negative control and untreated group, both groups of nanoparticles did not affect plasma coagulation time in vitro under the tested conditions (Fig.13). Prolongation of the plasma coagulation time samples that were exposed to nanoparticles suggests that these test-particles either deplete or inhibit the coagulation factor, and both 3WJ and 3WJ-FA-siRNA RNA nanoparticles clearly do not inhibit coagulation factors as tested (Prothrombin time, Activated Partial Thromboplastin time, and Thrombin time). This result clearly indicates the safety when administrating our RNA nanoparticles, and RNA nanoparticles alone or RNA nanoparticles with targeting and therapeutic groups clearly did not trigger plasma coagulation issues at any of the tested concentrations.TH Docket No.321502-2140 Complement activation study on RNA nanoparticles

[0311] Complement activation can be caused by some nanoparticles63like PEGylated nanoparticles64. The activation can cause acute inflammatory reactions and chronic Immunol responses63, and both may cause concerns about patients’ safety and the therapeutic efficacy of nanoparticles. An investigation of the in vitro complement activation against RNA nanoparticles is a critical part to understand the overall immune responses profile. Compared to the positive control group triggered by cobra venom factor (CVF) and Doxil, weak activation of complement (about 2-fold above baseline) of ic3b was detected in 3WJ nanoparticles treated plasma at the highest tested concentration (Fig.14A). However, the observed activation was more than 50% below that induced by Doxil, which is known to cause CARPA in sensitive patients in the clinic. The rest of the concentrations were undetectable or similar to the negative control group. Complement activation was not detected in plasma treated with 3WJ-FA-siRNA RNA nanoparticles even at the highest tested concentration (Fig.14A). Herein, we believe the immunogenicity of our RNA nanoparticles is relatively low.

[0312] Interferon (IFN) induction by RNA nanoparticles

[0313] The human body aims to target and destroy foreign materials through immune responses. Cytokines and chemokines, including interferons are induced and secreted by the immune system resulting in inflammation and destruction of foreign materials, primarily viral components. Viral RNA has shown to induce cytokine responses65; however, our previous results revealed that cytokine responses towards RNA nanoparticles are limited9, and the intensity of immune response to RNA nanoparticles are tunable based on size, shape, and sequence8, 66. It is important to further demonstrate our RNA nanoplatform is capable to be safe in cytokine responses, and specifically not induce interferon responses. Here, the 3WJ nanoparticles were tested for interferon induction using blood three different human donors. Compared to the negative control which is inducted by PBS and positive control which is inducted by either cobra venom factor or Doxil, the induction of all three types of interferons: type I (IFN α, β, and ω), type II (IFN γ) and type III (IFN λ) was evaluated on 3WJ-FA-siRNA nanoparticles. The induction of all interferons was detected in the positive control samples on every individual donor, however, IFN α, β, ω, and γ inductions are not detected in 3WJ-FA- siRNA treated patient samples (Fig.14B), which is similar to the negative control group. Limited IFN λ induction was detected on one donor group only at a relative low administration concentration (0.72 µg / ml) (Fig.14B) in 3WJ group, and we expect that is due to heterogenous response between individual donors. These results match with our past findings that RNA nanoparticles has limited IFN induction9.TH Docket No.321502-2140 In vivo safety profile of SN38 conjugated RNA nanoparticles: a case study

[0314] SN-38 is an active metabolite of the Topoisomerase I inhibitor Irinotecan, and SN-38 can inhibit both DNA and RNA synthesis. However, SN38 itself may cause adverse effects include severe diarrhea, myelosuppression, and neutropenia67. SN38 was a case study to understand the overall safety profile of RNA nanoparticles carrying chemotherapeutics while examining organ function and assayed for interactions with blood and organs. On a gross level of toxicity, organ weight change upon RNA nanoparticles carrying SN38 delivery was carried out. Heart, lung, or kidney weight changes were not observed compared to the PBS control group (Fig.15A). Reduced spleen weight and increased liver weights were observed in the 4WJ-SN38 group compared to the PBS group, and it may contribute to the toxic nature of SN38 itself (Fig.15A). To further investigate possible organ toxicities and to explain liver and spleen weight changes, H&E staining was used to examine possible lesions caused by RNA nanoparticles carrying chemotherapeutics. H&E staining is the histological assay combining Hematoxylin and Eosin. The H (Hematoxylin) stains cell nuclei into a purplish-blue color, and E (Eosin) stains the extracellular matrix and cytoplasm into pink color with other structures displaying different shades. Interestingly, no obvious tissue-level lesions were found in 4WJ- SN38 treatment groups when compared to the PBS control group (Fig.15B). Drug toxicity may cause necrotic and karyolysis cells that will show up in H&E staining68. Our data revealed that the morphology of cell nuclei is similar between both the PBS and 4WJ-SN38 treatment groups, and the unchanged nuclear morphology may indicate the limited amount of karyolysis. The cytoplasmic component distribution changes can be used to indicate the necrotic regions. No significant changes were detected in the 4WJ-SN38 treated and the PBS control groups.

