CN118434452A - Therapeutic compounds for red blood cell mediated delivery of active pharmaceutical ingredients to target cells - Google Patents

Abstract

Therapeutic compounds are described for use in red blood cell mediated delivery of active pharmaceutical ingredients to target cells. The therapeutic compound is configured to bind CD47 on the surface of erythrocytes and subsequently transfer to CD47 on the surface of target cells, which is ultimately internalized by the target cells by endocytosis. The target cell may be a cancer cell, a virus-infected cell, or a fibrotic cell.

Description

Therapeutic compounds for erythrocyte-mediated delivery of active pharmaceutical ingredients to target cells

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/281,370 filed on November 19, 2021 and U.S. Provisional Application No. 63/392,323 filed on July 26, 2022, each of which is incorporated herein by reference in its entirety.

Technical Field

The present invention generally relates to therapeutic compounds configured to bind CD47, and more particularly, to such compounds configured to bind CD47 on the surface of erythrocytes and subsequently translocate to CD47 on the surface of target cells, where the therapeutic compounds are ultimately internalized by the target cells via endocytosis.

Background technique

Cluster of differentiation 47 ("CD47") is an integrin-associated protein, a multi-spanning plasma membrane protein that is involved in the process of inhibiting phagocytic clearance or neutrophil motility. Signal regulatory protein alpha ("SIRPα") is a transmembrane protein expressed by innate immune cells (such as macrophages and dendritic cells) and is the main receptor for CD47. Binding of SIRPα to CD47 triggers the SIRPα inhibitory signal, which acts as a "don't eat me" signal to the recipient macrophage, preventing the activation of its phagocytosis. Therefore, the SIRPα-CD47 interaction acts as a negative checkpoint for innate immunity and subsequent adaptive immunity. Other proteins, such as signal regulatory protein γ ("SIRPγ") and thrombospondin-1 ("TSP-1"), can also bind to CD47, thereby inhibiting aspects of the immune response.

Mammalian cells usually express low levels of CD47 to protect them from being phagocytosed. However, cancer cells overexpress CD47 as an escape mechanism to escape immune surveillance and phagocytic attack. Some human solid tumors overexpress CD47, that is, the cells of these solid tumors express more CD47 than normal cells on average (Willingham et al. PNAS 109 (17): 6662-6667 (2012), the entire text of which is incorporated herein by reference). Therefore, CD47 has become a promising new therapeutic target for cancer immunotherapy (Willingham et al. PNAS 109 (17): 6662-6667 (2012); Weiskopf, Eur. J. Cancer 76: 100-109 (2017); Weiskopf et al. J Clin Invest 126 (7): 2610-2620 (2016), each of which is incorporated herein by reference in its entirety).

In addition, virus-infected cells also express high levels of CD47. These virus-infected cells include cells infected with SARS-CoV-2, which causes COVID-19 (Cham et al. Cell Rep 14; 31(2): 107494 (2020) doi: 10.1016/j.celrep.2020.03.058 and McLaughlin et al. bioRxiv 2021.03.01.433404 (2021) doi: 10.1101/2021.03.01.433404, each of which is incorporated herein by reference in its entirety). Blockade of CD47 inhibitory signaling has been shown to enhance innate and adaptive immune responses to viral infections.

In addition, increased CD47 expression has been observed in fibrotic fibroblasts, and blocking CD47 reverses fibrosis by enhancing phagocytosis of pro-fibrotic fibroblasts and by abolishing the inhibitory effects on adaptive immunity (Cui et al. Nat Commun 11:2795 (2020); Wernig et al. PNAS 2017; 114(18):4757-62; Boyd J Cyst Fibros Suppl 1:S54-S59 (2020); Lerbs et al. JCI Insight 2020; 5(16):e140458 (2020), each of which is incorporated herein by reference in its entirety).

Therefore, CD47 provides a promising target for the treatment of cancer, viral infections, and fibrotic diseases such as cystic fibrosis.

Summary of the invention

According to one embodiment of the present invention, a therapeutic compound for RBC-mediated delivery to a target cell expressing CD47 in a mammalian subject, the therapeutic compound comprising: a CD47 binding protein conjugated to an active pharmaceutical ingredient ("API") to form a conjugate; wherein the CD47 binding protein is selected from the group consisting of wild-type SIRPα (SEQ ID NO: 1), vSIRPα (SEQ ID NO: 3), wild-type thrombospondin-1 (TSP-1) (SEQ ID NO: 7), wild-type SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5), vSIRPγ-2 (SEQ ID NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), and ALX148 (SEQ ID NO: 965). NO:964), homologues of any of the foregoing, and combinations thereof, and configured to bind the conjugate to CD47 of the subject's erythrocytes to enable the conjugate to be transported to the target cells through the subject's circulatory system, so that (i) the CD47 binding protein configured to bind the conjugate to CD47 of the erythrocytes binds to CD47 of the target cells, thereby transferring the conjugate from the erythrocytes to the target cells to form a conjugate-CD47 complex on the target cells, thereby blocking CD47 and inhibiting CD47 activity as an immune escape mechanism of the target cells, and (ii) the conjugate is taken up by the target cells through endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune escape mechanism of the target cells and delivering the API to the target cells. The mammalian subject may be a human.

According to another embodiment of the present invention, a therapeutic compound for RBC-mediated delivery to a target cell expressing CD47 in a mammalian subject, the therapeutic compound comprising: a CD47 binding protein conjugated to an API to form a conjugate; wherein the CD47 binding protein is selected from the group consisting of wild-type thrombospondin-1 (TSP-1) (SEQ ID NO: 7), wild-type SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5), vSIRPγ-2 (SEQ ID NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), and ALX148 (SEQ ID NO: 965). NO:964), homologues of any of the foregoing, and combinations thereof, and configured to bind the conjugate to CD47 of the subject's erythrocytes to enable the conjugate to be transported to the target cells through the subject's circulatory system, so that (i) the CD47 binding protein configured to bind the conjugate to CD47 of the erythrocytes binds to CD47 of the target cells, thereby transferring the conjugate from the erythrocytes to the target cells to form a conjugate-CD47 complex on the target cells, thereby blocking CD47 and inhibiting CD47 activity as an immune escape mechanism of the target cells, and (ii) the conjugate is taken up by the target cells through endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune escape mechanism of the target cells and delivering the API to the target cells. The mammalian subject may be a human.

According to another embodiment of the present invention, a therapeutic compound for RBC-mediated delivery to a target cell expressing CD47 in a mammalian subject, the therapeutic compound comprising: a CD47 binding protein conjugated to an API to form a conjugate; wherein the CD47 binding protein is an anti-CD47 antibody, the anti-CD47 antibody comprising: (a) a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementary determining regions CDR1, CDR2 and CDR3 containing SEQ ID NO: 932, SEQ ID NO: 933 and SEQ ID NO: 934, respectively, and the light chain variable region comprising complementary determining regions CDR1, CDR2 and CDR3 containing SEQ ID NO: 935, SEQ ID NO: 936 and SEQ ID NO: 937, respectively; (b) a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementary determining regions CDR1, CDR2 and CDR3 containing SEQ ID NO: 940, SEQ ID NO: 941 and SEQ ID NO: 942, respectively, and the light chain variable region comprising complementary determining regions CDR1, CDR2 and CDR3 containing SEQ ID NO: 943, SEQ ID NO: 944, respectively, and the light chain variable region comprising complementary determining regions CDR1, CDR2 and CDR3 containing SEQ ID NO: 945, SEQ ID NO: 946 and SEQ ID NO: 947, respectively. (c) a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementary determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 948, SEQ ID NO: 949 and SEQ ID NO: 950, respectively, and the light chain variable region comprising complementary determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 951, SEQ ID NO: 952 and SEQ ID NO: 953, respectively; or (d) a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementary determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 956, SEQ ID NO: 957 and SEQ ID NO: 958, respectively, and the light chain variable region comprising complementary determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 959, SEQ ID NO: 960 and SEQ ID NO: 961, SEQ ID NO: 962, SEQ ID NO: 963, SEQ ID NO: 964, SEQ ID NO: 965, SEQ ID NO: 966, SEQ ID NO: 967, SEQ ID NO: 968, SEQ ID NO: 969, SEQ ID NO: 970, SEQ ID NO: 971, SEQ ID NO: 972, SEQ ID NO: 973, SEQ ID NO: 974, SEQ ID NO: 975, SEQ ID NO: 976, SEQ ID NO: 977, SEQ ID NO: 978, SEQ ID NO: 979, SEQ ID NO: 980, SEQ ID NO: 981, SEQ ID NO: NO:961 complementary determining regions CDR1, CDR2 and CDR3; and configured to bind the conjugate to CD47 of the subject's erythrocytes, so that the conjugate can be transported to the target cells through the subject's circulatory system, so that (i) the CD47 binding protein configured to bind the conjugate to CD47 of the erythrocytes binds to CD47 of the target cells, thereby transferring the conjugate from the erythrocytes to the target cells, so that a conjugate-CD47 complex is formed on the target cells, thereby blocking CD47 and inhibiting CD47 activity as a mechanism of target cell immune escape, and (ii) the conjugate is taken up by the target cells through endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune escape mechanism of the target cells and delivering the API to the target cells. The mammalian subject can be a human.

The CD47 binding protein can be conjugated to the API via a bond selected from the group consisting of: a covalent bond, a hydrogen bond, an ionic bond, a van der Waals interaction, and a combination thereof. The CD47 binding protein can be conjugated to the API via a linker, and the linker can be cleavable. The linker can be configured to be cleaved by a lysosomal degrading enzyme.

In some embodiments, the API is selected from the group consisting of RNA, DNA, RNA derivatives, DNA derivatives, proteins and small molecules. The RNA can be selected from the group consisting of siRNA, shRNA, miRNA, antimiR and mRNA.

The target cell can be a cell selected from the group consisting of cancer cells, virus-infected cells, fibrotic cells, and combinations thereof. In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell is a virus-infected cell. In some embodiments, the target cell is a fibrotic cell.

In some embodiments, the cancer cells are in a tumor attributable to a cancer selected from the group consisting of: brain tumors, spinal cord tumors, retinoblastoma, oral cancer, nasal cancer, paranasal sinus cancer, pharyngeal cancer, laryngeal cancer, neck cancer, head and neck cancer, melanoma, skin cancer, breast cancer, thyroid cancer, malignant adrenal tumors, endocrine cancer, lung cancer, pleural tumors, respiratory tract cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer, anal cancer, liver cancer, bile duct cancer, pancreatic cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, penis cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer, blood cancers including acute/chronic leukemias, malignant lymphomas and multiple myeloma, bone tumors, soft tissue tumors, childhood leukemias and childhood cancers.

In some embodiments, the cancer cell can be attributed to a cancer selected from the group consisting of: ovarian serous cystadenocarcinoma, lung adenocarcinoma, cervical and endocervical cancer, head and neck squamous cell carcinoma, thyroid cancer, uterine corpus endometrioid carcinoma, prostate adenocarcinoma, mesothelioma, diffuse large B-cell lymphoma, acute leukemia, lung squamous cell carcinoma, acute lymphocytic leukemia, esophageal cancer, myxofibrosarcoma, pancreatic adenocarcinoma, rectal adenocarcinoma, colon adenocarcinoma, acute megakaryocytic leukemia, breast invasive cancer, gastric adenocarcinoma, bladder urothelial carcinoma, bile duct cancer, leukemia, thymic carcinoma, leiomyosarcoma, thymoma, undifferentiated pleomorphic sarcoma, uterine carcinosarcoma, acute myeloid leukemia, glioblastoma multiforme, sarcoma, skin melanoma, renal clear cell carcinoma, dedifferentiated liposarcoma, lymphoma, retinoblastoma, neuroblastoma, osteosarcoma, juvenile myelomonocytic leukemia, gastrointestinal stromal tumor, embryonic development Undesirable neuroepithelial tumors, adrenocortical carcinoma, acute leukemia of unknown lineage, pheochromocytoma and paraganglioma, glioma, testicular germ cell tumor, supratentorial embryonal tumor NOS, neurofibroma, renal papillary cell carcinoma, hepatocellular carcinoma, renal chromophobe cell carcinoma, malignant peripheral nerve sheath tumor, ependymoma, adrenocortical carcinoma, nasopharyngeal carcinoma, spindle cell/sclerosing rhabdomyosarcoma, melanoma, choroid plexus carcinoma, undifferentiated spindle cell carcinoma, myositis Carcinoma of the skin, alveolar rhabdomyosarcoma, rhabdomyosarcoma, atypical teratoma/rhabdomyosarcoma, desmoplastic small round cell tumor, fibromatosis, synovial sarcoma, Wilms tumor, myofibromyxoma, fibrolamellar hepatocellular carcinoma, undifferentiated sarcoma NOS, embryonal rhabdomyosarcoma, uveal melanoma, Ewing sarcoma, hepatoblastoma, infantile fibrosarcoma, INI-deficient soft tissue sarcoma NOA, undifferentiated hepatic sarcoma, and medulloblastoma.

In some embodiments, the virus-infected cells are infected with SARS-CoV-2 virus. In some embodiments, the fibrotic cells are associated with cystic fibrosis.

