Antibody oligonucleotide conjugates for treating disease

Abstract

Disclosed herein are antibody-oligonucleotide conjugates, pharmaceutical compositions, and methods for treating Sickle Cell Disease. The disclosure contemplates compositions and methods capable of binding a human hematopoietic stem cell marker conjugated to an oligonucleotide capable of hybridizing a target sequence. In some embodiments, the target sequence is a BCL11A transcript sequence. In some embodiments of the disclosure, the antibody-oligonucleotide conjugate promotes antisense- and / or RNA interference-mediated inhibition of BCL11A.

Description

[0001] ANTIBODY OLIGONUCLEOTIDE CONJUGATES FOR TREATING DISEASE CROSS-REFERENCE TO RELATED APPLICATION The present application is related to and claims priority under 35 U.S.C. § 119(e) to U. S. provisional patent application No. 63 / 718,130, entitled “Antibody Oligonucleotide Conjugates for Treating Disease,’’ filed November 8, 2024 The entire content of the aforementioned patent application is incorporated herein by this reference.

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to compositions and methods for modulating BCL11A, which is a gene associated with hemoglobinopathies, e.g., Sickle Cell Disease (SCD). BCL11A-targeting compositions of the disclosure include oligonucleotides (e.g., siRNAs) and antibody-oligonucleotide conjugates (AOCs).

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been filed electronically in eXtensible Markup Language format and is hereby incorporated by reference in its entirety. Said XML file, created on November 5, 2025, is named 808955_000180.xml and is 716,587 bytes in size.

[0006] BACKGROUND

[0007] Gene suppression by RNA-induced gene silencing provides several levels of control: transcription inactivation, small interfering RNA (siRNA)-induced mRNA degradation, and siRNA-induced transcriptional attenuation. In some instances, RNA interference (RNAi) provides long lasting effect over multiple cell divisions. As such, RNAi represents a viable method useful for drug target validation, gene function analysis, pathway analysis, and disease therapeutics.

[0008] SUMMARY OF THE DISCLOSURE

[0009] Disclosed herein, in certain embodiments, are oligonucleotides and pharmaceutical compositions for modulating a gene associated with hemoglobinopathies, especially Sickle Cell Disease (SCD). In some embodiments, also described herein are methods of treating hemoglobinopathies, especially Sickle Cell Disease (SCD), with a nucleic acid molecule or an antibody-oligonucleotide conjugate disclosed herein.

[0010] Disclosed herein, in certain embodiments, is an antibody-oligonucleotide conjugate comprising an antibody or antigen binding fragment thereof conjugated to a nucleic acid molecule that hybridizes to a target sequence of BCL11A, and the antibody-oligonucleotide conjugate mediates RNA interference against the BCL11 A.

[0011] In some embodiments, the antibody-oligonucleotide conjugate includes an antibody or antigen binding fragment thereof capable of binding a human hematopoietic stem cell marker conjugated to an oligonucleotide capable of hybridizing to a target sequence of a BCL11A transcript and mediating antisense and / or RNA interference-mediated inhibition of the BCL11A transcript. In some embodiments, the human hematopoietic stem cell marker is selected from the group: HLA-DR, CD11a, CD18, CD34, CD41 / 61, CD43, CD45, CD49d (VLA-4), CD49f (VLA-6), CD51, CD58, CD71, CD84, CD90, CD97, CD117 (c-kit), CD 133. CD134, CD162, CDI66, CD 184 (CXCR4), CD205 and CD361.

[0012] In some embodiments, the oligonucleotide of the antibody-oligonucleotide conjugate is a strand of a double-stranded nucleic acid. Optionally, the oligonucleotide of the antibody-oligonucleotide conjugate is a small interfering RNA (siRNA). Optionally, the oligonucleotide includes one or more of the following respective siRNA strand sequences: SEQ ID NO: 23 and SEQ ID NO: 121; SEQ ID NO: 19 and SEQ ID NO: 117; SEQ ID NO: 25 and SEQ ID NO: 123; SEQ ID NO: 27 and SEQ ID NO: 125; SEQ ID NO: 29 and SEQ ID NO: 127; and SEQ ID NO: 32 and SEQ ID NO: 130.

[0013] In some embodiments, the antibody or antigen binding fragment thereof capable of binding the human hematopoietic stem cell marker includes a variable heavy chain (VH) region comprising at least one sequence selected from Table 1 or Table 3, a variable light chain (VL) region comprising at least one sequence selected from Table 2 or Table 4, at least one sequence selected from Table 5, a heavy chain sequence selected from Table 6, and / or a light chain sequence selected from Table 7

[0014] In some embodiments, a conjugate of the disclosure includes a cell penetrating peptide conjugated to an oligonucleotide capable of hybridizing to a target sequence of a BCL11A transcript and mediating antisense and / or RNA interference-mediated inhibition of the BCL11A transcript. In still further embodiments, the cell penetrating peptide includes a sequence selected from Table 9.

[0015] In certain embodiments, the antibody or antigen binding fragment thereof includes a nonhuman antibody or binding fragment thereof, a human antibody or antigen binding fragment thereof, a humanized antibody or antigen binding fragment thereof chimeric antibody or antigen binding fragment thereof, monoclonal antibody or antigen binding fragment thereof, monovalent Fab', divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, singledomain antibody (sdAb), or camelid antibody or antigen binding fragment thereof.

[0016] In certain embodiments, the oligonucleotide includes a sense strand and / or an antisense strand, and wherein the sense strand and / or the antisense strand each independently includes at least one 2' modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety In certain embodiments, the oligonucleotide hybridizes to at least 8 contiguous bases of the target sequence of BCL11A. In certain embodiments, the oligonucleotide is from about 8 to about 50 nucleotides in length or from about 10 to about 30 nucleotides in length. In certain embodiments, the nucleic acid molecule includes a sense strand and / or an antisense strand, and the sense strand includes a sequence of at least 12 nucleotides in length that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence selected from Table 10, Table 11, Table 12, Table 13, Table 14, SEQ ID NOs: 1-98; SEQ ID NOs: 197-231; SEQ ID NOs: 267-277: SEQ ID NO: 289; and SEQ ID NOs: 291-580. Alternatively and / or additionally, the oligonucleotide includes a sense strand and / or an antisense strand, and the antisense strand includes a sequence of at least 12 nucleotides in length that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence selected from Table 10, Table 11, Table 12, Table 13, Table 14, SEQ ID NOs: 99-196; SEQ ID NOs: 232-266; SEQ ID NOs: 278-288; SEQ ID NO: 290; and SEQ ID NOs: 291-580.