[0315] Following 4WJ conjugated with SN38 delivery to mice, serum biochemistry studies were completed to examine compatibility with organ functionality. Serum biochemistry results showed free SN38 significantly increased the level of Glutamic Oxaloacetic Transaminase (GOT) and Glutamate Pyruvate Transaminase (GPT), but 4WJ-SN38 did not increase these two transaminases (Fig.16A). Both GOT and GPT elevated levels indicate a possible sign of liver damage, confirming liver toxicity as a common adverse effect from free SN3869. Furthermore, the Lactate dehydrogenase (LDH) levels were also observed to increase comparing SN38 free drug group with the PBS control group, and increased LDH level may indicate tissue damage70. SN38-carried RNA nanoparticles may mitigate tissue damage caused by SN38 (Fig.16A). Using 4WJ-SN38 may overcome liver toxicity and tissue damage, which is the advantage of using RNA nanoparticles to deliver chemotherapeutics. Hematological results further demonstrated that the White Blood Cell, Platelet, Hemoglobin, and Mean CorpuscularTH Docket No.321502-2140 Hemoglobin concentration levels are significantly reduced for SN38 free drug group compared to the PBS control group while the 4WJ-SN38 group shows negligible effect (Fig.16B). The reduction of WBC and Platelet matches other groups’ findings when administering SN3871, and the reduction of Hemoglobin level is common indication of anemia when administering chemotherapy drugs72. Especially, considering neutropenia as one main side effect of SN38, 4WJ-SN38 does not cause neutropenia since there is no reduction on White blood cell count. These results demonstrate SN38 conjugated RNA nanoparticles can mitigate abnormal conditions at the hematological level. These results further support 4WJ-SN38 RNA nanoparticles may improve the overall safety profile of SN38 for in vivo application, while further enhancing therapeutic delivery to tumors as we have previously demonstrated. Discussion

[0316] The parameters of the pathology, safety, and side effects of RNA nanoparticles including 3WJ and 4WJ bound to chemotherapeutic drugs were investigated. Studies on in vitro and in vivo hemolysis, Platelet aggregation, complement activation, plasma coagulation, interferon induction, cytokine induction, organ weight, H&E staining, serum biochemistry, and hematology revealed that no significant detected toxicity, side effects or immune responses. To our best knowledge, this is the first comprehensive hemolysis level, Platelet aggregation level, complement activation level, and plasma coagulation level study on RNA nanoparticles further advancing the field of RNA nanotechnology towards the clinic. Immunogenicity level studies match our past data8, 9. In vivo study further verifies the advantage of using RNA nanoparticles to deliver chemotherapeutics compared to free drug.

[0317] Recently, protein corona formation at the surface of nanoparticles has been identified73. The protein corona formation can affect the biodistribution and safety of nanoparticles. Whether such corona formation can occur with RNA nanoparticles is an intriguing question. The immediate effect of protein corona will be the change in morphology, size, and Zeta potential of the RNA nanoparticles74, 75. The shielding effect of protein corona could lead to a longer circulation time and a slow release of payload from the RNA nanoparticles as the corona may block the binding between RNA nanoparticles and RNase76. The attachment of the albumin onto the RNA nanoparticles may also increase the retention of RNA nanoparticles at the tumor sites77, however, it might also hinder the renal excretion and body clearance. The protein corona formation may further change the surface charge of the RNA nanoparticles74, and it might lead to an unfavorable healthy organ accumulation which is negligible among negatively charged RNA nanoparticles37. To summarize, the protein corona formation may have both positive and negative effects on the RNA nanoparticles. The application of artificialTH Docket No.321502-2140 intelligence in understanding protein corona formation on RNA nanoparticles has also been reported73, 78, 79, and a more detailed investigation of the relationship between RNA nanoparticles and protein corona is important. Our preliminary studies indicated that serum albumin binding is RNA shape, size, and stoichiometry dependent, and the corona formation of 4WJ might not be a major concern. References

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[0397] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0398] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

TH Docket No.321502-2140 CLAIMS 1. An RNA nanoparticle comprising at least three synthetic RNA nucleotides coupled to each other, wherein the at least three synthetic RNA oligonucleotides form a central ore domain and at least three double-stranded arms arranged around the core domain and extending away from the central core domain, wherein at least one of the three double-stranded arms is conjugated with Irinotecan and / or SN-38 (7-Ethyl-10-hydroxycamptothecin) with an ester-bond that is cleavable by esterase in cancer tissue or cancer cells.