In some embodiments, the API is an siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence is selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 747 and SEQ ID NO: 771 to SEQ ID NO: 824, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 22 to SEQ ID NO: 747 and SEQ ID NO: 771 to SEQ ID NO: 824. In some embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 22 to SEQ ID NO: 37. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 38 to SEQ ID NO: 39. In some embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 40 to SEQ ID NO: 43. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 44 to SEQ ID NO: 51.

In some embodiments, the API is an siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence is selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 21, SEQ ID NO: 482 to SEQ ID NO: 486, and SEQ ID NO: 748 to SEQ ID NO: 765, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 482 to SEQ ID NO: 486 and SEQ ID NO: 748 to SEQ ID NO: 765.

In some embodiments, the API is an siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence is selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 21, SEQ ID NO: 40 to SEQ ID NO: 43, and SEQ ID NO: 766 to SEQ ID NO: 770, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 40 to SEQ ID NO: 43 and SEQ ID NO: 766 to SEQ ID NO: 770.

The API is shRNA, which is a single-stranded RNA molecule with a length of 44 to 71 nucleotides and has, in the 5'-3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, and (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from SEQ ID NO: 8 to SEQ ID NO: 747 and SEQ ID NO: 771 to SEQ ID NO:824, and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO:22 to SEQ ID NO:747 and SEQ ID NO:771 to SEQ ID NO:824. In some embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO:22 to SEQ ID NO:37. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO:38 to SEQ ID NO:39. In some embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO:40 to SEQ ID NO:43. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO:44 to SEQ ID NO:51.

In some embodiments, the API is an shRNA, which is a single-stranded RNA molecule with a length of 44 to 71 nucleotides and has in the 5'-3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, and (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from SEQ ID NO: 8 to SEQ ID NO: 21, SEQ ID NO: 482 to SEQ ID NO: 486, and SEQ ID NO: 748 to SEQ ID NO: NO:765, and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO:482 to SEQ ID NO:486 and SEQ ID NO:748 to SEQ ID NO:765.

In some embodiments, the API is an shRNA, which is a single-stranded RNA molecule with a length of 44 to 71 nucleotides and has in the 5'-3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, and (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from SEQ ID NO: 8 to SEQ ID NO: 21, SEQ ID NO: 40 to SEQ ID NO: 43, and SEQ ID NO: 766 to SEQ ID NO: In another embodiment, the mRNA sequence is selected from the group consisting of SEQ ID NO: 40 to SEQ ID NO: 43 and SEQ ID NO: 766 to SEQ ID NO: 770, and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 40 to SEQ ID NO: 43 and SEQ ID NO: 766 to SEQ ID NO: 770.

In some embodiments, the API is a miRNA selected from the group consisting of SEQ ID NO:825 to SEQ ID NO:844, SEQ ID NO:849 to SEQ ID NO:851, SEQ ID NO:853, SEQ ID NO:855, SEQ ID NO:857, SEQ ID NO:864, SEQ ID NO:865, and SEQ ID NO:867 to SEQ ID NO:883.

In some embodiments, the API is an antimiR, which is a single-stranded nucleic acid molecule with a length of 12 to 25 nucleotides, and the antimiR has a sequence of 12 to 25 consecutive nucleotides that are complementary to consecutive nucleotides in the target mature miRNA product sequence, and the mature miRNA product sequence is selected from the group consisting of SEQ ID NO: 884 to SEQ ID NO: 908, wherein the consecutive nucleotides in the mature miRNA product sequence include the 2nd to 8th nucleotides of the mature miRNA product sequence in the 5' to 3' direction.

In some embodiments, the API is a small molecule selected from the group consisting of methotrexate; doxorubicin; vinca alkaloids; camptothecin analogs; microtubule disrupting agents such as auristatins (e.g., MMAE and MMAF) and maytansinoids (e.g., DM1 and DM4); and DNA damaging agents such as DNA topoisomerase I inhibitors (e.g., SN-38 and exatecan), double-strand break agents (e.g., calicheamicin), cross-linking agents (e.g., pyrrolobenzodiazepine dimer - PBD) and alkylating agents (e.g., duocarmycin and indolebenzodiazepine dimer - IGN).

In some embodiments, the API is a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 909 to SEQ ID NO: 929 and homologues thereof. In other embodiments, the protein consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 909 to SEQ ID NO: 929 and homologues thereof.

In some embodiments, the API is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 909 to SEQ ID NO: 929 and homologues thereof, and the mRNA is configured to be translated in a target cell to produce a protein comprising the amino acid sequence. In other embodiments, the mRNA is configured to be translated in a target cell to produce a protein consisting of the amino acid sequence. In some embodiments, the mRNA is codon optimized.

According to one embodiment of the present invention, a method of treating cancer in a mammalian subject in need thereof comprises administering a therapeutically effective amount of a therapeutic compound described herein. The mammalian subject may be a human.

According to another embodiment of the present invention, a method of treating a viral infection in a mammalian subject in need thereof comprises administering a therapeutically effective amount of a therapeutic compound described herein. The mammalian subject may be a human.

According to one embodiment of the present invention, a method of treating a fibrotic disease in a mammalian subject in need thereof comprises administering a therapeutically effective amount of a therapeutic compound described herein. The mammalian subject may be a human.

According to another embodiment of the present invention, a pharmaceutical composition comprises a therapeutic compound as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Features of the foregoing embodiments will be more readily understood by reference to the following detailed description taken in conjunction with the accompanying drawings, in which:

Figure 1A shows a FAM-labeled vSIRPα-siRNA conjugate according to an embodiment of the present invention. Figure 1B is a diagram of incubating erythrocytes with the FAM-labeled vSIRPα-siRNA conjugate of Figure 1A. Figure 1C shows fluorescence microscopy images of erythrocytes not incubated with the FAM-labeled vSIRPα-siRNA conjugate shown in Figure 1A (left) and erythrocytes incubated with the FAM-labeled vSIRPα-siRNA conjugate shown in Figure 1A (right).

Figure 2A shows the flow cytometry results of CaCO2 cells before and after incubation with red blood cells bound to FAM-labeled vSIRPα-siRNA according to an embodiment of the present invention. Figure 2B shows the flow cytometry results of CT26.CL25 cells before and after incubation with red blood cells bound to FAM-labeled vSIRPα-siRNA.

FIG. 3A shows a CaCO2 cell coupled to an Alexa Fluor®-containing Figure 3B shows the flow cytometry results of CT26.CL25 cells before and after incubation with Alexa Fluor 647 anti-mouse CD47 monoclonal antibody. Flow cytometry results of erythrocytes before and after incubation with 647 anti-mouse CD47 monoclonal antibody.

4 shows flow cytometry results of CaCO2 cells and CT26.CL25 cells before ("unstained") and after incubation with erythrocytes bound to FAM-labeled vSIRPα-siRNA according to an embodiment of the present invention.

5 shows flow cytometry results of CaCO2 cells and CT26.CL25 cells before ("unstained") and after incubation with erythrocytes bound to Cy5.5-labeled vSIRPα, according to an embodiment of the present invention.

FIG. 6 shows a graph showing the expression of CT26.CL25 cells in combination with Alexa Fluor Flow cytometry results of erythrocytes before (“unstained”) and after incubation with 647 anti-mouse CD47 monoclonal antibody.

7 shows flow cytometry results of CT26.CL25 cells before ("unstained") and after incubation with erythrocytes bound to a CD47mAb-miR21-Cy5 conjugate, according to an embodiment of the present invention.

8 shows flow cytometry results of CaCO2 cells and CT26.CL25 cells before ("unstained") and after incubation with erythrocytes conjugated with Cy5.5-labeled mouse thrombospondin-1, according to an embodiment of the present invention.

9 shows red blood cells isolated from untreated mice, mice injected with fluorescently labeled siRNA conjugates, and mice injected with fluorescently labeled vSIRPα-siRNA conjugates, according to an embodiment of the present invention.

Detailed ways

As used in this specification and the appended claims, the following terms shall have the indicated meanings unless the context requires otherwise:

The terms "a" and "an" and "the" and similar references used in the context of describing the present invention (particularly in the context of the claims) should be interpreted as covering both the singular and the plural, unless otherwise indicated herein or the context is clearly contradictory. The ranges of values listed herein are intended only to be used as a shorthand method of individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually listed herein. Any text in the specification cannot be interpreted as indicating that any non-claimed element is essential to the implementation of the present invention.

A "set" contains at least one member.

The term "mammal" and the like refers to any animal species of the class mammals. Examples of mammals include humans; experimental animals such as rats, mice, simians, and guinea pigs; domestic animals such as rabbits, cows, sheep, goats, cats, dogs, horses, pigs, etc.

"Active pharmaceutical ingredient", "API", etc., means the non-CD47 binding protein portion and non-linker portion of a therapeutic compound according to an embodiment of the present invention, which is biologically active. Suitable active pharmaceutical ingredients ("API") include RNA (siRNA, miRNA, shRNA and mRNA), DNA, antimiR oligonucleotides (RNA, DNA and their derivatives), RNA and DNA derivatives (including but not limited to modified RNA and DNA containing modified backbones, sugars and/or bases), proteins and small molecules.

As used herein, a "homolog" or the like of a given protein shall mean a protein having at least 95% sequence identity with the given protein.

As used herein, "complementarity" with respect to nucleic acid sequences refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence through Watson-Crick base pairing or wobble base pairing. The complementarity percentage represents the percentage of nucleotides in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with nucleotides of a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementarity). "Complete complementarity" and the like means that all consecutive nucleotides of a nucleic acid sequence will form hydrogen bonds with the same number of consecutive nucleotides in a second nucleic acid sequence (i.e., the nucleic acid sequence has 100% complementarity). As used herein, "complementary" is not further defined, meaning that consecutive nucleotides of a nucleic acid sequence have a complementary percentage selected from the group consisting of 95%, 96%, 97%, 98%, 99% and 100% complementarity with consecutive nucleotides of a second nucleic acid sequence over a region of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or more nucleotides in the nucleic acid sequence. For example, a nucleic acid sequence is 19 nucleotides in length and is complementary to 14 nucleotides in a second nucleic acid sequence, meaning that 14 consecutive nucleotides of the nucleic acid sequence have a complementary percentage selected from the group consisting of 95%, 96%, 97%, 98%, 99% and 100% complementarity with consecutive nucleotides of the second nucleic acid sequence. The nucleic acid sequence is 19 nucleotides in length and is complementary to consecutive nucleotides in a second nucleic acid sequence, meaning that the 19 consecutive nucleotides of the nucleic acid sequence have a complementarity percentage with consecutive nucleotides of the second nucleic acid sequence selected from the group consisting of: 95%, 96%, 97%, 98%, 99% and 100% complementarity.

"Codon-optimized" means that for a given target cell/host cell species, for at least 60% of the codons of the coding sequence of the mRNA transcript, the coding sequence comprises the most or second most preferred codons, such that the codon-optimized sequence is more efficiently translated in the target cell/host cell relative to a non-optimized sequence.

The term "antibody" refers to an immunoglobulin molecule, which generally comprises two pairs of identical polypeptide chains, each pair having one "heavy" (H) chain and one "light" (L) chain. Human light chains are classified as kappa (κ) and lambda (λ). Heavy chains are classified as μ, δ, γ, α, or ε, and define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region of IgD, IgG, and IgA comprises three domains, CH1, CH2, and CH3, and the heavy chain constant region of IgM and IgE comprises four domains, CH1, CH2, CH3, and CH4. Each light chain comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various types of cells of the immune system (e.g., effector cells). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy chain/light chain pair (VH/VL) typically form the antigen binding site of the antibody. The term "antibody" is not limited to any particular method for producing an antibody. For example, it includes monoclonal antibodies, recombinant antibodies, and polyclonal antibodies.

The term "human antibody" refers to an antibody consisting solely of the amino acid sequence of a human immunoglobulin sequence. If produced in a mouse, mouse cell, or hybridoma derived from a mouse cell, a human antibody may contain murine carbohydrate chains. Human antibodies can be prepared in a variety of ways known in the art.

The term "humanized antibody" refers to an antibody comprising some or all of the CDRs from non-human animal antibodies, while the framework and constant region of the antibody contain amino acid residues derived from human antibody sequences. Humanized antibodies are usually produced by grafting CDRs from mouse antibodies into human framework sequences, and then replacing some human framework residues with corresponding mouse residues from source antibodies. The term "humanized antibody" also refers to an antibody from non-human sources, in which one or more variable regions have been removed one or more epitopes with a high tendency to constitute human T cells and/or B cell epitopes, in order to reduce immunogenicity. The amino acid sequence of an epitope can be removed in whole or in part. However, the amino acid sequence is usually changed by replacing one or more other amino acids with one or more amino acids constituting the epitope, thereby changing the amino acid sequence to a sequence that does not constitute a human T cell and/or B cell epitope. Amino acid is replaced by the amino acid at the corresponding position present in the corresponding human variable heavy chain or variable light chain (as the case may be).

The term "pharmaceutically acceptable carrier" means solvents, carriers, diluents, etc., which are usually used for administration of pharmaceutical compounds.

CD47 is overexpressed in various cancer cells, virus-infected cells, and fibrotic cells, providing a promising target for the treatment of various cancers, viral infections, and fibrotic diseases. By blocking CD47 signaling in these cells, and even blocking the expression of CD47 itself, the escape of these cells from the immune system can be inhibited, achieving their elimination and subsequent recovery of patients from the disease.