[0017] In certain embodiments, the oligonucleotide includes at least one 2' modified nucleotide, and optionally the 2' modified nucleotide includes 2’-O-methyl, 2'-O-methoxyethyl (2'-0-M0E), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fiuoro, 2'-O-antinopropyl (2-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2-O-DMAEOE), or 2'-0-N-m ethyl acetamido (2-0-NMA) modified nucleotide, or includes locked nucleic acid (LNA) or ethylene nucleic acid (ENA), or includes a combination thereof. In certain embodiments, the at least one modified internucleotide linkage includes a phosphorothioate linkage or a phosphorodithioate linkage.

[0018] In certain embodiments, the oligonucleotide includes 3 or more 2' modified nucleotides selected from 2-O-methyl and 2'-deoxy-2'-fluoro. In certain embodiments, the nucleic acid molecule includes a 5'-terminal vinylphosphonate modified nucleotide, such as those described in U. S. Publication No. 2019 / 0192681.

[0019] In certain embodiments, the 2' modified nucleotide is 2'-O-methyl modified nucleotide, and 2'-O-methyl modified nucleotide is at the 5'-end of the sense strand and / or the antisense strand. In some embodiments, the 2'-O-methyl modified nucleotide is a purine nucleotide, or the2'-O-methyl modified nucleotide is a pyridine nucleotide In certain embodiments, the sense and / or antisense strands include at least two, three, four consecutive the 2'-O-methyl modified nucleotides at the 5'-end. In certain embodiments, the antibody-oligonucleotide conjugate includes a linker connecting the target cell binding moiety to the nucleic acid moiety. In such embodiments, the linker is Cl-C6 alkyl linker, or the linker is a homo-bifunctional linker or heterobifunctional linker, and includes a maleimide group, a dipeptide moiety, a benzoic acid group, or its derivative thereof. Alternatively and / or additionally, the linker is a cleavable or non-cleavable linker. In certain embodiments, a ratio between the oligonucleotide moiety and the target cell binding moiety is about 1:1, 2:1, 3:1, or 4:1.

[0020] In certain embodiments, the oligonucleotide moiety mediates RNA interference against the human BCL11A and modulates symptoms of hemoglobinopathy in a subject. In some embodiments, the RNA interference includes reducing expression of the mRNA transcript of BCL11A gene at least 50%, at least 60%, or at least 70% or more compared to a quantity of the mRNA transcript of BCL11A gene in an untreated cell. In some embodiments, the hemoglobinopathy is Sickle Cell Disease (SCD).

[0021] In certain embodiments, antibody-oligonucleotide conjugate includes a molecule of Formula (I): A-X-B, where A is the antibody or antigen binding fragment thereof, B is the nucleic acid molecule that hybridizes to a target sequence of BCL I 1A, X is a bond or a non-polymeric linker, which is conjugated to a cysteine residue of A.

[0022] Disclosed herein are methods and compositions that are useful for ablating selected cell populations and conditioning a subject's tissues for engraftment or transplant, as well as assays and methods of identifying candidate antibodies or antigen binding fragments thereof that are useful for conditioning a subject's tissues for engraftment or transplant. In certain embodiments, the methods and compositions disclosed herein are non-myeloablative. Also disclosed are methods of delivering an oligonucleotide to a cell, e.g., by targeting one or more markers (e.g., the cell surface CD45 or CD117 markers), such that oligonucleotide is internalized; such methods are useful for effectively conditioning a subject for engraftment or transplant (e.g., conditioning a human subject for hematopoietic stem cell transplant).

[0023] In some embodiments, the inventions disclosed herein are directed to methods of selectively depleting or ablating an endogenous hematopoietic stem cell or progenitor cell population in a target tissue of a subject, the methods comprising administering to the subject an effective amount of an antibody or antigen binding fragment thereof coupled (e.g., functionally coupled) to an oligonucleotide; wherein the antibody or antigen binding fragment thereof selectively binds to CD 117 and the oligonucleotide is internalized by the endogenous HSC or progenitor cell population, thereby depleting or ablating the endogenous HSC or progenitor cell population in the target tissue.

[0024] In some embodiments, the anti-CD117 antibody or antigen binding fragment thereof (or the anti-CD7I antibody or antigen-binding fragment, or other antibody or antigen-binding fragment disclosed herein) is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a known CD! 17-binding antibody sequence (or to a sequence of a known anti-CD71 antibody or antigen-binding fragment, or to a sequence of a known other antibody or antigen-binding fragment disclosed herein). Optionally, the anti-CD117 antibody or antigen binding fragment thereof (or the anti-CD71 antibody or antigen-binding fragment, or other antibody or antigen-binding fragment disclosed herein) is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a commercially available CD 117-binding antibody sequence (or to a sequence of a corn merci ally available anti-CD71 antibody or antigenbinding fragment, or to a sequence of a commercially available other antibody or antigen-binding fragment disclosed herein).

[0025] In certain embodiments, disclosed herein are methods of stem cell transplant (e.g., hematopoietic stem cell transplant), such methods comprising: administering to a subject an effective amount of an internalizing antibody which specifically or selectively binds to GDI 17 and is coupled to an oligonucleotide and thereby ablating an endogenous stem cell population in a target tissue, and administering an exogenous stem cell population in the target tissue of the subject. In certain aspects, also disclosed are methods of treating or curing a hemoglobinopathy (e.g., sickle cell anemia) in a subject, the methods comprising: administering to the subject an effective amount of an internalizing antibody that specifically or selectively binds to CD71 or CD117 and is coupled to an oligonucleotide and thereby ablating an endogenous stem cell (e.g., hematopoietic stem cell) or progenitor cell population in a target tissue of the subject; followed by a step of administering an exogenous stem cell population to the target tissue of the subject.