2. The RNA nanoparticle of claim 1, wherein at least one of the synthetic RNA oligonucleotides comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1-236, or a variant thereof having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1-236.

3. The RNA nanoparticle of claim 2, wherein at least one of the synthetic RNA oligonucleotides comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOS:160-214, or a variant thereof having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 160-214.

4. The RNA nanoparticle of claim 2, comprising (a) a first synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-XXGYYAXGXGXAXGXGGG-3’; 5’-XXGYYAXGXGXAXGXGGGGGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGG C-3’ (SEQ ID NO:1); 5’-XXGYYAXGXGXAXGXGGGGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUU UCCC-3’ (SEQ ID NO:2); 5’-XXGYYXGXGXAXGXGGGGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:3); 5’-XXGYYAXGXGXAXGXGGGCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:4); 5’-GalNAc-XXGYYAXGXGXAXGXGGG-3’; 5’-FA-XXGYYAXGXGXAXGXGGG-3’; 5’- DCL-XXGYYAXGXGXAXGXGGG-3’; and 5’-UAMC1110-XXGYYAXGXGXAXGXGGG-3’; (b) a second synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-YYYAYAXAYXXXGXXGAXYY-3’; 5’ YYYAYAXAYXXXGXXGAXYYGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC- 3’ (SEQ ID NO:5); 5’ YYYAYAXAYXXXGXXGAXYYGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUU UCCC-3’ (SEQ ID NO:6); 5’-YYYAYAXAYXXXGXXGAXYYGCGACUGGUUACCCGGUCG-3’TH Docket No.321502-2140 (SEQ ID NO:7); 5’-YYYAYAXAYXXXGXXGAXYYCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:8); 5’-GalNAc-YYYAYAXAYXXXGXXGAXYY-3’; 5’-FA-YYYAYAXAYXXXGXXGAXYY-3’; 5’- DCL-YYYAYAXAYXXXGXXGAXYY-3’; and 5’-UAMC1110-YYYAYAXAYXXXGXXGAXYY-3’; and (c) a third synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-GGAXYAAXYAXGGYAA-3’ (SEQ ID NO:9); 5’ GGAXYAAXYAXGGYAAGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:10); 5’ GGAXYAAXYAXGGYAAGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUUUCC C-3’ (SEQ ID NO:11); 5’-GGAXYAAXYAXGGYAAGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:12); 5’-GGAXYAAXYAXGGYAACAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:13); 5’- GalNAc-GGAXYAAXYAXGGYAA-3’ (SEQ ID NO:9); 5’-FA-GGAXYAAXYAXGGYAA-3’ (SEQ ID NO:9); 5’-DCL-GGAXYAAXYAXGGYAA-3’ (SEQ ID NO:9); and 5’-UAMC1110- GGAXYAAXYAXGGYAA-3’ (SEQ ID NO:9), wherein X is SN38 and wherein Y is Irinotecan.

5. The RNA nanoparticle of claim 2, comprising (a) a first synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAA-3’ (SEQ ID NO:14); 5’ XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAAGCCUUAGUAACGUGCUUUGA UGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:15); 5’ XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAAGGGACCGAAAAAGACCUGAC UUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:16); 5’ XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAAGCGACUGGUUACCCGGUCG- 3’ (SEQ ID NO:17); 5’ XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAACAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:18); 5’-GalNAc-XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAA-3’ (SEQ ID NO:14); 5’-FA-XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAA-3’ (SEQ ID NO:14); 5’-DCL-XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAA-3’ (SEQ ID NO:14); 5’-UAMC1110-XXAGGXAAAGYYAYYXGYAGGXGYXAYYGAXGXAAXXYAA-3’ (SEQ ID NO:14); (b) a second synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of:TH Docket No.321502-2140 5’ XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAG-3’ (SEQ ID NO:19); 5’ XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAGGCCUUAGUAACGUGCUUU GAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:20); 5’ XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAGGGGACCGAAAAAGACCUGA CUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:21); 5’ XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAGGCGACUGGUUACCCGGUC G-3’ (SEQ ID NO:22); 5’ XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAGCAGAACGUAUACUAUUCUG -3’ (SEQ ID NO:23); 5’ GalNAc XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAG-3’ (SEQ ID NO:19); 5’-FA- XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAG-3’ (SEQ ID NO:19); 5’-DCL- XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAG-3’ (SEQ ID NO:19); and 5’- UAMC1110-XXGAAXXAYAXYGGXAGYAYGGGYXGXGYGAGGYXGAAYAG-3’ (SEQ ID NO:19); (c) a third synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGG-3’ (SEQ ID NO:20); 5 YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGGGCCUUAGUAACGUGCUUUGAUG UCGAUUCGACAGGAGGC-3’ (SEQ ID NO:21); 5’ YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGGGGGACCGAAAAAGACCUGACUU CUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:22); 5’ YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGGGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:23); 5’ YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGGCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:24); 5’ GalNAc-YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGG-3’ (SEQ ID NO: 20); 5’ FA-YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGG-3’ (SEQ ID NO: 20); 5’ DCL-YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGG-3’ (SEQ ID NO: 20); and 5’ UAMC1110-YXGXXYAGYYXYGYAYAGYYAGYAYGYAYYXGAAXAGG-3’ (SEQ ID NO: 20); and (d) a fourth synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAA-3’ (SEQ ID NO:25); 5’ YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAAGCCUUAGUAACGUGCUUU GAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:26); 5’TH Docket No.321502-2140 YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAAGGGACCGAAAAAGACCUGA CUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:27); 5’ YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAAGCGACUGGUUACCCGGUC G-3’ (SEQ ID NO:28); 5’ YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAACAGAACGUAUACUAUUCUG -3’ (SEQ ID NO:29); 5’ GalNAc- YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAA-3’ (SEQ ID NO:25); 5’ FA- YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAA-3’ (SEQ ID NO:25); 5’ DCL- YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAA-3’ (SEQ ID NO:25); and 5’ UAMC1110-YYXAXXYAGGXGYGXGYXGGGYXGYAGGXGGYXXXAYYXAA-3’ (SEQ ID NO:25), wherein X is SN38 and wherein Y is Irinotecan.