For example, it has been found that a variant SIRPα ("vSIRPα") that has a high affinity for CD47 and is conjugated to a probe can be taken up by tumorigenic cells in mice after intravenous injection. In addition, it has been shown that vSIRPα conjugated to siRNA targeting CD47 enhances phagocytosis of CT26.CL25 cancer cells in culture (Ko et al. Control Release 323:376-386 (2020) and U.S. Publication No. 2021/0015931, each of which is incorporated herein by reference in its entirety).

We have unexpectedly discovered that CD47, which is present on the surface of red blood cells ("RBCs") and target cells (eg, cancer cells, virus-infected cells, and fibrotic cells), can be exploited to deliver a variety of APIs to the target cells through a novel mechanism.

Here, we describe novel therapeutic compounds for delivering APIs to target cells expressing CD47 via RBCs in mammalian subjects. The therapeutic compound is a conjugate comprising a CD47 binding protein conjugated to an API. The CD47 binding protein of the conjugate binds the conjugate to CD47 present on the surface of the red blood cells of the subject, thereby allowing the conjugate to be transported to the target cells through the circulatory system of the subject. The conjugate is transferred from the RBC to the CD47 present on the surface of the target cell. After the conjugate binds to the CD47 of the target cell, the ability of the target cell to escape the immune system attack of the subject is reduced. In addition, after the conjugate binds to the CD47 of the target cell, the conjugate-CD47 complex is internalized by endocytosis, further reducing the ability of the target cell to escape the immune system attack of the subject, and delivering the API to the target cell. Surprisingly, when the conjugate binds to CD47 on the surface of the RBC, the conjugate-CD47 is not internalized by the RBC. In some embodiments, the mammalian subject is a human.

The CD47 binding protein can be conjugated to the API via a linker. The linker connects the CD47 binding protein to the API. The linker can be a cleavable linker that is cleaved when the conjugate is internalized by the target cell, thereby releasing the API from the CD47 binding protein.

In some embodiments, the target cell is a cancer cell and the therapeutic compound can be used to treat cancer in a mammalian subject.

The cancer cells may be in a tumor attributable to a cancer selected from the group consisting of brain tumors, spinal cord tumors, retinoblastoma, oral cancer, nasal cavity cancer, paranasal sinus cancer, pharyngeal cancer, laryngeal cancer, neck cancer, head and neck cancer, melanoma, skin cancer, breast cancer, thyroid cancer, malignant adrenal tumors, endocrine cancer, lung cancer, pleural tumors, respiratory tract cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer, anal cancer, liver cancer, bile duct cancer, pancreatic cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, penis cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer, blood cancers including acute/chronic leukemias, malignant lymphomas and multiple myeloma, bone tumors, soft tissue tumors, childhood leukemias and childhood cancers.

The cancer cells may be attributed to a cancer selected from the group consisting of: ovarian serous cystadenocarcinoma, lung adenocarcinoma, cervical and endocervical cancer, head and neck squamous cell carcinoma, thyroid cancer, uterine corpus endometrioid carcinoma, prostate adenocarcinoma, mesothelioma, diffuse large B-cell lymphoma, acute leukemia, lung squamous cell carcinoma, acute lymphocytic leukemia, esophageal cancer, myxofibrosarcoma, pancreatic adenocarcinoma, rectal adenocarcinoma, colon adenocarcinoma, acute megakaryocytic leukemia, breast invasive cancer, gastric adenocarcinoma, bladder urothelial carcinoma, bile duct cancer, leukemia, thymic carcinoma, leiomyosarcoma, thymoma, undifferentiated pleomorphic sarcoma, uterine carcinosarcoma, acute myeloid leukemia, glioblastoma multiforme, sarcoma, skin melanoma, renal clear cell carcinoma, dedifferentiated liposarcoma, lymphoma, retinoblastoma, neuroblastoma, osteosarcoma, juvenile myelomonocytic leukemia, gastrointestinal stromal tumor, dysembryoplastic neuroblastoma. Transepithelial neoplasms, adrenocortical carcinoma, acute leukemia of undetermined lineage, pheochromocytoma and paraganglioma, glioma, testicular germ cell tumor, supratentorial embryonal tumor NOS, neurofibroma, renal papillary cell carcinoma, hepatocellular carcinoma, renal chromophobe cell carcinoma, malignant peripheral nerve sheath tumor, ependymoma, adrenocortical carcinoma, nasopharyngeal carcinoma, spindle cell/sclerosing rhabdomyosarcoma, melanoma, choroid plexus carcinoma, undifferentiated spindle cell carcinoma, myoepithelial carcinoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, atypical teratoma/rhabdomyosarcoma, desmoplastic small round cell tumor, fibromatosis, synovial sarcoma, Wilms tumor, myofibromyxoma, fibrolamellar hepatocellular carcinoma, undifferentiated sarcoma NOS, embryonal rhabdomyosarcoma, uveal melanoma, Ewing sarcoma, hepatoblastoma, infantile fibrosarcoma, INI-deficient soft tissue sarcoma NOA, undifferentiated hepatic sarcoma, and medulloblastoma. See Gupta et al. Cancer Drug Resist 3:550-62 (2020), which is incorporated herein by reference in its entirety.

In other embodiments, the target cell is a virally infected cell and the therapeutic compound can be used to treat a viral infection in a mammalian subject. The virally infected cell can be infected with the SARS-CoV-2 virus.

In some embodiments, the target cell is a fibrotic cell and the therapeutic compound can be used to treat a fibrotic disease in a mammalian subject. The fibrotic cell can be a fibrotic fibroblast. In some embodiments, the fibrotic disease is cystic fibrosis.

CD47 binding protein

CD47 binding proteins suitable for use in the conjugates described herein include wild-type ("wt") SIRPα (SEQ ID NO: 1), variant SIRPα ("vSIRPα") (SEQ ID NO: 3), wt TSP-1 (SEQ ID NO: 7), wt SIRPγ (SEQ ID NO: 4), variant SIRPγ-1 ("vSIRPγ-1") (SEQ ID NO: 5), variant SIRPγ-2 ("vSIRPγ-2") (SEQ ID NO: 6), and homologs of any of the foregoing. ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964) and homologs thereof are also CD47 binding proteins suitable for use in the conjugates described herein. ALX148 is a variant of SIRPαD1 fused to an Fc domain monomer. See, e.g., U.S. Pat. No. 10,696,730, the entirety of which is incorporated herein by reference. TTI-661 is the IgV domain of human SIRPα variant 2 fused to a human IgG1 antibody constant region, and TTI-662 is the IgV domain of human SIRPα variant 2 fused to a human IgG4 antibody constant region. See, e.g., U.S. Pat. No. 9,969,789, which is incorporated herein by reference in its entirety.

Other suitable CD47 binding proteins include anti-CD47 antibodies.

In some embodiments, the CD47 binding protein is an anti-CD47 antibody, such as B6H12, 5F9, 8B6, C3, and Hu5F9-G4, described in U.S. Pat. No. 9,017,675 and U.S. Pat. No. 9,623,079, each of which is incorporated herein by reference in its entirety.

In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising SEQ ID NO: 930 and a light chain variable region comprising SEQ ID NO: 931. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising SEQ ID NO: 938 and a light chain variable region comprising SEQ ID NO: 939. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising SEQ ID NO: 946 and a light chain variable region comprising SEQ ID NO: 947. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising SEQ ID NO: 954 and a light chain variable region comprising SEQ ID NO: 955.

In some embodiments, an anti-CD47 antibody comprises a heavy chain variable region comprising complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:932, SEQ ID NO:933, and SEQ ID NO:934, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:935, SEQ ID NO:936, and SEQ ID NO:937, respectively.

In some embodiments, an anti-CD47 antibody comprises a heavy chain variable region comprising complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:940, SEQ ID NO:941, and SEQ ID NO:942, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:943, SEQ ID NO:944, and SEQ ID NO:945, respectively.

In some embodiments, an anti-CD47 antibody comprises a heavy chain variable region comprising complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:948, SEQ ID NO:949, and SEQ ID NO:950, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:951, SEQ ID NO:952, and SEQ ID NO:953, respectively.

In some embodiments, an anti-CD47 antibody comprises a heavy chain variable region comprising complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:956, SEQ ID NO:957, and SEQ ID NO:958, respectively, and a light chain variable region comprising complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:959, SEQ ID NO:960, and SEQ ID NO:961, respectively.

In some embodiments, the anti-CD47 antibody is a human antibody. In some embodiments, the anti-CD47 antibody is a humanized antibody.

Each of these CD47 binding proteins binds to CD47 present on the surface of a target cell.

In some embodiments, the CD47 binding protein is conjugated to the API via a bond selected from the group consisting of: a covalent bond, a hydrogen bond, an ionic bond, a van der Waals interaction, and a combination thereof. Examples of linkers that covalently bind the CD47 binding protein to the API are described below.

Linker

Suitable linkers include, but are not limited to, cleavable linkers (e.g., hydrazone linkers, imine linkers, oxime linkers, carbonate linkers, acetal linkers, orthoester linkers, silyl ether linkers, disulfide linkers, trioxolane linkers, β-glucuronide linkers, β-galactoside linkers, pyrophosphate linkers, phosphoramidate linkers, aryl sulfate linkers, heptamethine cyanine linkers, nitrobenzyl linkers, arylboronic acid linkers, boronic acid linkers, thioether linkers, maleimidocaproyl-containing linkers, enzymatically cleavable peptide linkers, and p-aminobenzylcarbamate-containing linkers), as well as non-cleavable linkers (e.g., polyethylene glycol).

The CD47 binding protein-API conjugate described herein can be prepared by connecting the CD47 binding protein to the API through a linker using a coupling reaction, such as bis(vinylsulfonyl)piperazine-disulfide coupling, N-methyl-N-phenylvinylsulfonamide-cysteine coupling, platinum (II) compound-histidine coupling, and tetrazine-trans-cyclooctene coupling. Suitable linkers and coupling reactions are known to those skilled in the art. See, for example, Su et al. ActaPharmaceutica Sinica B (2021), ISSN 2211-3835; Pan et al. Med Res Rev. 40:2682-2713 (2020) Khongorzul et al. Mol Cancer Res 18:3-19 (2020); Bargh et al. Chem Soc Rev 48:4361-4374 (2019); and Smith et al. Pharm Res 32:3526-3540 (2015), each of which is incorporated herein by reference in its entirety.

According to embodiments of the present invention, various enzymatically cleavable peptide linkers (e.g., as described below) can be used to couple CD47 binding proteins to APIs to form CD47 binding protein-API conjugates. These linkers comprise amino acid residues and are cleaved by specific enzymes in cells (e.g., lysosomal degrading enzymes). See, e.g., Kon et al. J Biol Chem 290:7160-7168 (2015); Poreba FEBS J 287:1936-1969 (2020); and Singh et al. Current Medicinal Chemistry 15 (18) (2008), each of which is incorporated herein by reference in its entirety. Exemplary peptide linkers suitable for use in embodiments of the present invention are described below.

For example, a dipeptide linker comprises two amino acid residues that serve as a recognition motif for cleavage by the enzyme cathepsin B, which cleaves the amide bond after the second amino acid residue between the carbonyl and the amine. Dipeptide linkers cleaved by cathepsin B include Phe-Arg, Phe-Cit, Phe-Lys, Ala-Arg, Ala-Cit, Val-Ala, Val-Arg, Val-Lys, Val-Cit, and Arg-Arg. Cathepsin B similarly recognizes and cleaves the tetrapeptide linkers Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu after the fourth amino acid residue.

In addition, the tripeptide linker Ala-Ala-Asn is cleaved after the last amino acid residue by asparagine endopeptidase (legumain). The tetrapeptide linkers Lys-Ala-Gly-Gly, Leu-Arg-Gly-Gly and Arg-Lys-Arg-Arg are cleaved by papain-like proteases.

The peptide linkers Arg-Arg-X, Ala-Leu-X, Gly-Leu-Phe-Gly-X, Gly-Phe-Leu-Gly-X, and Ala-Leu-Ala-Leu-X (where X is any amino acid) are cleaved by the enzymes cathepsins B, H, and L. Cathepsins B, H, and L are responsible for lysosomal degradation of proteins.

The peptide linkers Phe-Ala-Ala-Phe( _NO2 )-Phe-Val-Leu-OM4P-X and Bz-Arg-Gly-Phe-Phe-Pro-4mβNA (where X is any amino acid) are cleaved by the enzyme cathepsin D.

Serum plasminogen activator is produced in many tumor cells. Plasminogen is converted to plasmin, resulting in high levels of plasmin in tumor cells. This plasmin is rapidly degraded in plasma, and therefore tissues away from the tumor are not exposed to plasmin. Plasmin is responsible for fibrinolysis and degradation of plasma proteins and cleaves the peptide linkers D-Val-Leu-Lys-X, D-Ala-Phe-Lys-X, and D-Ala-Trp-Lys-X, where X is any amino acid.

Tissue plasminogen activator (tPA) and urokinase (uPA) are responsible for activating the formation of plasmin and each can cleave the peptide linker Gly-Gly-Gly-Arg-Arg-Arg-Val-X, where X is any amino acid.

Prostate-specific antigen is responsible for the liquefaction of semen and cleaves the peptide linker morpholinecarbonyl-His-Ser-Ser-Lys-Leu-Gln-Leu-X, where X is any amino acid.