[0026] Any markers that are capable of being used to discriminate the target cell population from the population of non-targeted cells, including any of the markers described herein, can be targeted by the antibodies or antigen binding fragments thereof that comprise the oligonucleotide described herein for delivery of oligonucleotide to the cell population. For example, in certain aspects of the present inventions, an antibody or antigen binding fragment, thereof that includes the oligonucleotide composition may selectively bind to one or more cell surface markers expressed by the cells of the target tissues.

[0027] In certain embodiments, the targeted hematopoietic stem cells or progenitor cells express one or more markers that may be targeted and to which the oligonucleotide selectively or preferentially binds, such markers selected from the group of markers consisting of CD71, CD117 (c-kit), HLA-DR, CD1 l a, CD 18, CD34, CD41 / 61, CD43, CD45, CD49d (VLA-4), CD49f (VLA-6), CD51. CD58, CD84, CD90, CD97, CD 133, CD 134, CD 162, CD 166, CD 184 (CXCR4), CD205 and CD36I. In certain embodiments, the targeted cells (e.g., the hematopoietic stem cells or progenitor cells) in the target tissue express one or more markers that may be targeted and to which the oligonucleotide selectively or preferentially binds, such markers selected from the group of markers consisting of: CD71, CD 1 17: cKit / SCF receptor, CD 13, CD33, CD34, CD44, CD45, CD49d: VLA-4, CD49f: VLA-6, CD59, CD84: CD150 family, CD90: Thyl, CD93, CD105: Endoglin, CD123' IL-3R, CD126: IL-6R, CD133, CD135: Flt3 receptor, CD166: ALCAM, CD 184: CXCR4, Prominin 2, Erythropoietin R, Endothelial Cell-Selective Adhesion Molecule, CD244, Tiel, Tie2, MPL, G-CSFRor CSF3R, IL-1R, gp!30, Leukemia inhibitory factor Receptor, oncostatin M receptor, Embigin and IL-18R. In still other embodiments, the targeted cells (e.g., hematopoietic stem cells or progenitor cells) in the target tissue express one or more markers that may be targeted and to which the antibodies or antigen binding fragments thereof that comprise the oligonucleotide selectively bind, such markers selected from the group of markers consisting of CD150, ( 1)27 and CD201. For example, in some embodiments, the hematopoietic stem cells or progenitor cells express CD45. Similarly, in some embodiments, the hematopoietic stem cells or progenitor cells express GDI 17. Similarly, in some embodiments, the hematopoietic stem cells or progenitor cells express CD34.

[0028] In certain embodiments, the marker is selected from the group consisting of CD71, CD117 (c-kit), HLA-DR, CD 11 a, CD 18, CD34, CD41 / 61, CD43, CD45, CD47, CD58, CD84, CD97, CD133, CD162, CD166, CD205 and CD361. In certain embodiments, the targeted cells comprise human hematopoietic stem cells expressing one or more markers that may be targeted and to which the antibodies or antigen binding fragments thereof that comprise the oligonucleotide bind, such markers selected from the group consisting of CD71, CD117, CD7, CDwl2, CD 13, CD 15, CD 19, CD21, CD22, CD29, CD30, CD33, CD34, CD36, CD38, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD48, CD49b, CD49d, CD49e, CD49f, CD50, CD53, CD55, CD64a, CD68, CD72, CD73, CD81, CD82, CD85A, CD85K, CD90, CD99, CD 104, CD 105, CD 109, GDI 10, GDI 11, CD 112, CD 114, CD115, GDI 23, CD 124, CD 126, CD127, CD130, CD131, CD133, CD135, CD138, CD151, CD157, CD 162, CD 164, CD168, CD 172a, CD 173, CD 174, CD 175, CD175s, CD 176, CD183, CD191, CD 200, CD201, CD205, CD217, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD235a, CD235b, CD236, CD236R, CD238, CD240, CD242, CD243, CD277, CD292, CDw293, CD295, CD298, CD309, CD318, CD324, CD325, CD338, CD344, CD349 and CD35O.

[0029] In some embodiments, the antibodies or antigen binding fragments thereof disclosed herein selectively bind to GDI 17, In certain aspects, the antibody or antigen binding fragment thereof is a CD117 antagonist. Alternatively, in certain aspects the antibody or antigen binding fragment thereof is not a CD 117 antagonist. In some embodiments, the toxin is internalized by a cell expressing CD 117 following binding of the antibody or antigen binding fragment thereof to an epitope of the GDI 17 cell surface marker.

[0030] In certain embodiments, the antibody or antigen binding fragment thereof is coupled to an oligonucleotide. In certain aspects, the antibodies or antigen binding fragments thereof disclosed herein are characterized as being internalizing. In certain aspects, such antibodies or antigen binding fragments thereof are internalized by a cell expressing a marker or moiety (e g., a cell surface marker or antigen) to which the antibody or antigen binding fragment thereof binds (including, but not limited to, CD71 and / or CD117) following binding of such antibody or antigen binding fragment thereof. In certain aspects, the methods disclosed herein further comprise a step of administering a stem cell population to the target tissues of the subject, wherein the administered stem cell population engrafts in the target tissues of the subject. In certain embodiments, the step of administering or transplanting a stem cell population is performed after the endogenous stem cells (e g., hematopoietic stem cells) or progenitor cells are depleted or ablated from the target tissues either partially or fully. In certain embodiments, such administering step is performed after the subject's target tissue (e.g., bone marrow tissue) has been conditioned in accordance with the methods and compositions disclosed herein In some embodiments, the stem cell population is administered to the target tissues of the subject after the oligonucleotide has cleared or dissipated from the subject's target tissues such that the level of oligonucleotide remaining in the target tissue of the subject does not induce significant adverse effects in the transplanted cel! population. For example, in some embodiments, the stem cell population is administered to the target tissue of the subject about two to about eighteen days after the administration of the oligonucleotide In some embodiments, the stem cell population is administered to the target tissue of the subject at least one, two, three, four, five, six, seven, eight, nine, ten, twelve, twelve, thirteen, fourteen, fifteen, eighteen, twenty one, thirty six, forty two, fifty six, sixty three, seventy, eighty, ninety, one hundred, one hundred and twenty days or more, after the oligonucleotide has cleared or dissipated from the target tissues of the subject.