6. The RNA nanoparticle of claim 2, comprising (a) a first synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYG-3’ (SEQ ID NO:30); 5’ GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYGGCCUUAGUAACGUGCUUU GAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:31); 5’ GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYGGGGACCGAAAAAGACCUG ACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:32); 5’ GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYGGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:33); 5’ GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYGCAGAACGUAUACUAUUCU G-3’ (SEQ ID NO:34); 5’ GalNAc GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYG-3’ (SEQ ID NO:30); 5’-FA- GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYG-3’ (SEQ ID NO:30); 5’-DCL- GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYG-3’ (SEQ ID NO:30); and 5’ UAMC1110 GAGXAXAXGXXAGGYYXGGGXGAGXYYXXGYGXYXXYXAYYG-3’ (SEQ ID NO:30); (b) a second synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYY-3’ (SEQ ID NO:35); 5’ YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYYGCCUUAGUAACGUGCUUU GAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:36); 5’TH Docket No.321502-2140 YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYYGGGACCGAAAAAGACCUG ACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:37); 5’ YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYYGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:38); 5’ YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYYCAGAACGUAUACUAUUCU G-3’ (SEQ ID NO:39); 5’ GalNAc YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYY-3’ (SEQ ID NO:35); 5’-FA- YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYY-3’ (SEQ ID NO:35); 5’-DCL- YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYY-3’ (SEQ ID NO:35); and 5’ UAMC1110 YGGXAGAAGAYGYAAGGAYXXGYXAGXXGXGGXAYXGXXYYY-3’ (SEQ ID NO:35); (c) a third synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYA-3’ (SEQ ID NO:40); 5’ GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYAGCCUUAGUAACGUGCUUU GAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:41); 5’ GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYAGGGACCGAAAAAGACCUG ACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:42); 5’ GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYAGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:43); 5’ GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYACAGAACGUAUACUAUUCU G-3’ (SEQ ID NO:44); 5’ GalNAc GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYA-3’ (SEQ ID NO:40); 5’-FA- GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYA-3’ (SEQ ID NO:40); 5’-DCL- GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYA-3’ (SEQ ID NO:40); and 5’ UAMC1110 GGGAAYAGXAYYAYAAYXAGXGXYYYGGGAXAGGGAYAXAYA-3’ (SEQ ID NO:40); and (d) a fourth synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXY-3’ (SEQ ID NO:45); 5’ XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXYGCCUUAGUAACGUGCUUU GAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:46); 5’ XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXYGGGACCGAAAAAGACCUGA CUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:47); 5’TH Docket No.321502-2140 XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXYGCGACUGGUUACCCGGUC G-3’ (SEQ ID NO:48); 5’ XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXYCAGAACGUAUACUAUUCUG -3’ (SEQ ID NO:49); 5’ GalNAc XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXY-3’ (SEQ ID NO:45); 5’-FA- XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXY-3’ (SEQ ID NO:45); 5’-DCL- XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXY-3’ (SEQ ID NO:45); or 5’- UAMC1110-XGXAXGXYYYXAXYYYGGGAXGYYYAGGYYXAAYAXAXAYXY-3’ (SEQ ID NO:45), wherein X is SN38 and wherein Y is Irinotecan.