Matrix metalloproteinases (MMP-2 and MM-9) are responsible for the degradation of the extracellular matrix and collagen and cleave the peptide linkers Ac-Pro-Leu-Gln-Leu-X and Gly-Pro-Leu-Leu-Ile-Ala-Gly-Gln-X, where X is any amino acid.

API

According to some embodiments, the API can be a small molecule. For example, small molecule APIs that can be used to treat cancer include, but are not limited to, methotrexate; doxorubicin; vinca alkaloids; camptothecin analogs; microtubule disruptors, such as auristatins (e.g., MMAE and MMAF) and maytansine alkaloids (e.g., DM1 and DM4); and DNA damaging agents, such as DNA topoisomerase I inhibitors (e.g., SN-38 and exitecan), double-strand breakers (e.g., calicheamicin), cross-linkers (e.g., pyrrolobenzodiazepine dimers-PBD) and alkylating agents (e.g., duocarmycin and indolebenzodiazepine dimers-IGN). See, e.g., Khongorzul et al. Mol Cancer Res 18:3-19 (2020); Salomon et al. Mol Pharm 16(12):4817-4825 (2019); and Drago et al. Nat Rev Clin Oncol 18, 327-344 (2021), each of which is incorporated herein by reference in its entirety.

According to other embodiments, the API can be a small interfering RNA ("siRNA"). siRNA is a double-stranded RNA molecule that can reduce the expression of a specific gene by causing degradation of the mRNA transcript of the gene that shares partial complementarity with the strands of the double-stranded siRNA molecule. The process of using siRNA to reduce gene expression is called RNA interference ("RNAi"). See U.S. Patent No. 7,056,704, U.S. Patent No. 7,078,196, U.S. Patent No. 8,372,968, each of which is incorporated herein by reference in its entirety.

Several genes have been implicated in promoting cancer progression and cancer cell proliferation through various mechanisms. These genes and their mRNA transcripts are listed in Table 1. By reducing the expression of one or more genes from Table 1, cancer progression and cancer cell proliferation can be inhibited. Therefore, the transcripts of the genes listed in Table 1 represent key targets for the use of siRNA-mediated RNAi to treat cancer.

In some embodiments, the API that can be used to treat cancer in a mammal is an siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 747 and SEQ ID NO: 771 to SEQ ID NO: 824, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length.

In some embodiments, the API that can be used to treat cancer in a mammal is an siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:22 to SEQ ID NO:747 and SEQ ID NO:771 to SEQ ID NO:824, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length.

In some embodiments, the API that can be used to treat cancer in a mammal is an siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:22 to SEQ ID NO:37, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length.

In some embodiments, the API that can be used to treat cancer in a mammal is an siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:38 to SEQ ID NO:39, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length.

In some embodiments, the API that can be used to treat cancer in a mammal is an siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:40 to SEQ ID NO:43, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length.

In some embodiments, the API that can be used to treat cancer in a mammal is an siRNA, which is a double-stranded RNA molecule comprising an antisense RNA chain and a sense RNA chain, wherein: (a) the antisense RNA chain is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:44 to SEQ ID NO:51, (b) the sense RNA chain is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides of the antisense RNA chain, and (c) the double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides.

Table 1: siRNA and shRNA cancer (cancer cell) target transcripts

Using siRNA-mediated RNAi to reduce gene expression can also be used for the treatment of viral infection. For example, reducing the expression of a group of genes that can make infected cells escape the host immune system, or reducing the expression of a group of genes required for viral replication can hinder the proliferation of viruses in the host. Such genes and their mRNA transcripts have been listed in Table 2. Therefore, the transcripts of the genes listed in Table 2 are key targets for using siRNA-mediated RNAi to treat viral infection.

Thus, in some embodiments, the API useful for treating a viral infection in a mammal is an siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:21, SEQ ID NO:482 to SEQ ID NO:486, and SEQ ID NO:748 to SEQ ID NO:765, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length.

In some embodiments, the API that can be used to treat a viral infection in a mammal is an siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, and the cDNA sequence is selected from the group consisting of SEQ ID NO:482 to SEQ ID NO:486 and SEQ ID NO:748 to SEQ ID NO:765, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length.

Table 2: siRNA and shRNA virus infection (virus-infected cells) target transcripts

Increased CD47 expression has been observed in fibrotic fibroblasts, and blocking CD47 reverses fibrosis by enhancing phagocytosis of pro-fibrotic fibroblasts and by eliminating the inhibitory effect on adaptive immunity. In addition to CD47, the expression of other genes listed in Table 3 is associated with promoting fibrosis. Using siRNA-mediated RNAi to reduce the expression of these genes can also be used for the treatment of fibrotic diseases. Such genes and their mRNA transcripts are listed in Table 3. Therefore, the transcripts of the genes listed in Table 3 represent key targets for the treatment of fibrotic diseases using siRNA-mediated RNAi.

In some embodiments, the API that can be used to treat a fibrotic disease in a mammal is an siRNA, which is a double-stranded RNA molecule comprising an antisense RNA chain and a sense RNA chain, wherein: (a) the antisense RNA chain is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:21, SEQ ID NO:40 to SEQ ID NO:43, and SEQ ID NO:766 to SEQ ID NO:770, (b) the sense RNA chain is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA chain, and (c) the double-stranded RNA molecule has a double-stranded region of 14 to 29 nucleotides in length and a 3' overhang region of 0 to 5 nucleotides in length.

In some embodiments, the API that can be used to treat a fibrotic disease in a mammal is an siRNA, which is a double-stranded RNA molecule comprising an antisense RNA chain and a sense RNA chain, wherein: (a) the antisense RNA chain is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:40 to SEQ ID NO:43 and SEQ ID NO:766 to SEQ ID NO:770, (b) the sense RNA chain is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA chain, and (c) the double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides.

Table 3: siRNA and shRNA fibrosis disease (fibrotic cells) target transcripts

According to some embodiments, the API can be a short hairpin RNA ("shRNA"). Short hairpin RNA is a single-stranded RNA molecule that forms a stem-loop structure and can reduce the expression of a specific gene by causing degradation of the mRNA transcript of the gene, which shares partial complementarity with a region of the shRNA molecule. As with siRNA, the process of using shRNA to reduce gene expression is called RNAi. See generally Rao et al. Adv Drug Deliv Rev 61(9):746-59 (2009), which is incorporated herein by reference in its entirety.

Several genes have been implicated in promoting cancer progression and cancer cell proliferation through various mechanisms. These genes and their mRNA transcripts are listed in Table 1. By reducing the expression of one or more genes from Table 1, cancer progression and cancer cell proliferation can be inhibited. Therefore, the transcripts of the genes listed in Table 1 represent key targets for the use of shRNA-mediated RNAi to treat cancer.

Therefore, in some embodiments, the API that can be used to treat cancer in a mammal is an shRNA, which is a single-stranded RNA molecule with a length of 44 to 71 nucleotides and has in the 5'-3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, and (c) the third region is complementary to consecutive nucleotides in a target mammal mRNA sequence, the mRNA sequence being selected from SEQ ID NO: 8 to SEQ ID NO: 747 and SEQ ID NO: 771 to SEQ ID NO: NO:824, and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

In some embodiments, the API that can be used to treat cancer in a mammal is an shRNA, which is a single-stranded RNA molecule with a length of 44 to 71 nucleotides and has in the 5'-3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, and (c) the third region is complementary to consecutive nucleotides in a target mammal mRNA sequence, the mRNA sequence being selected from SEQ ID NO: 22 to SEQ ID NO: 747 and SEQ ID NO: 771 to SEQ ID NO: NO:824, and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

In some embodiments, the API that can be used to treat cancer in a mammal is an shRNA, which is a single-stranded RNA molecule with a length of 44 to 71 nucleotides and has in the 5'-3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, and (c) the third region is complementary to consecutive nucleotides in a target mammal mRNA sequence, the mRNA sequence being selected from SEQ ID NO: 22 to SEQ ID NO: NO:37, and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

In some embodiments, the API that can be used to treat mammalian cancer is shRNA, which is a single-stranded RNA molecule with a length of 44 to 71 nucleotides and has in the 5'-3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, and (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, and the mRNA sequence is selected from SEQ ID NO: 38 to SEQ ID NO:39, and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

In some embodiments, the API that can be used to treat cancer in a mammal is an shRNA, which is a single-stranded RNA molecule with a length of 44 to 71 nucleotides and has in the 5'-3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, and (c) the third region is complementary to consecutive nucleotides in a target mammal mRNA sequence, and the mRNA sequence is selected from SEQ ID NO: 40 to SEQ ID NO: NO:43, and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

In some embodiments, the API that can be used to treat cancer in a mammal is an shRNA, which is a single-stranded RNA molecule with a length of 44 to 71 nucleotides and has in the 5'-3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, and (c) the third region is complementary to consecutive nucleotides in a target mammal mRNA sequence, the mRNA sequence being selected from SEQ ID NO: 44 to SEQ ID NO: NO:51, and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

Using shRNA-mediated RNAi to reduce gene expression can also be used for the treatment of viral infection. For example, reducing the expression of a group of genes that can enable infected cells to escape the host immune system, or reducing the expression of a group of genes required for viral replication can hinder the proliferation of viruses in the host. Such genes and their mRNA transcripts are listed in Table 2. Therefore, the transcripts of the genes listed in Table 2 are key targets for using shRNA-mediated RNAi to treat viral infection.

Therefore, in some embodiments, the API that can be used to treat viral infection in a mammal is an shRNA, which is a single-stranded RNA molecule with a length of 44 to 71 nucleotides and has in the 5'-3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, and (c) the third region is complementary to consecutive nucleotides in a target mammal mRNA sequence, the mRNA sequence being selected from SEQ ID NO: 8 to SEQ ID NO: 21, SEQ ID NO: 482 to SEQ ID NO: 486, and SEQ ID NO: 748 to SEQ ID NO: NO:765, and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

In some embodiments, the API that can be used to treat viral infection in a mammal is an shRNA, which is a single-stranded RNA molecule with a length of 44 to 71 nucleotides and has in the 5'-3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, and (c) the third region is complementary to consecutive nucleotides in a target mammal mRNA sequence, the mRNA sequence being selected from SEQ ID NO: 482 to SEQ ID NO: 486 and SEQ ID NO: 748 to SEQ ID NO: NO:765, and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

As described above, increased CD47 expression has been observed in fibrotic fibroblasts, and blocking CD47 reverses fibrosis by enhancing phagocytosis of pro-fibrotic fibroblasts and by eliminating the inhibitory effect on adaptive immunity. In addition to CD47, the expression of other genes listed in Table 3 is associated with promoting fibrosis. Using shRNA-mediated RNAi to reduce the expression of these genes can also be used for the treatment of fibrotic diseases. Such genes and their mRNA transcripts are listed in Table 3. Therefore, the transcripts of the genes listed in Table 3 represent key targets for the treatment of fibrotic diseases using shRNA-mediated RNAi.

Therefore, in some embodiments, the API for treating a fibrotic disease in a mammal is an shRNA, which is a single-stranded RNA molecule with a length of 44 to 71 nucleotides and has in the 5'-3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, and (c) the third region is complementary to consecutive nucleotides in a target mammal mRNA sequence, the mRNA sequence being selected from SEQ ID NO: 8 to SEQ ID NO: 21, SEQ ID NO: 40 to SEQ ID NO: 43, and SEQ ID NO: 766 to SEQ ID NO: NO:770, and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

In some embodiments, the API that can be used to treat a fibrotic disease in a mammal is an shRNA, which is a single-stranded RNA molecule with a length of 44 to 71 nucleotides and has in the 5'-3' direction: a first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, and (c) the third region is complementary to consecutive nucleotides in a target mammal mRNA sequence, the mRNA sequence being selected from SEQ ID NO: 40 to SEQ ID NO: 43 and SEQ ID NO: 766 to SEQ ID NO: NO:770, and (d) the single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

MicroRNA ("miRNA")-based therapeutics include miRNAs (and miRNA mimics) and miRNA inhibitors ("antimiRs").

MiRNA is transcribed as a single-stranded RNA precursor with a stem-loop structure and subsequently processed in the cytosol by the Dicer enzyme to produce a mature double-stranded product ("mature miRNA product"). These mature miRNA products are believed to have regulatory effects, including post-transcriptional regulation of RNA silencing and gene expression through their complementarity with mRNA.

It is known that the expression of the miRNAs shown in Table 4 is downregulated in various cancers, and supplementation of these downregulated miRNAs provides a promising therapy for cancer treatment.

Therefore, in some embodiments, the API that can be used to treat cancer in a mammal is a miRNA selected from the group consisting of SEQ ID NO:825 to SEQ ID NO:844, SEQ ID NO:849 to SEQ ID NO:851, SEQ ID NO:853, SEQ ID NO:855, SEQ ID NO:857, SEQ ID NO:864, SEQ ID NO:865 and SEQ ID NO:867 to SEQ ID NO:883.

Table 4: miRNA API for cancer treatment (cancer cells)

Compared to miRNA, antimiR (also called "antagomir") is a single-stranded antisense oligonucleotide ("ASO") having a sequence complementary to the sequence of a region of the target mature miRNA product. The mature miRNA product is a short single-stranded RNA molecule produced after the miRNA molecule is processed in the cytosol. Typically, two mature miRNA products are produced from one miRNA molecule: a 5p RNA molecule (so named because it is processed from the 5' end of the duplex as the miRNA stem) and a 3p RNA molecule (so named because it is processed from the 3' end of the duplex as the miRNA stem). The 5p and 3p molecules can base pair with each other to form a duplex, and each molecule can be functional in the cell through its complementarity with the mRNA, and can actually play a separate function. In some cases, a miRNA molecule can only produce a single functional mature miRNA product after processing.