[0031] In some embodiments, the cell is a human cell. In some embodiments, the cell is a mouse cell. In certain embodiments, the cell is a stem cell. In certain aspects, such cells comprise hematopoietic stem cells or progenitor cells. In some embodiments, the hematopoietic stem cells or progenitor cells express one or more markers selected from the group of markers consisting of CD71, CD117 (c-kit), HLA-DR, CDlIa, CD 18, CD34, CD4I / 6I, CD43, CD45, CD49d (VLA-4), CD49f (VLA-6), CD51, CD58, CD84, CD90, CD97, CD133, CD134, CD162, CD166, CD184 (CXCR4), CD205 and CD361. In some embodiments, the human hematopoietic stem cells or progenitor cells express CD34.

[0032] In certain embodiments, the targeted cells comprise human hematopoietic stem cells expressing one or more markers that may be targeted and to which the antibodies or antigen binding fragments thereof that comprise the oligonucleotide selectively bind, such markers selected from the group consisting of CD71, CD 117, CD7, CD l2, CD13, CD15, CD 19, CD21, CD22, CD29, CD30, CD33, CD34, CD36, CD38, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD48, CD49b, CD49d, CD49e, CD49f, CD50, CD53, CD55, CD64a, CD68, CD72, CD73, CD81, CD82, CD85A, CD85K, CD90, CD99, CD104, CD 105, CD109, CD110, CD111, CD112, CD 114, CD115, CD123, CD124, CD126, CD127, CD130, CD13I, CD133, CD135, CD138, CD151, CD157, CD162, CD164, CD168, CD 172a, CD173, CD 174, CD 175, CD175s, CD 176, CD183, CD191, CD200, CD201, CD205, CD217, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD235a, CD235b, CD236, CD236R, CD238, CD240, CD242, CD243, CD277, CD292, CDw293, CD295, CD298, CD309, CD318, CD324, CD325, CD338, CD344, CD349, and CD35O.

[0033] In certain embodiments, the endogenous stem cell population includes hematopoietic stem ceils. In certain embodiments, the hematopoietic stem cells or progenitor cells comprise or express one or more markers. For example, in certain embodiments the hematopoietic stem cells or progenitor cells express one or more markers selected from the group of markers consisting of: CD13, CD33, CD34, CD44, CD45, CD49d: VLA-4, CD49f: VLA-6, CD59, CD84: CD150family, CD90: Thyl, CD93, CD105: Endoglin, CD117: cKit / SCF receptor, CD123: IL-3R, CD126: IL-611, CD133, CD135: Flt3 receptor, CD166: ALC M, CD184: CXCR4, Prominin 2, Erythropoietin R, Endothelial Cell -Selective Adhesion Molecule, CD244, Tiel, Tie2, MPL, G-CSFR or CSF3R, IL-1R, gpl30, Leukemia inhibitory factor Receptor, oncostatin M receptor, Embigin and IL-18R In certain embodiments, the hematopoietic stem cells or progenitor cells express one or more markers selected from the group consisting of HLA-DR, CDlla, CD 18, CD34, CD41 / 61, CD43, CD45, CD47, CD58, CD71, CD84, CD97, CD 1 17 (c-kit), GDI 33, CD 162. CD166, CD205 and CD361. In certain aspects, the antibody or antigen binding fragment thereof selectively binds to the marker. In certain aspects, upon binding of the antibody or antigen binding fragment thereof to the marker, the oligonucleotide is internalized by the cells expressing such marker.

[0034] Another aspect of the disclosure provides an oligonucleotide conjugate including an endosomolytic moiety and / or a cell penetrating peptide conjugated to an oligonucleotide capable of hybridizing to a target sequence of a BCL11 A transcript and mediating antisense and / or RNA interference-mediated inhibition of the BCL11 A transcript.

[0035] In one embodiment, the endosomolytic moiety includes a sequence selected from Table 8. In another embodiment, the cell penetrating peptide includes a sequence selected from Table 9.

[0036] Disclosed herein, in certain embodiments, is a pharmaceutical composition comprising an antibody -oligonucleotide conjugate or other oligonucleotide conjugate as described herein, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated as a nanoparticle formulation In some embodiments, the pharmaceutical composition is formulated for parenteral, oral, intranasal, buccal, rectal, transderrnal, or intravenous, subcutaneous, or intrathecal administration.

[0037] In another aspect, the present disclosure relates to methods of treating a hemoglobinopathy-related disease in a subject in need thereof, including but not limited to sickle-cell disease or betathalassemia in which repression or elimination of expression of the BCL 11 A protein by modifying the BCL11 A gene in target cells of the subject ameliorates the signs, symptoms, or effects of the disease, notwithstanding that the subject may still be afflicted with the underlying disease.

[0038] Disclosed herein, in certain embodiments, is a method for treating a hemoglobinopathy-related disease in a subject in need thereof by providing an antibody-oligonucleotide conjugate as described herein, and administering the nucleic acid conjugate to the subject in need thereof to treat the hemoglobinopathy-related disease In certain embodiments, the nucleic acid conjugate reduces a quantity of the mRNA transcript of human BCL II A In some embodiments, the oligonucleotide moiety mediates RNA interference against the human BCL1 lA.

[0039] Disclosed herein, in certain embodiments, is a use of the antibody-oligonucleotide conjugate or a pharmaceutical composition as described herein for treating a subject diagnosed with or suspected to have Sickle Cell Disease (SCD). Also disclosed herein, in certain embodiments, is a use of the antibody-oligonucleotide conjugate or the pharmaceutical composition as described herein for manufacturing a medicament for treating a subject diagnosed with or suspected to have Sickle Cell Disease (SCD).