7. The RNA nanoparticle of claim 2, comprising (a) a first synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-UUGYYAUGUGUAUGUGGG-3’ (SEQ ID NO:50); 5’ UUGYYAUGUGUAUGUGGGGGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC -3’ (SEQ ID NO:51); 5’ UUGYYAUGUGUAUGUGGGGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUUU CCC-3’ (SEQ ID NO:52); 5’-UUGYYAUGUGUAUGUGGGGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:53); 5’-UUGYYAUGUGUAUGUGGGCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:54); 5’-GalNAc-UUGYYAUGUGUAUGUGGG-3’ (SEQ ID NO:50); 5’-FA- UUGYYAUGUGUAUGUGGG-3’ (SEQ ID NO:50); 5’-DCL-UUGYYAUGUGUAUGUGGG-3’ (SEQ ID NO:50); and 5’-UAMC1110-UUGYYAUGUGUAUGUGGG-3’ (SEQ ID NO:50); (b) a second synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’ YYYAYAUAYUUUGUUGAUYY-3’ (SEQ ID NO:55); 5’ YYYAYAUAYUUUGUUGAUYYGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC -3’ (SEQ ID NO:56); 5’ YYYAYAUAYUUUGUUGAUYYGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGU UUCCC-3’ (SEQ ID NO:57); 5’ YYYAYAUAYUUUGUUGAUYYGCGACUGGUUACCCGGUCG- 3’ (SEQ ID NO:58); 5’ YYYAYAUAYUUUGUUGAUYYCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:59); 5’ GalNAc-YYYAYAUAYUUUGUUGAUYY-3’ (SEQ ID NO:55); 5’ FA YYYAYAUAYUUUGUUGAUYY-3’ (SEQ ID NO:55); 5’ DCL YYYAYAUAYUUUGUUGAUYY-3’ (SEQ ID NO:55); and 5’ UAMC1110 YYYAYAUAYUUUGUUGAUYY-3’ (SEQ ID NO:55); andTH Docket No.321502-2140 (c) a third synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-GGAUYAAUYAUGGYAA-3’ (SEQ ID NO:60); 5’ GGAUYAAUYAUGGYAAGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:61); 5’ GGAUYAAUYAUGGYAAGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUUUCC C-3’ (SEQ ID NO:62); 5’ GGAUYAAUYAUGGYAAGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:63); 5’ GGAUYAAUYAUGGYAACAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:64); 5’ GalNAc-GGAUYAAUYAUGGYAA-3’ (SEQ ID NO: 60); 5’ FA-GGAUYAAUYAUGGYAA-3’ (SEQ ID NO: 60); 5’ DCL-GGAUYAAUYAUGGYAA-3’ (SEQ ID NO: 60); and 5’ UAMC1110 GGAUYAAUYAUGGYAA-3’ (SEQ ID NO: 60), wherein Y is Irinotecan.

8. The RNA nanoparticle of claim 2, comprising (a) a first synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAA-3’ (SEQ ID NO:65); 5’ UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAAGCCUUAGUAACGUGCUUUG AUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:66); 5’ UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAAGGGACCGAAAAAGACCUGA CUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:67); 5’ UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAAGCGACUGGUUACCCGGUCG -3’ (SEQ ID NO:68); 5’ UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAACAGAACGUAUACUAUUCUG- 3’ (SEQ ID NO:69); 5’ GalNAc UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAA- 3’ (SEQ ID NO: 65); 5’-FA-UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAA-3’ (SEQ ID NO: 65); 5’ DCL-UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAA-3’ (SEQ ID NO: 65); and 5’ UAMC1110- UUAGGUAAAGYYAYYUGYAGGUGYUAYYGAUGUAAUUYAA-3’ (SEQ ID NO: 65); (b) a second synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAG-3’ (SEQ ID NO:70); 5’ UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAGGCCUUAGUAACGUGCUUU GAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:71); 5’ UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAGGGGACCGAAAAAGACCUGTH Docket No.321502-2140 ACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:72); 5’ UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAGGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:73); 5’ UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAGCAGAACGUAUACUAUUCU G-3’ (SEQ ID NO:74); 5’ GalNAc UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAG-3’ (SEQ ID NO:70); 5’-FA- UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAG-3’ (SEQ ID NO:70); 5’-DCL- UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAG-3’ (SEQ ID NO:70); and 5’- UAMC1110-UUGAAUUAYAUYGGUAGYAYGGGYUGUGYGAGGYUGAAYAG-3’ (SEQ ID NO:70); (c) a third synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGG-3’ (SEQ ID NO:75); 5’ YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGGGCCUUAGUAACGUGCUUUGAU GUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:76); 5’ YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGGGGGACCGAAAAAGACCUGACUU CUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:77); 5’ YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGGGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:78); 5’ YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGGCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:79); 5’-GalNAc-YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGG-3’ (SEQ ID NO:75); 5’-FA-YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGG-3’ (SEQ ID NO:75); 5’-DCL-YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGG-3’ (SEQ ID NO:75); and 5’-UAMC1110-YUGUUYAGYYUYGYAYAGYYAGYAYGYAYYUGAAUAGG-3’ (SEQ ID NO:75); and (d) a fourth synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAA-3’ (SEQ ID NO:80); 5’ YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAAGCCUUAGUAACGUGCUUU GAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:81); 5’ YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAAGGGACCGAAAAAGACCUG ACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:82); 5’ YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAAGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:83); 5’TH Docket No.321502-2140 YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAACAGAACGUAUACUAUUCU G-3’ (SEQ ID NO:84); 5’ GalNAc YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAA-3’ (SEQ ID NO:80); 5’-FA- YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAA-3’ (SEQ ID NO:80); 5’-DCL- YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAA-3’ (SEQ ID NO:80); 5’ UAMC1110 YYUAUUYAGGUGYGUGYUGGGYUGYAGGUGGYUUUAYYUAA-3’ (SEQ ID NO:80), wherein Y is Irinotecan.