AntimiR binds to its target mature miRNA through base pairing, and can block the binding of mature miRNA products to their targets, thereby inhibiting the function of mature miRNA products. The use of antimiR to inhibit mature miRNA products is called miRNA knockdown. See generally Quemener et al. Wiley Interdiscip Rev RNA (5): e1594 (2020), which is incorporated herein by reference in its entirety.

The length of antimiR is 12 to 25 nucleotides and is complementary to the continuous nucleotides of the target mature miRNA product. Different types of nucleic acids can be used to produce antimiR. Preferably, antimiR comprises RNA because RNA/RNA hybrids are very stable. In addition, antimiR can comprise DNA, or both RNA and DNA nucleotides (referred to herein as "chimeras"). AntimiR should bind with high affinity to the "seed region" of the mature miRNA product through complementary base pairing, and the "seed region" spans 2 to 8 nucleotides from the 5' end of the mature miRNA product (Lennox et al. Gene Therapy 18: 1111-20 (2011), which is incorporated herein by reference in its entirety).

Over the years, significant improvements in antimiR binding affinity, stability and target modulation have been achieved through chemical modifications of the oligonucleotide backbone. Thus, antimiRs can be RNA derivatives or DNA derivatives. In some embodiments, the antimiR comprises modifications that provide the antimiR with additional properties, such as tolerance to endonucleases and RNase H, stability (e.g., in body fluids), and reduced toxicity. In some embodiments, the modifications are 2'-O-methyl-phosphorothioate oligonucleotide modifications, 2'-O-methoxyethyl oligonucleotide modifications, and combinations thereof. In some embodiments, the antimiR comprises a peptide nucleic acid, a locked nucleic acid, or a morpholinophosphodiamidate. See generally Wahlestedt et al. PNAS 97, 5633-5638 (2000); Elayadi et al. Curr Opin Investig Drugs 2, 558-61 (2001); Larsen et al. Biochim Biophys Acta 1489, 159-166 (1999); Braasch et al. Biochemistry 41, 4503-4510 (2002); Summerton et al. Antisense Nucleic Acid Drug Dev 7, 187-195 (1997), each of which is incorporated herein by reference in its entirety.

The expression of the miRNAs shown in Table 5 is known to be upregulated in various cancers, and as shown in Table 6, their mature miRNA products are preferred targets for miRNA knockdown therapy for cancer.

Therefore, in some embodiments, the API that can be used to treat cancer in a mammal is an antimiR, which is a single-stranded nucleic acid molecule with a length of 12 to 25 nucleotides, and the antimiR has a sequence of 12 to 25 consecutive nucleotides that are complementary to the consecutive nucleotides in the target mature miRNA product sequence, and the mature miRNA product sequence is selected from the group consisting of SEQ ID NO: 884 to SEQ ID NO: 908, wherein the consecutive nucleotides in the mature miRNA product sequence include the 2nd to 8th nucleotides in the 5' to 3' direction.

Table 5: Upregulated miRNA expression in cancer

Table 6: AntimiR cancer (cancer cell) target mature miRNA products

In other embodiments, the API useful for treating cancer in a mammal is a protein with anti-cancer properties. Anti-cancer properties include inhibiting the proliferation of cancer cells, inhibiting the proliferation of tumors, causing the death of cancer cells, reducing the size of tumors, or causing the elimination of tumors. The proteins listed in Table 7 have anti-cancer properties. Therefore, a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 909 to SEQ ID NO: 929 and homologues thereof is an API suitable for use according to embodiments of the present invention. In other embodiments, a protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 909 to SEQ ID NO: 929 and homologues thereof is an API suitable for use according to embodiments of the present invention.

In some embodiments, the API suitable for treating cancer in a mammal is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 909 to SEQ ID NO: 929 and homologues thereof, and the mRNA is configured to be translated in a target cell of a mammal to produce a protein comprising the amino acid sequence. In other embodiments, the API suitable for treating cancer in a mammal is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 909 to SEQ ID NO: 929 and homologues thereof, and the mRNA is configured to be translated in a target cell of a mammal to produce a protein consisting of the amino acid sequence. The mRNA may be codon-optimized for translation in a target cell of a mammal.

Table 7: Protein API for cancer treatment (cancer cells)

Example 1: vSIRPα-siRNA conjugate binds to red blood cells

FAM-labeled siRNA ( FIG. 1A ) (SEQ ID NO: 965 (sense strand) and SEQ ID NO: 966 (antisense strand)) was mixed with vSIRPα at a 1:1 molar ratio (50 pmol vSIRPα to 50 pmol siRNA). siRNA was modified with maleimide at its 3' end. vSIRPα was designed with cysteine near its C-terminus. The thiol groups from cysteine and maleimide were reacted at neutral pH by click chemistry. The reaction was kept in a shaker at 4°C overnight to obtain the vSIRPα-siRNA conjugate shown in FIG. 1A .

vSIRPα-siRNA conjugates were isolated using NAP-5 columns using the manufacturer's protocol.

To evaluate the ability of vSIRPα-siRNA conjugates to bind to red blood cells ("RBC"), vSIRPα-siRNA conjugates were mixed with mouse RBCs as depicted in FIG. 1B. For this experiment, 6x10 ⁶ red blood cells in a volume of 2 μl phosphate buffered saline ("PBS") were mixed with 2.5 μl PBS containing vSIRPα-siRNA conjugates (a total of 50 pmol vSIRPα-siRNA conjugates) and 5.5 μl PBS. A negative control was prepared by mixing 6x10 ⁶ red blood cells in a volume of 2 μl PBS with 8 μl of PBS. Each mixture was then incubated at room temperature (20°C to 25°C) for 2 hours (FIG. 1B).

After incubation, 5 μl of each mixture was diluted 20-fold with PBS to a total volume of 100 μl, and each dilution was placed in a glass bottom dish and imaged fluorescently at 494 nm to 567 nm using a microscope (488 nm excitation wavelength).

As can be observed in FIG. 1C , the negative control showed no fluorescence, whereas the mixture containing the vSIRPα-siRNA conjugate showed a strong fluorescence signal associated with erythrocytes, indicating that the vSIRPα-siRNA conjugate bound to erythrocytes.

Example 2: Transfer of vSIRPα-siRNA conjugates from RBCs to cancer cells as demonstrated by flow cytometry

500 pmol of vSIRPα-siRNA conjugate from Example 1 was incubated with 5×10 ³ mouse RBCs in Dulbecco's phosphate buffered saline (“DPBS”) in a total volume of 20 μl for 30 minutes at room temperature (20° C. to 25° C.).

After 30 minutes, the mixture was washed with DPBS by centrifugation at 500 xg for 10 minutes, and the supernatant was removed. The RBC bound to the vSIRPα-siRNA conjugate was then resuspended in 20 μl PBS.

Two cell lines were used: CT26.CL25 (ATC CCRL-2639) and CaCO2 (ATCC HTB-37). CT26.CL25 is a mouse colon cancer cell line. CaCO2 is a human colorectal adenocarcinoma cell line deficient in CD47 expression and was therefore used as a negative control (Liu et al. J Biol Chem 276(43):40156-66 (2001), which is incorporated herein by reference in its entirety).

Each cell line grown separately in a cell culture dish was detached from the dish with trypsin-EDTA. The cells from each dish were then washed with DPBS, counted and diluted to 10 ⁵ cells/100 μl PBS. 4 μl FAM-vSIRPα-siRNA/RBC resuspension (a total of 10 ³ RBCs and 100 pmol vSIRPα-siRNA) was mixed with 100 μl of each dilution of CT26.CL25 and CaCO2 cells, respectively. Each mixture was incubated at room temperature (20°C to 25°C) for 30 minutes.

After 30 minutes, each mixture was centrifuged and the supernatant was removed. The cell pellet was resuspended with 200 μl of flow cytometry buffer for flow cytometry analysis.

Figures 2A and 2B show the flow cytometry results of CaCO2 cells and CT26.CL25 cells, respectively. Flow cytometry was performed using a 488nm laser to excite the FAM label and detect at a wavelength of 525nm to 565nm. After incubation with RBCs bound to FAM-labeled vSIRPα-siRNA, each cancer cell line showed different levels of fluorescence shift. These results show a significant fluorescence shift after incubation of CT26.CL25 with RBCs and almost no shift after incubation of CaCO2 cells with RBCs, indicating that the extent of fluorescence shift depends on the CD47 level on the cancer cells. These results strongly indicate that FAM-labeled vSIRPα-siRNA has been transferred from RBCs to CD47 present on the surface of CT26.CL25 cells.

Example 3: Anti-CD47 antibody transfer from RBCs to cancer cells as demonstrated by flow cytometry

First, 0.1 μg Alexa 647 anti-mouse CD47 monoclonal antibody (Biolegend, #127510) was incubated with 4x10 ³ mouse RBCs in a total volume of 100 μL DPBS at room temperature for 30 minutes. After incubation, the RBC-antibody mixture was centrifuged at 500 g for 5 minutes. The supernatant was then poured off and the cells were resuspended in 20 μl DPBS.

Then 2x10 ⁵ CT26.CL25 cells (in 100 μL DPBS) were mixed with 10 μL of resuspended RBC-antibody mixture in a total volume of 110 μL (Mixture #1). Similarly, 2x10 ⁵ CaCO2 cells (in 100 μL DPBS) were mixed with 10 μL of resuspended RBC-antibody mixture in a total volume of 110 μL (Mixture #2).

Mixture #1 and mixture #2 were incubated at room temperature for 30 minutes. After incubation, 1 ml of DPBS was added to each mixture, and then centrifuged at 500 g for 5 minutes. For each mixture, after centrifugation, the supernatant was poured out, and the remaining cells were resuspended in 100 μl DPBS and subjected to flow cytometry (Beckman, laser 488 nm, detection 650 nm to 670 nm).

Figures 3A and 3B show the flow cytometry results for CaCO2 cells and CT26.CL25 cells, respectively. Although not as pronounced as the fluorescence shift observed in Example 2, a greater fluorescence shift was observed after incubation of CT26.CL25 cells with RBCs than after incubation of CaCO2 cells with RBCs. This again suggests that the extent of the fluorescence shift depends on the level of CD47 on cancer cells. These results suggest that Alexa 647 anti-mouse CD47 monoclonal antibody is transferred from RBC to CD47 present on the surface of CT26.CL25 cells.

Example 4: Transfer of vSIRPα-siRNA conjugates from RBCs to cancer cells as demonstrated by flow cytometry

The ability of vSIRPα-siRNA-FAM of Example 1 to transfer from RBC to CD47 present on the surface of CT26.CL25 cells was further tested. The experiment was performed as in Example 2, except that 100 pmol of the conjugate was incubated with RBC instead of 500 pmol.

As shown in the flow cytometry results of FIG. 4 , a significantly greater percentage of CT26.CL25 cells showed FAM fluorescence compared to CaCO 2 cells, again strongly suggesting that FAM-labeled vSIRPα-siRNA had been transferred from RBCs to CD47 present on the surface of CT26.CL25 cells.

Example 5: Cy5.5-labeled vSIRPα is transferred from RBCs to cancer cells as demonstrated by flow cytometry

The ability of vSIRPα labeled with Cy5.5 to transfer from RBCs to CD47 present on the surface of CT26.CL25 cells was tested. The experiment was performed as in Example 2, except that 200 pmol of labeled vSIRPα was incubated with RBCs instead of 500 pmol of the conjugate.

As shown in the flow cytometry results of FIG. 5 , a significantly greater percentage of CT26.CL25 cells showed Cy5.5 fluorescence compared to CaCO 2 cells, strongly suggesting that Cy5.5-labeled vSIRPα had been transferred from RBCs to CD47 present on the surface of CT26.CL25 cells.

Example 6: Anti-CD47 antibody transfer from RBCs to cancer cells as demonstrated by flow cytometry

Tested with Alexa The ability of 647-labeled anti-mouse CD47 monoclonal antibody (Biolegend, #127510) to transfer from RBC to CD47 present on the surface of CT26.CL25 cells. The experiment was performed as in Example 3, except that 1 μg of the labeled antibody was incubated with RBC instead of 0.1 μg.

As shown in the flow cytometry results of Figure 6, a significantly greater percentage of CT26.CL25 cells showed Alexa Fluor 484 expression compared to unstained cells. 647 fluorescence strongly indicates the 647-bound anti-CD47 antibodies have been transferred from RBCs to CD47 present on the surface of CT26.CL25 cells. This experiment also demonstrated that monoclonal antibodies against CD47 can be transferred from RBCs to CD47 present on the surface of cancer cells.

Example 7: Antibody-miR21 conjugate transfer from RBCs to cancer cells as demonstrated by flow cytometry

The ability of anti-CD47 monoclonal antibody (Bioxcell, #BE0270) conjugated to Cy5-labeled miR21 (SEQ ID NO: 878) to transfer from RBC to CD47 present on the surface of CT26.CL25 cells was tested. The experiment was performed as in Example 3, except that 15.8 μg of the antibody conjugate was incubated with RBC instead of 0.1 μg.