[0040] Disclosed herein, in certain embodiments, is a kit comprising the antibody-oligonucleotide conjugate (or cell penetrating peptide-oligonucleotide conjugate) or the pharmaceutical composition as described herein.

[0041] Another aspect of the disclosure provides an agent capable of inhibiting BCL11A mRNA, the agent including one or more of the following respective siRNA strand sequences: SEQ ID NO: 23 and SEQ ID NO: 121; SEQ ID NO: 19 and SEQ ID NO: 117; SEQ ID NO: 25 and SEQ ID NO: 123, SEQ ID NO: 27 and SEQ ID NO: 125, SEQ ID NO: 29 and SEQ ID NO: 127, and SEQ ID NO: 32 and SEQ ID NO: 130. In certain embodiments, HbF levels are induced at least two-fold in a target cell, tissue, or subject. Optionally, HbF levels are induced at least three-fold in a target cell, tissue, or subject Optionally, HbF levels are induced at least four-fold in a target cell, tissue, or subject.

[0042] DETAILED DESCRIPTION OF THE DISCLOSURE

[0043] A hemoglobinopathy is a disease or disorder characterized by one or more mutation(s) in the genome that results in abnormal structure of one or more of the globin chains of the hemoglobin molecule. Exemplary hemoglobinopathies include hemolytic anemia, sickle cell disease, and thalassemia. Sickle cell disease is characterized by the presence of abnormal, sickle-shaped hemoglobins, which can result in severe infections, severe pain, stroke, and an increased risk of death. Subjects having sickle cell disease can be identified, e.g., using one or more of a complete blood count, a blood film, hemoglobin electrophoresis, and genetic testing. Thalassemias are a group of autosomal recessive diseases characterized by a reduction in the amount of hemoglobin produced. Symptoms include iron overload, infection, bone deformities, enlarged spleen, and cardiac disease. The subgroups of thalassemias include alpha-thalassemia, beta-thalassemia, and delta thalassemia. Additional hemoglobinopathies such as hemophilia A and hemophilia B may be treated using any of the particles, vectors, virions, expression systems, compositions, and host cells described herein.

[0044] BCL11 A is a transcription factor that plays many roles in development and hematopoiesis. Genome-wide association and functional follow-up studies in cell and animal models have shown that BCL11A is an important silencer of fetal hemoglobin (HbF) expression. In a seminal study, disruption of BCL11A by erythroid-specific conditional knockout in a transgenic humanized mouse model of sickle cell disease (SCD) lead to failure of hemoglobin switching, maintenance of high-levels of HbF, and significant improvements in the hematologic and pathologic characteristics associated with SCD (Xu et al. (2011) Science 334(6058):993-6). Thus, inhibition of BCL11 A appears to be a potentially effective strategy for treating β-globin disorders such as TDT and SCD in humans. However, targeting the BCL11 A gene for therapeutic approaches poses challenges due to the crucial role of BCL11A in development and hematopoiesis (Brendel et al. (2016) J Clin Invest 126(10:3868-3878). An alternative strategy targets an erythroid-specific enhancer (ESE) element that is located in the second intron of the BCL11A and that is required for BCL11A expression in erythroid cells but not. in other lineages. The enhancer element was found to contain a common genetic variation associated with higher' HbF levels (Bauer et al. (2013) Science 342(6155):253-7). It is therefore hypothesized that modification of this erythroid-specific enhancer of the BCL11A gene could boost endogenous HbF levels in erythroid cells without deleterious effects on global BCL11A function (Hardison & Blobel (2013) Science 342(6155):2067).

[0045] Nucleic acid (e.g., RNAi) therapy is a targeted therapy with high selectivity and specificity. However, in some instances, nucleic acid therapy is also hindered by poor intracellular uptake, limited blood stability and non-specific immune stimulation. To address these issues, various modifications of the nucleic acid composition are explored, such as for example, novel linkers for better stabilizing and / or lower toxicity, optimization of binding moiety for increased target specificity and / or target delivery', and nucleic acid polymer modifications for increased stability and / or reduced off-target effect.

[0046] In some embodiments, the arrangement or order of the different components that make up the nucleic acid composition further affects intracellular uptake, stability, toxicity, efficacy, and / or non-specific immune stimulation. For example, if the nucleic acid component includes a binding moiety, a polymer, and a nucleic acid molecule (or polynucleotide), the order or arrangement of the binding moiety, the polymer, and / or the nucleic acid molecule (or polynucleotide) (e.g., binding moiety-nucleic acid molecule-polymer, binding moiety-polymer-nucleic acid molecule, or polymer -binding moiety-nucleic acid molecule) further affects intracellular uptake, stability, toxicity, efficacy, and / or non-specific immune stimulation.

[0047] In some embodiments, described herein are nucleic acid molecules and antibody-oligonucleotide conjugates for the treatment of hemoglobinopathies, especially Sickle Cell Disease (SCD), In some instances, the antibody-oligonucleotide conjugates described herein enhance intracellular uptake, stability, and / or efficacy In some cases, the antibody-oligonucleotide conjugates comprise an antibody or antigen binding fragment thereof conjugated to an oligonucleotide molecule. In some cases, the nucleic acid molecules that hybridize to target sequences of BCL11A, optionally humanBCL11A.

[0048] Additional embodiments described herein include methods of treating SCD, comprising administering to a subject a nucleic acid molecule or an antibody-oligonucleotide conjugate described herein. Oligonucleotides

[0049] In certain embodiments, an oligonucleotide hybridizes to a target sequence of B-cell lymphoma / leukemia HA (BCL11A) gene In some instances, an oligonucleotide described herein hybridizes to a target sequence of human BCL11A gene (BCL11 / X) and reduces BCL11A mRNA in erythroid cells.

[0050] In some embodiments, the oligonucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Table 10, Table 11, Table 12, Table 13, Table 14, and SEQ ID NOs: 291-580.