9. The RNA nanoparticle of claim 2, comprising (a) a first synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYG-3’ (SEQ ID NO:85); 5’ GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYGGCCUUAGUAACGUGCU UUGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:86); 5’ GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYGGGGACCGAAAAAGACCU GACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:87); 5’ GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYGGCGACUGGUUACCCGG UCG-3’ (SEQ ID NO:88); 5’ GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYGCAGAACGUAUACUAUUC UG-3’ (SEQ ID NO:89); 5’ GalNAc GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYG-3’ (SEQ ID NO:85); 5’-FA- GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYG-3’ (SEQ ID NO:85); 5’- DCL-GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYG-3’ (SEQ ID NO:85); and 5’ UAMC1110 GAGUAUAUGUUAGGYYUGGGUGAGUYYUUGYGUYUUYUAYYG-3’ (SEQ ID NO:85); (b) a second synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYY-3’ (SEQ ID NO:90); 5’ YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYYGCCUUAGUAACGUGCUU UGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:91); 5’ YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYYGGGACCGAAAAAGACCU GACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:92); 5’ YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYYGCGACUGGUUACCCGG UCG-3’ (SEQ ID NO:93); 5’TH Docket No.321502-2140 YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYYCAGAACGUAUACUAUUC UG-3’ (SEQ ID NO:94); 5’ GalNAc YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYY-3 (SEQ ID NO:90); 5’-FA- YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYY-3’ (SEQ ID NO:90); 5’- DCL-YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYY-3’ (SEQ ID NO:90); and 5’ UAMC1110 YGGUAGAAGAYGYAAGGAYUUGYUAGUUGUGGUAYUGUUYYY-3’ (SEQ ID NO:90); (c) a third synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYA-3’ (SEQ ID NO:95); 5’ GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYAGCCUUAGUAACGUGCUU UGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:96); 5’ GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYAGGGACCGAAAAAGACCU GACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:97); 5’ GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYAGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:98); 5’ GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYACAGAACGUAUACUAUUCU G-3’ (SEQ ID NO:99); 5’ GalNAc GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYA-3’ (SEQ ID NO:95); 5’-FA- GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYA-3’ (SEQ ID NO:95); 5’-DCL- GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYA-3’ (SEQ ID NO:95); and 5’ UAMC1110 GGGAAYAGUAYYAYAAYUAGUGUYYYGGGAUAGGGAYAUAYA-3’ (SEQ ID NO:95); and (d) a fourth synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUY-3’ (SEQ ID NO:100); 5’ UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUYGCCUUAGUAACGUGCUU UGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:101); 5’ UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUYGGGACCGAAAAAGACCUG ACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:102); 5’ UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUYGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:103); 5’ UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUYCAGAACGUAUACUAUUCU G-3’ (SEQ ID NO:104); 5’ GalNAcTH Docket No.321502-2140 UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUY-3’ (SEQ ID NO:100); 5’-FA- UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUY-3’ (SEQ ID NO:100); 5’- DCL-UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUY-3’ (SEQ ID NO:100); and 5’ UAMC1110 UGUAUGUYYYUAUYYYGGGAUGYYYAGGYYUAAYAUAUAYUY-3’ (SEQ ID NO:100), wherein Y is Irinotecan.