As shown in the flow cytometry results of Figure 7, a greater percentage of CT26.CL25 cells showed Cy5 fluorescence compared to unstained cells, strongly indicating that the CD47mAb-miR21-Cy5 conjugate had been transferred from RBCs to CD47 present on the surface of CT26.CL25 cells.

Example 8: Transfer of thrombospondin-1 from RBCs to cancer cells as demonstrated by flow cytometry

Thrombospondin-1 (TSP-1) is a matricellular protein that inhibits angiogenesis and endothelial cell proliferation. TSP-1 binds CD47. The TSP-1 signaling pathway has been found to be involved in various conditions, such as kidney disease, cardiovascular disease, inflammation, and cancer. The potential mechanisms and pathways of TSP-1 have not yet been fully elucidated. However, the functions of TSP-1 on several key receptors CD36/VEGF and CD47 have been confirmed. In cancer in particular, activated TSP-1 and CD47 pathways have been found to reduce tumor growth and metastasis. See Kale et al. Int J Mol Sci, 22 (8) (2021) and Kaur et al. J Biol Chem, 285 (50), 38923-38932 (2010). Here, we show that labeled mouse TSP-1 is transferred from RBC to CT26.CL25 cancer cells.

The ability of mouse TSP-1 labeled with Cy5.5 (7859-TH, R&D Systems) to transfer from RBC to CD47 present on the surface of CT26.CL25 cells was tested. The experiment was performed as in Example 2, except that 5 μg of Cy5.5 labeled TSP-1 was incubated with RBC instead of 500 pmol of the conjugate.

As shown in the flow cytometry results of FIG8 , a significantly greater percentage of CT26.CL25 cells showed Cy5.5 fluorescence compared to CaCO 2 cells, strongly suggesting that Cy5.5-labeled TSP-1 had been transferred from RBCs to CD47 present on the surface of CT26.CL25 cells.

Example 9: vSIRPα-siRNA conjugate binds to RBCs in vivo

5 nmol of vSIRPα-siRNA conjugate from Example 1 and 5 nmol of unconjugated siRNA from Example 1 were stained with intercalating dye YOYO ^™ -1 iodide at a molar ratio of 1:1 in a total volume of 120 μl of RNase-free water. After incubation at room temperature for 30 minutes, the stained conjugate and siRNA were injected into mice as detailed below.

Experiments were performed using 3 strains of Balb/c mice: 1 untreated mouse was used as a negative control; 1 mouse was injected intravenously at the tail vein with 5 nmol of unconjugated dyed siRNA, and 1 mouse was injected intravenously at the tail vein with 5 nmol of dyed vSIRPα-siRNA conjugate.

After injection 45 minutes, blood was collected from mice. 50 μl whole blood from each sample was washed twice by centrifugation at 500 x g for 10 minutes using 1 ml DPBS, and resuspended in DPBS. Using confocal dish (SPL 100350), resuspended blood was imaged by confocal microscopy, and the YOYO ^™ -1 iodide fluorescence signal related to RBC between different groups (control, siRNA and conjugate) was compared. 488 nm laser (YOYO ^™ -1 iodide has an excitation wavelength of 491 nm) was used.

As shown in FIG. 9 , RBCs from mice injected with vSIRPα-siRNA conjugates showed fluorescent spots (arrows in FIG. 9 ), indicating that binding of vSIRPα-siRNA conjugates to RBCs occurred in vivo.

Potential claims

Various embodiments of the present invention may be characterized by the potential claims listed in the paragraphs following this paragraph (and before the actual claims provided at the end of this application). These potential claims form part of the written description of this application. Therefore, in subsequent proceedings involving this application or any application claiming priority based on this application, the subject matter of the following potential claims may be presented as actual claims. The inclusion of such potential claims should not be interpreted as meaning that the actual claims do not cover the subject matter of the potential claims. Therefore, the decision not to present these potential claims in subsequent proceedings should not be interpreted as donating the subject matter to the public.

Without limitation, potential subject matter that may be claimed (prefaced by the letter "P" to avoid confusion with the actual claims set forth below) includes:

P1. A therapeutic compound for RBC-mediated delivery to a target cell expressing CD47 in a mammalian subject, the therapeutic compound comprising:

A CD47 binding protein, which is conjugated to the API to form a conjugate;

wherein the CD47 binding protein is selected from the group consisting of wild-type SIRPα (SEQ ID NO: 1), vSIRPα (SEQ ID NO: 3), wild-type thrombospondin-1 (TSP-1) (SEQ ID NO: 7), wild-type SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5), vSIRPγ-2 (SEQ ID NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), and TTI-663 (SEQ ID NO: 965). NO:964), homologues of any of the foregoing, and combinations thereof, and are configured to bind the conjugate to CD47 of the subject's erythrocytes so as to be transported to the target cells through the subject's circulatory system, so that (i) the CD47 binding protein configured to bind the conjugate to CD47 of the erythrocytes binds to CD47 of the target cells, thereby transferring the conjugate from the erythrocytes to the target cells to form a conjugate-CD47 complex on the target cells, thereby blocking CD47 and inhibiting CD47 activity as an immune escape mechanism of the target cells, and (ii) the conjugate is taken up by the target cells through endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune escape mechanism of the target cells and delivering the API to the target cells.

P2. A therapeutic compound for RBC-mediated delivery to a target cell expressing CD47 in a mammalian subject, the therapeutic compound comprising:

A CD47 binding protein, which is conjugated to the API to form a conjugate;

wherein the CD47 binding protein is selected from the group consisting of wild-type thrombospondin-1 (TSP-1) (SEQ ID NO: 7), wild-type SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5), vSIRPγ-2 (SEQ ID NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), and TTI-663 (SEQ ID NO: 965). NO:964), homologues of any of the foregoing, and combinations thereof, and are configured to bind the conjugate to CD47 of the subject's erythrocytes so as to be transported to the target cells through the subject's circulatory system, so that (i) the CD47 binding protein configured to bind the conjugate to CD47 of the erythrocytes binds to CD47 of the target cells, thereby transferring the conjugate from the erythrocytes to the target cells to form a conjugate-CD47 complex on the target cells, thereby blocking CD47 and inhibiting CD47 activity as an immune escape mechanism of the target cells, and (ii) the conjugate is taken up by the target cells through endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune escape mechanism of the target cells and delivering the API to the target cells.

P3. A therapeutic compound for RBC-mediated delivery to a target cell expressing CD47 in a mammalian subject, the therapeutic compound comprising:

CD47 binding protein, which is conjugated to the API to form a conjugate:

Wherein the CD47 binding protein is an anti-CD47 antibody, and the anti-CD47 antibody comprises:

(a) a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 932, SEQ ID NO: 933 and SEQ ID NO: 934, respectively, and the light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 935, SEQ ID NO: 936 and SEQ ID NO: 937, respectively;

(b) a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 940, SEQ ID NO: 941 and SEQ ID NO: 942, respectively, and the light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 943, SEQ ID NO: 944 and SEQ ID NO: 945, respectively;

(c) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementary determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO:948, SEQ ID NO:949 and SEQ ID NO:950, respectively, and the light chain variable region comprises complementary determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO:951, SEQ ID NO:952 and SEQ ID NO:953, respectively; or

(d) a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementary determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 956, SEQ ID NO: 957 and SEQ ID NO: 958, respectively, and the light chain variable region comprising complementary determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 959, SEQ ID NO: 960 and SEQ ID NO: 961, respectively; and

The conjugate is configured to bind to CD47 of the subject's erythrocytes so as to be transported to the target cells through the subject's circulatory system, so that (i) the CD47 binding protein configured to bind to CD47 of the erythrocytes binds to CD47 of the target cells, thereby transferring the conjugate from the erythrocytes to the target cells to form a conjugate-CD47 complex on the target cells, thereby blocking CD47 and inhibiting CD47 activity as an immune escape mechanism of the target cells, and (ii) the conjugate is taken up by the target cells through endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune escape mechanism of the target cells and delivering the API into the target cells.

P3.5 A therapeutic compound according to claim P3, wherein the anti-CD47 antibody is a humanized antibody.

P4. The therapeutic compound according to any one of the preceding claims, wherein the CD47 binding protein is conjugated to the API via a bond selected from the group consisting of: covalent bonds, hydrogen bonds, ionic bonds, van der Waals interactions, and combinations thereof.

P5. A therapeutic compound according to any of the preceding potential claims, wherein the CD47 binding protein is conjugated to the API via a linker.

P6. The therapeutic compound according to claim P5, wherein the linker is cleavable.

P7. A therapeutic compound according to any one of claims P5 and P6, wherein the linker is configured to be cleaved by a lysosomal degradative enzyme.

P8. A therapeutic compound according to any of the preceding potential claims, wherein the API is selected from the group consisting of RNA, DNA, RNA derivatives, DNA derivatives, proteins and small molecules.

P9. The therapeutic compound according to any one of claims PI to P7, wherein the API is selected from the group consisting of siRNA, shRNA, miRNA, antimiR and mRNA.

P10. The therapeutic compound according to any of the preceding potential claims, wherein the target cell is a cell selected from the group consisting of cancer cells, virus-infected cells, fibrotic cells, and combinations thereof.

P11. The therapeutic compound according to any one of claims PI to P9, wherein the target cell is a cancer cell.

P12. The therapeutic compound according to any one of claims PI to P9, wherein the target cell is a virus-infected cell.

P13. The therapeutic compound according to any one of claims PI to P9, wherein the target cell is a fibrotic cell.

P14. A therapeutic compound according to claim P11, wherein the cancer cell is in a tumor attributable to a cancer selected from the group consisting of: brain tumors, spinal cord tumors, retinoblastoma, oral cancer, nasal cancer, paranasal sinus cancer, pharyngeal cancer, laryngeal cancer, neck cancer, head and neck cancer, melanoma, skin cancer, breast cancer, thyroid cancer, malignant adrenal tumors, endocrine cancer, lung cancer, pleural tumors, respiratory tract cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer, anal cancer, liver cancer, bile duct cancer, pancreatic cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, penis cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer, blood cancer including acute/chronic leukemia, malignant lymphoma and multiple myeloma, bone tumors, soft tissue tumors, childhood leukemia and childhood cancer.

P15. The therapeutic compound of claim P11, wherein the cancer cell is attributable to a cancer selected from the group consisting of: ovarian serous cystadenocarcinoma, lung adenocarcinoma, cervical and endocervical cancer, head and neck squamous cell carcinoma, thyroid cancer, uterine corpus endometrioid carcinoma, prostate adenocarcinoma, mesothelioma, diffuse large B-cell lymphoma, acute leukemia, lung squamous cell carcinoma, acute lymphocytic leukemia, esophageal cancer, myxofibrosarcoma, pancreatic adenocarcinoma, rectal cancer. Adenocarcinoma, colon adenocarcinoma, acute megakaryocytic leukemia, invasive breast cancer, gastric adenocarcinoma, bladder urothelial carcinoma, bile duct cancer, leukemia, thymic carcinoma, leiomyosarcoma, thymoma, undifferentiated pleomorphic sarcoma, uterine carcinosarcoma, acute myeloid leukemia, glioblastoma multiforme, sarcoma, skin melanoma, renal clear cell carcinoma, dedifferentiated liposarcoma, lymphoma, retinoblastoma, neuroblastoma, osteosarcoma, juvenile myelomonocytic leukemia, gastrointestinal stromal tumor, dysembryoplastic neuroepithelial tumor, adrenocortical carcinoma, acute leukemia of unidentified lineage, pheochromocytoma and paraganglioma, glioma, testicular germ cell tumor, supratentorial embryonal tumor NOS, neurofibroma, renal papillary cell carcinoma, hepatocellular carcinoma, renal chromophobe cell carcinoma, malignant peripheral nerve sheath tumor, ependymoma, adrenocortical carcinoma, nasopharyngeal carcinoma, spindle cell/sclerosing rhabdomyosarcoma, melanoma, choroid plexus carcinoma, undifferentiated spindle cell carcinoma, myoepithelial carcinoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, atypical teratoma/rhabdomyosarcoma, desmoplastic small round cell tumor, fibromatosis, synovial sarcoma, Wilms tumor, myofibromyxoma, fibrolamellar hepatocellular carcinoma, undifferentiated sarcoma NOS, embryonal rhabdomyosarcoma, uveal melanoma, Ewing sarcoma, hepatoblastoma, infantile fibrosarcoma, INI-deficient soft tissue sarcoma NOA, undifferentiated hepatic sarcoma, and medulloblastoma.

P16. A therapeutic compound according to claim P12, wherein the virus-infected cells are infected with the SARS-CoV-2 virus.

P17. The therapeutic compound according to claim P13, wherein the fibrotic cells are associated with cystic fibrosis.

P18. The therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand,

in:

(a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 747 and SEQ ID NO: 771 to SEQ ID NO: 824,

(b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and

(c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides.

P19. The therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand,

in:

(a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:22 to SEQ ID NO:747 and SEQ ID NO:771 to SEQ ID NO:824,

(b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and

(c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides.

P20. The therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand,

in:

(a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NO: 22 to SEQ ID NO: 37,

(b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and

(c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides.

P21. The therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand,

in:

(a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NO: 38 to SEQ ID NO: 39,

(b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and

(c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides.

P22. The therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand,

in:

(a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NO: 40 to SEQ ID NO: 43,

(b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and

(c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides.