[0051] In some embodiments, the oligonucleotide comprises a first polynucleotide and a second polynucleotide. In some instances, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Table 10, Table 11, Table 12, Table 13, Table 14, and SEQ ID NOs: 291-580. In some cases, the second polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Table 10, Table 11, Table 12, Table 13, Table 14, and SEQ ID NOs: 291-580, In some cases, the oligonucleotide comprises a first polynucleotide and a second polynucleotide.

[0052] In some embodiments, the oligonucleotide comprises a sense strand (e.g., a passenger strand) and an antisense strand (e.g., a guide strand). In some instances, the sense strand (e.g., the passenger strand) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Table 10, Table 11, Table 12, Table 13, Table 14, SEQ ID NOs: 1-98; SEQ ID NOs: 197-231; SEQ ID NOs: 267-277; SEQ ID NO: 289, and SEQ ID NOs: 291-580 In some instances, the antisense strand (e.g., the guide strand) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Table 10, Table 11, Table 12, Table 13, Table 14, SEQ ID NOs: 99-196; SEQ ID NOs: 232-266; SEQ ID NOs: 278-288, SEQ ID NO: 290; and SEQ ID NOs: 291-580 In some embodiments, the oligonucleotide comprises a sense strand (e.g., a passenger strand) and an antisense strand (e g., a guide strand). In some instances, the sense strand (e.g., the passenger strand) comprises a sequence having at least 50%, 55%, 60%, 65%. 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence starting 13 nucleotides (nt), 15 nt, 16 nt, 23 nt, 25 nt, 38 nt, 39 nt, 41 nt. 46 nt, 49 nt, 54 nt, 62 nt, 65 nt, 66 nt, 67 nt, 68 nt, 69 nt, 78 nt, 81 nt, 88 nt, 91 nt, 94 nt, 97 nt, 100 nt, 107 nt, 141 nt, 147 nt, 157 nt, 185 nt, 190 nt, 193 nt, 199 nt, 205 nt, 208 nt, 211 nt, 216 nt, 220 nt, 224 nt, 225 nt, 229 nt, 231 nt, 238 nt, 244 nt, 250 nt, 254 nt, 261 nt, 262 nt, 265 nt, 269 nt, 273 nt, 275 nt, 281 nt, 285 nt, 292 nt, 305 nt, 306 nt, 418 nt, 449 nt, 667 nt, 1301 nt, 1417 nt, 1462 nt, 1518 nt, 1528 nt, 1545 nt, 1581 nt, 1596 nt, 1736 nt, 1746 nt, 1755 nt, 1761 nt, 1774 nt, 1809 nt, 1818 nt, 1830 nt, 1837 nt, 1858 nt, 1901 nt, 1907 nt, 1908 nt, 1942 nt, 1951 nt, 1982 nt, 1989 nt, 1992 nt, 2129 nt, 2160 nt, 2172 nt, 2273 nt, 2330 nt, 2420 nt, 2434 nt, 2513 nt, 2565 nt, 2572 nt, 2624 nt, 2636 nt, 2683 nt, 2706 nt, 2772 nt, 2797 nt, 2834 nt, 3083 nt, 3094 nt, 3175 nt, 3200 nt, 3239 nt, 3251 nt, 3313 nt, 3342 nt, 3351 nt, 3363 nt, 3396 nt, 3447 nt, 3507 nt, 3598 nt, 3612 nt, 3623 nt, 3693 nt, 3845 nt, 3868 nt, 3898 nt, 4048 nt, 4099 nt, 4252 nt, 4265 nt, 4284 nt, 4290 nt, 4300 nt, 4427 nt, 4494 nt, 4537 nt, 4668 nt, 4683 nt, 4788 nt, 4798 nt, 4814 nt, 4824 nt, 4830 nt, 4986 nt, 5099 nt, or 5159 nt from the first (5'-most) nucleotide of NM_022893. In some instances, the antisense strand (e.g, the guide strand) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence starting 13 nt. 15 nt, 16 nt, 23 nt, 25 nt, 38 nt, 39 nt, 41 nt, 46 nt, 49 nt, 54 nt, 62 nt, 65 nt, 66 nt, 67 nt, 68 nt, 69 nt, 78 nt, 81 nt, 88 nt, 91 nt, 94 nt, 97 nt, 100 nt, 107 nt, 141 nt, 147 nt, 157 nt, 185 nt, 190 nt, 193 nt, 199 nt. 205 nt, 208 nt, 211 nt, 216 nt, 220 nt, 224 nt, 225 nt, 229 nt, 231 nt, 238 nt, 244 nt, 250 nt, 254 nt, 261 nt, 262 nt, 265 nt, 269 nt, 273 nt, 275 nt, 281 nt, 285 nt, 292 nt, 305 nt, 306 nt, 418 nt, 449 nt, 667 nt, 1301 nt, 1417 nt, 1462 nt, 1518 nt, 1528 nt, 1545 nt, 1581 nt, 1596 nt, 1736 nt, 1746 nt, 1755 nt, 1761 nt, 1774 nt, 1809 nt, 1818 nt, 1830 nt, 1837 nt, 1858 nt, 1901 nt, 1907 nt, 1908 nt, 1942 nt, 1951 nt, 1982 nt, 1989 nt, 1992 nt, 2129 nt, 2160 nt, 2172 nt, 2273 nt, 2330 nt, 2420 nt, 2434 nt, 2513 nt, 2565 nt, 2572 nt, 2624 nt, 2636 nt, 2683 nt, 2706 nt, 2772 nt, 2797 nt, 2834 nt, 3083 nt, 3094 nt, 3175 nt, 3200 nt, 3239 nt, 3251 nt, 3313 nt, 3342 nt, 3351 nt, 3363 nt, 3396 nt, 3447 nt, 3507 nt, 3598 nt, 3612 nt, 3623 nt, 3693 nt, 3845 nt, 3868 nt, 3898 nt, 4048 nt, 4099 nt, 4252 nt, 4265 nt, 4284 nt, 4290 nt, 4300 nt, 4427 nt, 4494 nt, 4537 nt, 4668 nt, 4683 nt, 4788 nt, 4798 nt, 4814 nt, 4824 nt, 4830 nt, 4986 nt, 5099 nt, or 5159 nt from the first (5‘-most) nucleotide of NM 022893. In some embodiments, the oligonucleotide described herein comprises RNA or DNA. In some cases, the oligonucleotide comprises RNA. In some instances, RNA comprises short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterogeneous nuclear RNA (hnRNA). In some instances, RNA comprises shRNA. In some instances, RNA comprises miRNA. In some instances, RNA comprises dsRNA. In some instances, RNA comprises tRNA. In some instances, RNA comprises rRNA. In some instances, RN A comprises hnRNA. In some instances, the oligonucleotide is a phosphorodiamidate morpholino oligomers (PMO), which are short singlestranded oligonucleotide analogs that are built upon a backbone of morpholine rings connected by phosphorodiamidate linkages In some instances, the RNA comprises siRNA. In some instances, the oligonucleotide comprises siRNA.