10. The RNA nanoparticle of claim 2, comprising (a) a first synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-XXGCCAXGXGXAXGXGGG-3’ (SEQ ID NO:105); 5’ XXGCCAXGXGXAXGXGGGGGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC- 3’ (SEQ ID NO:106); 5’ XXGCCAXGXGXAXGXGGGGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUUU CCC-3’ (SEQ ID NO:107); 5’-XXGCCAXGXGXAXGXGGGGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:108); 5’-XXGCCAXGXGXAXGXGGGCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:109); 5’-GalNAc-XXGCCAXGXGXAXGXGGG-3’ (SEQ ID NO:105); 5’ FA XXGCCAXGXGXAXGXGGG-3’ (SEQ ID NO:105); 5’-DCL-XXGCCAXGXGXAXGXGGG-3’ (SEQ ID NO:105); and 5’-UAMC1110-XXGCCAXGXGXAXGXGGG-3’ (SEQ ID NO:105); (b) a second synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-CCCACAXACXXXGXXGAXCC-3’ (SEQ ID NO:110); 5’ CCCACAXACXXXGXXGAXCCGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC -3’ (SEQ ID NO:111); 5’ CCCACAXACXXXGXXGAXCCGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGU UUCCC-3’ (SEQ ID NO:112); 5’ CCCACAXACXXXGXXGAXCCGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:113); 5’ CCCACAXACXXXGXXGAXCCCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:114); 5’ GalNAc- CCCACAXACXXXGXXGAXCC-3’ (SEQ ID NO:110); 5’ FA CCCACAXACXXXGXXGAXCC-3’ (SEQ ID NO:110); 5’ DCL CCCACAXACXXXGXXGAXCC-3’ (SEQ ID NO:110); and 5’ UAMC1110 CCCACAXACXXXGXXGAXCC-3’ (SEQ ID NO:110); (c) a third synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-GGAXCAAXCAXGGCAA-3’ (SEQ ID NO:115); 5’ GGAXCAAXCAXGGCAAGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC-3’TH Docket No.321502-2140 (SEQ ID NO:116); 5’ GGAXCAAXCAXGGCAAGGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUUUCC C-3’ (SEQ ID NO:117); 5’-GGAXCAAXCAXGGCAAGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:118); 5’-GGAXCAAXCAXGGCAACAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:119); 5’ GalNAc-GGAXCAAXCAXGGCAA-3’ (SEQ ID NO:115); 5’-FA-GGAXCAAXCAXGGCAA-3’ (SEQ ID NO:115); 5’-DCL-GGAXCAAXCAXGGCAA-3’ (SEQ ID NO:115); 5’-UAMC1110- GGAXCAAXCAXGGCAA-3’ (SEQ ID NO:115), wherein X is SN38.

11. The RNA nanoparticle of claim 2, comprising (a) a first synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAA-3’ (SEQ ID NO:120); 5’ XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAAGCCUUAGUAACGUGCUUUG AUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:121); 5’ XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAAGGGACCGAAAAAGACCUGAC UUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:122); 5’ XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAAGCGACUGGUUACCCGGUCG- 3’ (SEQ ID NO:123); 5’ XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAACAGAACGUAUACUAUUCUG- 3’ (SEQ ID NO:124); 5’ GalNAc XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAA-3’ (SEQ ID NO:120); 5’-FA- XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAA-3’ (SEQ ID NO:120); 5’ DCL- XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAA-3’ (SEQ ID NO:120); and 5’- UAMC1110-XXAGGXAAAGCCACCXGCAGGXGCXACCGAXGXAAXXCAA-3’ (SEQ ID NO:120); (b) a second synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAG-3’ (SEQ ID NO:125); 5’ XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAGGCCUUAGUAACGUGCUUU GAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:126); 5’ XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAGGGGACCGAAAAAGACCUG ACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:127); 5’ XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAGGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:128); 5’TH Docket No.321502-2140 XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAGCAGAACGUAUACUAUUCU G-3’ (SEQ ID NO:129); 5’ GalNAc XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAG-3’ (SEQ ID NO:125); 5’-FA- XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAG-3’ (SEQ ID NO:125); 5’-DCL- XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAG-3’ (SEQ ID NO:125); and 5’ UAMC1110 XXGAAXXACAXCGGXAGCACGGGCXGXGCGAGGCXGAACAG-3’ (SEQ ID NO:125); (c) a third synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGG-3’ (SEQ ID NO:130); 5’ CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGGGCCUUAGUAACGUGCUUUGAU GUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:131); 5’ CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGGGGGACCGAAAAAGACCUGACU UCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:132); 5’ CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGGGCGACUGGUUACCCGGUCG-3’ (SEQ ID NO:133); 5’ CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGGCAGAACGUAUACUAUUCUG-3’ (SEQ ID NO:134); 5’-GalNAc-CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGG-3’ (SEQ ID NO:130); 5’-FA-CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGG-3’ (SEQ ID NO:130); 5’-DCL-CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGG-3’ (SEQ ID NO:130); and 5’-UAMC1110-CXGXXCAGCCXCGCACAGCCAGCACGCACCXGAAXAGG-3’ (SEQ ID NO:130); and (d) a fourth synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAA-3’ (SEQ ID NO:135); 5’ CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAAGCCUUAGUAACGUGCUUU GAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:136); 5’ CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAAGGGACCGAAAAAGACCUG ACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:137); 5’ CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAAGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:138); 5’ CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAACAGAACGUAUACUAUUCU G-3’ (SEQ ID NO:139); 5’ GalNAc CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAA-3’ (SEQ ID NO:135); 5’-FA-TH Docket No.321502-2140 CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAA-3’ (SEQ ID NO:135); 5’-DCL- CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAA-3’ (SEQ ID NO:135); and 5’ UAMC1110 CCXAXXCAGGXGCGXGCXGGGCXGCAGGXGGCXXXACCXAA-3’ (SEQ ID NO:135), wherein X is SN38.