P23. A therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand,

in:

(a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NO: 44 to SEQ ID NO: 51,

(b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and

(c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides.

P24. The therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand,

in:

(a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:21, SEQ ID NO:482 to SEQ ID NO:486, and SEQ ID NO:748 to SEQ ID NO:765,

(b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and

(c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides.

P25. The therapeutic compound according to any one of claims PI to P7, P10, P12 and P16, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand,

in:

(a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:482 to SEQ ID NO:486 and SEQ ID NO:748 to SEQ ID NO:765,

(b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and

(c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides.

P26. A therapeutic compound according to any one of claims PI to P7, P10, P13 and P17, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand,

in:

(a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:21, SEQ ID NO:40 to SEQ ID NO:43, and SEQ ID NO:766 to SEQ ID NO:770,

(b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and

(c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides.

P27. A therapeutic compound according to any one of claims PI to P7, P10, P13 and P17, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand,

in:

(a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:40 to SEQ ID NO:43 and SEQ ID NO:766 to SEQ ID NO:770,

(b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and

(c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides.

P28. A therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is a shRNA, which is a single-stranded RNA molecule having a length of 44 to 71 nucleotides and having in the 5'-3' direction:

A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence;

a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence;

a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and

In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence,

in:

(a) the first region has the same number of nucleotides as the third region,

(b) the third sequence is the reverse complementary sequence of the first sequence,

(c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 747 and SEQ ID NO: 771 to SEQ ID NO: 824, and

(d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

P29. The therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is a shRNA, which is a single-stranded RNA molecule having a length of 44 to 71 nucleotides and having in the 5'-3' direction:

A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence;

a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence;

a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and

In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence,

in:

(a) the first region has the same number of nucleotides as the third region,

(b) the third sequence is the reverse complementary sequence of the first sequence,

(c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:22 to SEQ ID NO:747 and SEQ ID NO:771 to SEQ ID NO:824, and

(d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

P30. The therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is a shRNA, which is a single-stranded RNA molecule having a length of 44 to 71 nucleotides and having in the 5'-3' direction:

A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence;

a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence;

a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and

In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence,

in:

(a) the first region has the same number of nucleotides as the third region,

(b) the third sequence is the reverse complementary sequence of the first sequence,

(c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:22 to SEQ ID NO:37, and

(d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

P31. A therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is a shRNA, which is a single-stranded RNA molecule having a length of 44 to 71 nucleotides and having in the 5'-3' direction:

A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence;

a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence;

a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and

In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence,

in:

(a) the first region has the same number of nucleotides as the third region,

(b) the third sequence is the reverse complementary sequence of the first sequence,

(c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:38 to SEQ ID NO:39, and

(d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

P32. The therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is a shRNA, which is a single-stranded RNA molecule having a length of 44 to 71 nucleotides and having in the 5'-3' direction:

A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence;

a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence;

a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and

In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence,

in:

(a) the first region has the same number of nucleotides as the third region,

(b) the third sequence is the reverse complementary sequence of the first sequence,

(c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:40 to SEQ ID NO:43, and

(d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

P33. A therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is a shRNA, which is a single-stranded RNA molecule having a length of 44 to 71 nucleotides and having in the 5'-3' direction:

A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence;

a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence;

a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and

In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence,

in:

(a) the first region has the same number of nucleotides as the third region,

(b) the third sequence is the reverse complementary sequence of the first sequence,

(c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:44 to SEQ ID NO:51, and

(d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

P34. A therapeutic compound according to any one of claims PI to P7, P10, P12 and P16, wherein the API is a shRNA, which is a single-stranded RNA molecule having a length of 44 to 71 nucleotides and having in the 5'-3' direction:

A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence;

a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence;

a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and

In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence,

in:

(a) the first region has the same number of nucleotides as the third region,

(b) the third sequence is the reverse complementary sequence of the first sequence,

(c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:21, SEQ ID NO:482 to SEQ ID NO:486, and SEQ ID NO:748 to SEQ ID NO:765, and

(d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

P35. A therapeutic compound according to any one of claims PI to P7, P10, P12 and P16, wherein the API is a shRNA, which is a single-stranded RNA molecule having a length of 44 to 71 nucleotides and having in the 5'-3' direction:

A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence;

a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence;

a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and

In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence,

in:

(a) the first region has the same number of nucleotides as the third region,

(b) the third sequence is the reverse complementary sequence of the first sequence,

(c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:482 to SEQ ID NO:486 and SEQ ID NO:748 to SEQ ID NO:765, and

(d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

P36. A therapeutic compound according to any one of claims PI to P7, P10, P13 and P17, wherein the API is a shRNA, which is a single-stranded RNA molecule having a length of 44 to 71 nucleotides and having in the 5'-3' direction:

A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence;

a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence;

a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and

In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence,

in:

(a) the first region has the same number of nucleotides as the third region,

(b) the third sequence is the reverse complementary sequence of the first sequence,

(c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:21, SEQ ID NO:40 to SEQ ID NO:43, and SEQ ID NO:766 to SEQ ID NO:770, and

(d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

P37. A therapeutic compound according to any one of claims PI to P7, P10, P13 and P17, wherein the API is a shRNA, which is a single-stranded RNA molecule having a length of 44 to 71 nucleotides and having in the 5'-3' direction:

A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence;

a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence;

a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and

In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence,

in:

(a) the first region has the same number of nucleotides as the third region,

(b) the third sequence is the reverse complementary sequence of the first sequence,

(c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:40 to SEQ ID NO:43 and SEQ ID NO:766 to SEQ ID NO:770, and

(d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang.

P38. A therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is a miRNA selected from the group consisting of: SEQ ID NO: 825 to SEQ ID NO: 844, SEQ ID NO: 849 to SEQ ID NO: 851, SEQ ID NO: 853, SEQ ID NO: 855, SEQ ID NO: 857, SEQ ID NO: 864, SEQ ID NO: 865 and

SEQ ID NO:867 to SEQ ID NO:883.

P39. A therapeutic compound according to any one of claims P1 to P7, P10, P11, P14 and P15, wherein the API is an antimiR, which is a single-stranded nucleic acid molecule with a length of 12 to 25 nucleotides, and the antimiR has a sequence of 12 to 25 consecutive nucleotides that are complementary to consecutive nucleotides in a target mature miRNA product sequence, and the mature miRNA product sequence is selected from the group consisting of SEQ ID NO: 884 to SEQ ID NO: 908, wherein the consecutive nucleotides in the mature miRNA product sequence include the 2nd to 8th nucleotides of the mature miRNA product sequence in the 5' to 3' direction.

P40. A therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is a small molecule selected from the group consisting of: methotrexate; doxorubicin; vinca alkaloids; camptothecin analogs; microtubule disrupting agents such as auristatins (e.g., MMAE and MMAF) and maytansine alkaloids (e.g., DM1 and DM4); and DNA damaging agents such as DNA topoisomerase I inhibitors (e.g., SN-38 and exitecan), double-strand break agents (e.g., calicheamicin), cross-linking agents (e.g., pyrrolobenzodiazepine dimer-PBD) and alkylating agents (e.g., duocarmycin and indolebenzodiazepine dimer-IGN).

P41. A therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 909 to SEQ ID NO: 929 and homologues thereof.

P42. A therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is a protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 909 to SEQ ID NO: 929 and homologues thereof.

P43. A therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NO:909 to SEQ ID NO:929 and homologs thereof, and the mRNA is configured to be translated in the target cell to produce a protein comprising the amino acid sequence.

P44. A therapeutic compound according to any one of claims PI to P7, P10, P11, P14 and P15, wherein the API is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NO:909 to SEQ ID NO:929 and homologs thereof, and the mRNA is configured to be translated in the target cell to produce a protein consisting of the amino acid sequence.

P45. A therapeutic compound according to any one of claims P43 and P44, wherein the mRNA is codon optimized.

P46. A method of treating cancer in a mammalian subject in need thereof, the method comprising administering a therapeutically effective amount of a therapeutic compound according to any one of claims Pl to P11, P14, P15, P18 to P23, P28 to P33, and P38 to P45.

P47. A method of treating a viral infection in a mammalian subject in need thereof, the method comprising administering a therapeutically effective amount of a therapeutic compound according to any one of claims PI to P10, P12, P16, P24, P25, P34 and P35.

P48. A method of treating a fibrotic disease in a mammalian subject in need thereof, the method comprising administering a therapeutically effective amount of a therapeutic compound according to any one of claims PI to P10, P13, P17, P26, P27, P36 and P37.

P49. A therapeutic compound according to any one of claims PI to P45, wherein the mammalian subject is a human.

P50. The method according to any one of claims P46 to P48, wherein the mammalian subject is a human.

P51. A pharmaceutical composition comprising a therapeutic compound according to any one of claims PI to P45 and P49 and a pharmaceutically acceptable carrier.

The embodiments of the present invention described above are intended to be exemplary only; many variations and modifications are apparent to those skilled in the art. All such variations and modifications are intended to be included within the scope of the present invention as defined by any appended claims.

Claims (51)