[0053] In some embodiments, the oligonucleotide is from about 8 to about 50 nucleotides in length. In some embodiments, the oligonucleotide is from about 10 to about 50 nucleotides in length In some instances, the oligonucleotide is from about 10 to about 30, from about 15 to about 30, from about 18 to about 25, form about 18 to about 24, from about 19 to about 23, or from about 20 to about 22 nucleotides in length.

[0054] In some embodiments, the oligonucleotide is about 50 nucleotides in length.

[0055] In some instances, the oligonucleotide is about 45 nucleotides in length. In some instances, the oligonucleotide is about 40 nucleotides in length. In some instances, the oligonucleotide is about 35 nucleotides in length. In some instances, the oligonucleotide is about 30 nucleotides in length. In some instances, the oligonucleotide is about 25 nucleotides in length In some instances, the oligonucleotide is about 20 nucleotides in length. In some instances, the oligonucleotide is about 19 nucleotides in length. In some instances, the oligonucleotide is about 18 nucleotides in length. In some instances, the oligonucleotide is about 17 nucleotides in length. In some instances, the oligonucleotide is about 16 nucleotides in length. In some instances, the oligonucleotide is about 15 nucleotides in length. In some instances, the oligonucleotide is about 14 nucleotides in length. In some instances, the oligonucleotide is about 13 nucleotides in length In some instances, the oligonucleotide is about 12 nucleotides in length. In some instances, the oligonucleotide is about 11 nucleotides in length. In some instances, the oligonucleotide is about 10 nucleotides in length. In some instances, the oligonucleotide is about 8 nucleotides in length. In some instances, the oligonucleotide is between about 8 and about 50 nucleotides in length. In some instances, the oligonucleotide is between about 10 and about 50 nucleotides in length. In some instances, the oligonucleotide is between about 10 and about 45 nucleotides in length In some instances, the oligonucleotide is between about 10 and about 40 nucleotides in length. In some instances, the oligonucleotide is between about 10 and about 35 nucleotides in length. In some instances, the oligonucleotide is between about 10 and about 30 nucleotides in length. In some instances, the oligonucleotide is between about 10 and about 25 nucleotides in length. In some instances, the oligonucleotide is between about 10 and about 20 nucleotides in length. In some instances, the oligonucleotide is between about 15 and about 25 nucleotides in length In some instances, the oligonucleotide is between about 15 and about 30 nucleotides in length. In some instances, the oligonucleotide is between about 12 and about 30 nucleotides in length

[0056] In some embodiments, the oligonucleotide comprises a first polynucleotide.

[0057] In some instances, the oligonucleotide comprises a second polynucleotide. In some instances, the oligonucleotide comprises a first polynucleotide and a second polynucleotide. In some instances, the first polynucleotide is a sense strand or passenger strand. In some instances, the second polynucleotide is an antisense strand or guide strand

[0058] In some embodiments, the oligonucleotide is a first polynucleotide. In some embodiments, the first polynucleotide is from about 8 to about 50 nucleotides in length. In some embodiments, the first polynucleotide is from about 10 to about 50 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 30, from about 15 to about 30, from about 18 to about 25, form about 18 to about 24, from about 19 to about 23, or from about 20 to about 22 nucleotides in length.

[0059] In some instances, the first polynucleotide is about 50 nucleotides in length. In some instances, the first polynucleotide is about 45 nucleotides in length. In some instances, the first polynucleotide is about 40 nucleotides in length. In some instances, the first polynucleotide is about 35 nucleotides in length. In some instances, the first polynucleotide is about 30 nucleotides in length. In some instances, the first polynucleotide is about 25 nucleotides in length. In some instances, the first polynucleotide is about 20 nucleotides in length. In some instances, the first polynucleotide is about 19 nucleotides in length. In some instances, the first polynucleotide is about 18 nucleotides in length. In some instances, the first polynucleotide is about 17 nucleotides in length. In some instances, the first polynucleotide is about 16 nucleotides in length. In some instances, the first polynucleotide is about 15 nucleotides in length In some instances, the first polynucleotide is about 14 nucleotides in length. In some instances, the first polynucleotide is about 13 nucleotides in length. In some instances, the first polynucleotide is about 12 nucleotides in length. In some instances, the first polynucleotide is about 11 nucleotides in length. In some instances, the first polynucleotide is about 10 nucleotides in length. In some instances, the first polynucleotide is about 8 nucleotides in length. In some instances, the first polynucleotide is between about 8 and about 50 nucleotides in length. In some instances, the first polynucleotide is between about 10 and about 50 nucleotides in length. In some instances, the first polynucleotide is between about. 10 and about 45 nucleotides in length. In some instances, the first polynucleotide is between about 10 and about 40 nucleotides in length. In some instances, the first polynucleotide is between about 10 and about 35 nucleotides in length. In some instances, the first polynucleotide is between about 10 and about 30 nucleotides in length. In some instances, the first polynucleotide is between about 10 and about 25 nucleotides in length. In some instances, the first polynucleotide is between about 10 and about 20 nucleotides in length. In some instances, the first polynucleotide is between about 15 and about 25 nucleotides in length. In some instances, the first polynucleotide is between about 15 and about 30 nucleotides in length. In some instances, the first polynucleotide is between about 12 and about 30 nucleotides in length.