12. The RNA nanoparticle of claim 2, comprising (a) a first synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCG-3’ (SEQ ID NO:140); 5’ GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCGGCCUUAGUAACGUGCUU UGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:141); 5’ GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCGGGGACCGAAAAAGACCU GACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:142); 5’ GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCGGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:143); 5’ GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCGCAGAACGUAUACUAUUC UG-3’ (SEQ ID NO:144); 5’ GalNAc GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCG-3’ (SEQ ID NO:140); 5’-FA- GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCG-3’ (SEQ ID NO:140); 5’- DCL-GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCG-3’ (SEQ ID NO:140); 5’ UAMC1110 GAGXAXAXGXXAGGCCXGGGXGAGXCCXXGCGXCXXCXACCG-3’ (SEQ ID NO:140); (b) a first synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCC-3’ (SEQ ID NO:145); 5’ CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCCGCCUUAGUAACGUGCUU UGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:146); 5’ CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCCGGGACCGAAAAAGACCU GACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:147); 5’ CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCCGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:148); 5’ CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCCCAGAACGUAUACUAUUC UG-3’ (SEQ ID NO:149); 5’ GalNAc CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCC-3’ (SEQ ID NO:145); 5’-FA-TH Docket No.321502-2140 CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCC-3’ (SEQ ID NO:145); 5’- DCL-CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCC-3’ (SEQ ID NO:145); and 5’ UAMC1110 CGGXAGAAGACGCAAGGACXXGCXAGXXGXGGXACXGXXCCC-3’ (SEQ ID NO:145); (c) a first synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACA-3’ (SEQ ID NO:150); 5’ GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACAGCCUUAGUAACGUGCUU UGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:151); 5’ GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACAGGGACCGAAAAAGACCU GACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:152); 5’ GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACAGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:153); 5’ GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACACAGAACGUAUACUAUUC UG-3’ (SEQ ID NO:154); 5’ GalNAc GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACA-3’ (SEQ ID NO:150); 5’-FA- GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACA-3’ (SEQ ID NO:150); 5’- DCL-GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACA-3’ (SEQ ID NO:150); and 5’ UAMC1110 GGGAACAGXACCACAACXAGXGXCCCGGGAXAGGGACAXACA-3’ (SEQ ID NO:150); and (d) a first synthetic RNA oligonucleotide having the nucleic acid sequence selected from the group consisting of: 5’-XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXC-3’ (SEQ ID NO:155); 5’ XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXCGCCUUAGUAACGUGCUU UGAUGUCGAUUCGACAGGAGGC-3’ (SEQ ID NO:156); 5’ XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXCGGGACCGAAAAAGACCU GACUUCUAUACUAAGUCUACGUUUCCC-3’ (SEQ ID NO:157); 5’ XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXCGCGACUGGUUACCCGGU CG-3’ (SEQ ID NO:158); 5’ XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXCCAGAACGUAUACUAUUCU G-3’ (SEQ ID NO:159); 5’ GalNAc XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXC-3’ (SEQ ID NO:155); 5’-FA- XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXC-3’ (SEQ ID NO:155); 5’- DCL-XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXC-3’ (SEQ ID NO:155);TH Docket No.321502-2140 or 5’ UAMC1110 XGXAXGXCCCXAXCCCGGGAXGCCCAGGCCXAACAXAXACXC-3’ (SEQ ID NO:155), wherein X is SN38.

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