1. A therapeutic compound for RBC-mediated delivery to a target cell expressing CD47 in a mammalian subject, the therapeutic compound comprising: A CD47 binding protein, which is conjugated to the API to form a conjugate; wherein the CD47 binding protein is selected from the group consisting of wild-type thrombospondin-1 (TSP-1) (SEQ ID NO: 7), wild-type SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5), vSIRPγ-2 (SEQ ID NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), and TTI-663 (SEQ ID NO: 965). NO:964), homologues of any of the foregoing, and combinations thereof, and are configured to bind the conjugate to CD47 of the subject's erythrocytes so as to be transported to the target cells through the subject's circulatory system, so that (i) the CD47 binding protein configured to bind the conjugate to CD47 of the erythrocytes binds to CD47 of the target cells, thereby transferring the conjugate from the erythrocytes to the target cells to form a conjugate-CD47 complex on the target cells, thereby blocking CD47 and inhibiting CD47 activity as an immune escape mechanism of the target cells, and (ii) the conjugate is taken up by the target cells through endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune escape mechanism of the target cells and delivering the API to the target cells. 2. A therapeutic compound for RBC-mediated delivery to a target cell expressing CD47 in a mammalian subject, the therapeutic compound comprising: A CD47 binding protein, which is conjugated to the API to form a conjugate; wherein the CD47 binding protein is selected from the group consisting of wild-type SIRPα (SEQ ID NO: 1), vSIRPα (SEQ ID NO: 3), wild-type thrombospondin-1 (TSP-1) (SEQ ID NO: 7), wild-type SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5), vSIRPγ-2 (SEQ ID NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), and TTI-663 (SEQ ID NO: 965). NO:964), homologues of any of the foregoing, and combinations thereof, and are configured to bind the conjugate to CD47 of the subject's erythrocytes so as to be transported to the target cells through the subject's circulatory system, so that (i) the CD47 binding protein configured to bind the conjugate to CD47 of the erythrocytes binds to CD47 of the target cells, thereby transferring the conjugate from the erythrocytes to the target cells to form a conjugate-CD47 complex on the target cells, thereby blocking CD47 and inhibiting CD47 activity as an immune escape mechanism of the target cells, and (ii) the conjugate is taken up by the target cells through endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune escape mechanism of the target cells and delivering the API to the target cells. 3. A therapeutic compound for RBC-mediated delivery to a target cell expressing CD47 in a mammalian subject, the therapeutic compound comprising: A CD47 binding protein, which is conjugated to the API to form a conjugate; Wherein the CD47 binding protein is an anti-CD47 antibody, and the anti-CD47 antibody comprises: (a) a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 932, SEQ ID NO: 933 and SEQ ID NO: 934, respectively, and the light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 935, SEQ ID NO: 936 and SEQ ID NO: 937, respectively; (b) a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 940, SEQ ID NO: 941 and SEQ ID NO: 942, respectively, and the light chain variable region comprising complementarity determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO: 943, SEQ ID NO: 944 and SEQ ID NO: 945, respectively; (c) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementary determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO:948, SEQ ID NO:949 and SEQ ID NO:950, respectively, and the light chain variable region comprises complementary determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO:951, SEQ ID NO:952 and SEQ ID NO:953, respectively; or (d) a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementary determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO:956, SEQ ID NO:957 and SEQ ID NO:958, respectively, and the light chain variable region comprising complementary determining regions CDR1, CDR2 and CDR3 comprising SEQ ID NO:959, SEQ ID NO:960 and SEQ ID NO:961, respectively; and The conjugate is configured to bind to CD47 of the subject's erythrocytes so as to be transported to the target cells through the subject's circulatory system, so that (i) the CD47 binding protein configured to bind to CD47 of the erythrocytes binds to CD47 of the target cells, thereby transferring the conjugate from the erythrocytes to the target cells to form a conjugate-CD47 complex on the target cells, thereby blocking CD47 and inhibiting CD47 activity as an immune escape mechanism of the target cells, and (ii) the conjugate is taken up by the target cells through endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune escape mechanism of the target cells and delivering the API into the target cells. 4. The therapeutic compound of any one of the preceding claims, wherein the CD47 binding protein is conjugated to the API via a bond selected from the group consisting of: covalent bonds, hydrogen bonds, ionic bonds, van der Waals interactions, and combinations thereof. 5. The therapeutic compound according to any one of the preceding claims, wherein the CD47 binding protein is conjugated to the API via a linker. 6. The therapeutic compound of claim 5, wherein the linker is cleavable. 7. The therapeutic compound of any one of claims 5 and 6, wherein the linker is configured to be cleaved by a lysosomal degradative enzyme. 8. The therapeutic compound according to any one of the preceding claims, wherein the API is selected from the group consisting of RNA, DNA, RNA derivatives, DNA derivatives, proteins and small molecules. 9. The therapeutic compound according to any one of claims 1 to 7, wherein the API is selected from the group consisting of siRNA, shRNA, miRNA, antimiR and mRNA. 10. The therapeutic compound according to any one of the preceding claims, wherein the target cell is a cell selected from the group consisting of cancer cells, virus-infected cells, fibrotic cells, and combinations thereof. 11. The therapeutic compound of any one of claims 1 to 8, wherein the target cell is a cancer cell. 12. The therapeutic compound of any one of claims 1 to 8, wherein the target cell is a virus-infected cell. 13. The therapeutic compound of any one of claims 1 to 8, wherein the target cell is a fibrotic cell. 14. The therapeutic compound of claim 11, wherein the cancer cell is in a tumor attributable to a cancer selected from the group consisting of brain tumors, spinal cord tumors, retinoblastoma, oral cancer, nasal cavity cancer, paranasal sinus cancer, pharyngeal cancer, laryngeal cancer, neck cancer, head and neck cancer, melanoma, skin cancer, breast cancer, thyroid cancer, malignant adrenal tumors, endocrine cancer, lung cancer, pleural tumors, respiratory tract cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer, anal cancer, liver cancer, bile duct cancer, pancreatic cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, penis cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer, blood cancers including acute/chronic leukemias, malignant lymphomas and multiple myeloma, bone tumors, soft tissue tumors, childhood leukemias and childhood cancers. 15. The therapeutic compound of claim 11, wherein the cancer cell is attributable to a cancer selected from the group consisting of: ovarian serous cystadenocarcinoma, lung adenocarcinoma, cervical and endocervical cancer, head and neck squamous cell carcinoma, thyroid cancer, uterine corpus endometrioid carcinoma, prostate adenocarcinoma, mesothelioma, diffuse large B-cell lymphoma, acute leukemia, lung squamous cell carcinoma, acute lymphocytic leukemia, esophageal cancer, myxofibrosarcoma, pancreatic adenocarcinoma, rectal adenocarcinoma, Cancer, colon adenocarcinoma, acute megakaryocytic leukemia, invasive breast cancer, gastric adenocarcinoma, bladder urothelial carcinoma, bile duct cancer, leukemia, thymic carcinoma, leiomyosarcoma, thymoma, undifferentiated pleomorphic sarcoma, uterine carcinosarcoma, acute myeloid leukemia, glioblastoma multiforme, sarcoma, skin melanoma, renal clear cell carcinoma, dedifferentiated liposarcoma, lymphoma, retinoblastoma, neuroblastoma, osteosarcoma, juvenile myelomonocytic leukemia, gastrointestinal Stromal tumors, dysembryoplastic neuroepithelial tumors, adrenocortical carcinoma, acute leukemia of unidentified lineage, pheochromocytoma and paraganglioma, glioma, testicular germ cell tumor, supratentorial embryonal tumor NOS, neurofibroma, renal papillary cell carcinoma, hepatocellular carcinoma, renal chromophobe cell carcinoma, malignant peripheral nerve sheath tumor, ependymoma, adrenocortical carcinoma, nasopharyngeal carcinoma, spindle cell/sclerosing rhabdomyosarcoma, melanoma, choroid plexus carcinoma, undifferentiated spindle cell carcinoma, myoepithelial carcinoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, atypical teratoma/rhabdomyosarcoma, desmoplastic small round cell tumor, fibromatosis, synovial sarcoma, Wilms tumor, myofibromyxoma, fibrolamellar hepatocellular carcinoma, undifferentiated sarcoma NOS, embryonal rhabdomyosarcoma, uveal melanoma, Ewing sarcoma, hepatoblastoma, infantile fibrosarcoma, INI-deficient soft tissue sarcoma NOA, undifferentiated hepatic sarcoma, and medulloblastoma. 16. The therapeutic compound of claim 12, wherein the virus-infected cells are infected with the SARS-CoV-2 virus. 17. The therapeutic compound of claim 13, wherein the fibrotic cells are associated with cystic fibrosis. 18. The therapeutic compound according to any one of claims 11, 14 and 15, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, in: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 747 and SEQ ID NO: 771 to SEQ ID NO: 824, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides. 19. The therapeutic compound according to any one of claims 11, 14 and 15, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, in: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:22 to SEQ ID NO:747 and SEQ ID NO:771 to SEQ ID NO:824, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides. 20. The therapeutic compound according to any one of claims 11, 14 and 15, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, in: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NO: 22 to SEQ ID NO: 37, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides. 21. The therapeutic compound according to any one of claims 11, 14 and 15, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, in: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NO: 38 to SEQ ID NO: 39, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides. 22. The therapeutic compound according to any one of claims 11, 14 and 15, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, in: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NO: 40 to SEQ ID NO: 43, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides. 23. The therapeutic compound according to any one of claims 11, 14 and 15, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, in: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, wherein the mRNA sequence is selected from the group consisting of SEQ ID NO: 44 to SEQ ID NO: 51, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides. 24. The therapeutic compound according to any one of claims 12 and 16, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, in: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 21, SEQ ID NO: 482 to SEQ ID NO: 486, and SEQ ID NO: 748 to SEQ ID NO: 765, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides. 25. The therapeutic compound according to any one of claims 12 and 16, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, in: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:482 to SEQ ID NO:486 and SEQ ID NO:748 to SEQ ID NO:765, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides. 26. The therapeutic compound according to any one of claims 13 and 17, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, in: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:21, SEQ ID NO:40 to SEQ ID NO:43, and SEQ ID NO:766 to SEQ ID NO:770, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides. 27. The therapeutic compound according to any one of claims 13 and 17, wherein the API is siRNA, which is a double-stranded RNA molecule comprising an antisense RNA strand and a sense RNA strand, in: (a) the antisense RNA strand is 19 to 29 nucleotides in length and is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:40 to SEQ ID NO:43 and SEQ ID NO:766 to SEQ ID NO:770, (b) the sense RNA strand is 19 to 29 nucleotides in length and is complementary to 14 to 29 nucleotides from the antisense RNA strand, and (c) The double-stranded RNA molecule has a double-stranded region with a length of 14 to 29 nucleotides and a 3' overhang region with a length of 0 to 5 nucleotides. 28. The therapeutic compound of any one of claims 11, 14 and 15, wherein the API is a shRNA, which is a single-stranded RNA molecule of 44 to 71 nucleotides in length and has in the 5'-3' direction: A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence, in: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 747 and SEQ ID NO: 771 to SEQ ID NO: 824, and (d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 29. The therapeutic compound of any one of claims 11, 14 and 15, wherein the API is a shRNA, which is a single-stranded RNA molecule of 44 to 71 nucleotides in length and has in the 5'-3' direction: A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence, in: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:22 to SEQ ID NO:747 and SEQ ID NO:771 to SEQ ID NO:824, and (d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 30. The therapeutic compound of any one of claims 11, 14 and 15, wherein the API is a shRNA, which is a single-stranded RNA molecule of 44 to 71 nucleotides in length and has in the 5'-3' direction: A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence, in: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:22 to SEQ ID NO:37, and (d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 31. The therapeutic compound of any one of claims 11, 14 and 15, wherein the API is a shRNA, which is a single-stranded RNA molecule of 44 to 71 nucleotides in length and has in the 5'-3' direction: A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence, in: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:38 to SEQ ID NO:39, and (d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 32. The therapeutic compound of any one of claims 11, 14 and 15, wherein the API is a shRNA, which is a single-stranded RNA molecule of 44 to 71 nucleotides in length and has in the 5'-3' direction: A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence, in: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:40 to SEQ ID NO:43, and (d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 33. The therapeutic compound of any one of claims 11, 14 and 15, wherein the API is a shRNA, which is a single-stranded RNA molecule of 44 to 71 nucleotides in length and has in the 5'-3' direction: A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence, in: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:44 to SEQ ID NO:51, and (d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 34. The therapeutic compound of any one of claims 12 and 16, wherein the API is a shRNA, which is a single-stranded RNA molecule of 44 to 71 nucleotides in length and has in the 5'-3' direction: A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence, in: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:21, SEQ ID NO:482 to SEQ ID NO:486, and SEQ ID NO:748 to SEQ ID NO:765, and (d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 35. The therapeutic compound of any one of claims 12 and 16, wherein the API is a shRNA, which is a single-stranded RNA molecule of 44 to 71 nucleotides in length and has in the 5'-3' direction: A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence, in: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:482 to SEQ ID NO:486 and SEQ ID NO:748 to SEQ ID NO:765, and (d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 36. The therapeutic compound of any one of claims 13 and 17, wherein the API is a shRNA, which is a single-stranded RNA molecule of 44 to 71 nucleotides in length and has in the 5'-3' direction: A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence, in: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:21, SEQ ID NO:40 to SEQ ID NO:43, and SEQ ID NO:766 to SEQ ID NO:770, and (d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 37. The therapeutic compound of any one of claims 13 and 17, wherein the API is a shRNA, which is a single-stranded RNA molecule of 44 to 71 nucleotides in length and has in the 5'-3' direction: A first region of 19 to 29 nucleotides at the 5' end of the single-stranded RNA molecule, wherein the first region has a first sequence; a second region of 4 to 11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19 to 29 nucleotides directly adjacent to the second region, the third region having a third sequence; and In a fourth region of 2 nucleotides directly adjacent to the third region at the 3' end of the single-stranded RNA molecule, the fourth region has a fourth sequence, in: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse complementary sequence of the first sequence, (c) the third region is complementary to consecutive nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO:40 to SEQ ID NO:43 and SEQ ID NO:766 to SEQ ID NO:770, and (d) The single-stranded RNA molecule is configured to form a stem-loop structure, the first region is base-paired with the third region to form a stem, the second region forms a loop, and the fourth region forms a 3' overhang. 38. The therapeutic compound of any one of claims 11, 14 and 15, wherein the API is a miRNA selected from the group consisting of SEQ ID NO: 825 to SEQ ID NO: 844, SEQ ID NO: 849 to SEQ ID NO: 851, SEQ ID NO: 853, SEQ ID NO: 855, SEQ ID NO: 857, SEQ ID NO: 864, SEQ ID NO: 865 and SEQ ID NO: 867 to SEQ ID NO: 883. 39. A therapeutic compound according to any one of claims 11, 14 and 15, wherein the API is an antimiR, which is a single-stranded nucleic acid molecule with a length of 12 to 25 nucleotides, and the antimiR has a sequence of 12 to 25 consecutive nucleotides that are complementary to consecutive nucleotides in a target mature miRNA product sequence, and the mature miRNA product sequence is selected from the group consisting of SEQ ID NO: 884 to SEQ ID NO: 908, wherein the consecutive nucleotides in the mature miRNA product sequence include the 2nd to 8th nucleotides of the mature miRNA product sequence in the 5' to 3' direction. 40. The therapeutic compound of any one of claims 11, 14, and 15, wherein the API is a small molecule selected from the group consisting of: methotrexate; doxorubicin; vinca alkaloids; camptothecin analogs; microtubule disrupting agents; and DNA damaging agents. 41. The therapeutic compound of any one of claims 11, 14 and 15, wherein the API is a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 909 to SEQ ID NO: 929 and homologs thereof. 42. The therapeutic compound of any one of claims 11, 14 and 15, wherein the API is a protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 909 to SEQ ID NO: 929 and homologs thereof. 43. The therapeutic compound of any one of claims 11, 14 and 15, wherein the API is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 909 to SEQ ID NO: 929 and homologs thereof, and the mRNA is configured to be translated in the target cell to produce a protein comprising the amino acid sequence. 44. The therapeutic compound of any one of claims 11, 14 and 15, wherein the API is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 909 to SEQ ID NO: 929 and homologs thereof, and the mRNA is configured to be translated in the target cell to produce a protein consisting of the amino acid sequence. 45. The therapeutic compound of any one of claims 43 and 44, wherein the mRNA is codon optimized. 46. A method of treating cancer in a mammalian subject in need thereof, the method comprising administering a therapeutically effective amount of a therapeutic compound according to any one of claims 1 to 11, 14, 15, 18 to 23, 28 to 33, and 38 to 45. 47. A method of treating a viral infection in a mammalian subject in need thereof, the method comprising administering a therapeutically effective amount of a therapeutic compound according to any one of claims 1 to 10, 12, 16, 24, 25, 34 and 35. 48. A method of treating a fibrotic disease in a mammalian subject in need thereof, the method comprising administering a therapeutically effective amount of a therapeutic compound according to any one of claims 1 to 10, 13, 17, 26, 27, 36 and 37. 49. The therapeutic compound of any one of claims 1 to 45, wherein the mammalian subject is a human. 50. The method of any one of claims 46 to 48, wherein the mammalian subject is a human. 51. A pharmaceutical composition comprising a therapeutic compound according to any one of claims 1 to 45 and 49 and a pharmaceutically acceptable carrier.