[0060] In some embodiments, the oligonucleotide is a second polynucleotide. In some embodiments, the second polynucleotide is from about 8 to about 50 nucleotides in length. In some embodiments, the second polynucleotide is from about 10 to about 50 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 30, from about 15 to about 30, from about 18 to about 25, form about 18 to about 24, from about 19 to about 23, or from about 20 to about 22 nucleotides in length.

[0061] In some instances, the second polynucleotide is about 50 nucleotides in length. In some instances, the second polynucleotide is about 45 nucleotides in length In some instances, the second polynucleotide is about 40 nucleotides in length. In some instances, the second polynucleotide is about 35 nucleotides in length. In some instances, the second polynucleotide is about 30 nucleotides in length. In some instances, the second polynucleotide is about 25 nucleotides in length. In some instances, the second polynucleotide is about 20 nucleotides in length. In some instances, the second polynucleotide is about 19 nucleotides in length In some instances, the second polynucleotide is about 18 nucleotides in length. In some instances, the second polynucleotide is about 17 nucleotides in length. In some instances, the second polynucleotide is about 16 nucleotides in length In some instances, the second polynucleotide is about 15 nucleotides in length. In some instances, the second polynucleotide is about 14 nucleotides in length. In some instances, the second polynucleotide is about 13 nucleotides in length. In some instances, the second polynucleotide is about 12 nucleotides in length. In some instances, the second polynucleotide is about 11 nucleotides in length. In some instances, the second polynucleotide is about 10 nucleotides in length. In some instances, the second polynucleotide is about 8 nucleotides in length. In some instances, the second polynucleotide is between about 8 and about 50 nucleotides in length In some instances, the second polynucleotide is between about 10 and about 50 nucleotides in length. In some instances, the second polynucleotide is between about 10 and about 45 nucleotides in length In some instances, the second polynucleotide is between about 10 and about 40 nucleotides in length. In some instances, the second polynucleotide is between about 10 and about 35 nucleotides in length. In some instances, the second polynucleotide is between about 10 and about 30 nucleotides in length. In some instances, the second polynucleotide is between about 10 and about 25 nucleotides in length. In some instances., the second polynucleotide is between about 10 and about 20 nucleotides in length. In some instances, the second polynucleotide is between about 15 and about 25 nucleotides in length. In some instances, the second polynucleotide is between about 15 and about 30 nucleotides in length. In some instances, the second polynucleotide is between about 12 and about 30 nucleotides in length.

[0062] In some embodiments, the oligonucleotide comprises a first polynucleotide and a second polynucleotide In some instances, the oligonucleotide further comprises a blunt terminus, an overhang, or a combination thereof. In some instances, the blunt, terminus is a 5' blunt terminus, a 3' blunt terminus, or both. In some cases, the overhang is a 5' overhang, 3' overhang, or both. In some cases, the overhang comprises I, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-base pairing nucleotides. In some cases, the overhang comprises 1, 2, 3, 4, 5, or 6 non base pairing nucleotides. In some cases, the overhang comprises 1, 2, 3, or 4 non-base pairing nucleotides. In some cases, the overhang comprises I non-base pairing nucleotide. In some cases, the overhang comprises 2 non-base pairing nucleotides. In some cases, the overhang comprises 3 non-base pairing nucleotides. In some cases, the overhang comprises 4 non-base pairing nucleotides. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, and the antisense strand includes two non-base pairing nucleotides as an overhang at the 3'-end while the sense strand has no overhang. Optionally, in such embodiments, the non-base pairing nucleotides have a sequence of TT, dTdT, or UU. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, and the sense strand has one or more nucleotides at the 5‘-end that are complementary to the antisense sequence.

[0063] In some embodiments, the sequence of the oligonucleotide is at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% complementary to a target sequence of BCL11A. In some embodiments, the target sequence of BCL11A is a nucleic acid sequence of about 10-50 base pair length, about 15-50 base pair length, 15-40 base pair length, 15-30 base pair length, or 15-25 base pair length sequences in BCL1 IA, in which the first nucleotide of the target sequence starts at any nucleotide in BCL11A mRNA transcript in the coding region, or in the 5' or 3'-untranslated region (UTR). For example, the first nucleotide of the target sequence can be selected so that it starts at the nucleic acid location (nal, number starting from the 5'-end of the full length of BCL11A RX-X e.g., the 5'-end first nucleotide is nal.l) 1, nal 2, nal 3, nal 4, nal 5, nal 6, nal 7, nal 8, nal 9, nal 10, nal 11, nal 12, nal 13, nal 14, nal 15, nal 15, nal 16, nal 17, or any other nucleic acid location in the coding or noncoding regions (5‘ or 3'-untraslated region) of BCL11 A mRNA. In some embodiments, the first nucleotide of the target sequence can be selected so that it starts at a location within, or between, nal 10-nal 15, nal 10-nal 20, nal 50-nal 60, nal 55-nal 65, nal 75 -nal 85, nal 95-nal 105, nal 135-nal 145, nal 155-nal 165, nal 225-nal 235, nal 265-nal 275, nal 275-nal 285, nal 285-nal 295, nal 325-nal 335, nal 335-nal 345, nal 385-nal 395, nal 515-nal 525, nal 665-nal 675, nal 675-nal 685, nal 695-nal 705, nal 705-nal 715, nal 875-nal 885, nal 885-nal 895, nal 895-nal 905, nal 1035-nal 1045, nal 1045-nal 1055, nal 1125-nal 1135, nal 1135-nal 1 145, nal 1145-nal 1155, nal 1155-nal 1165, nal 1125-nal 1135, nal 1155-nal 1165, nal 1225-nal 1235, nal 1235-nal 1245, nal 1275-nal 1285, nal 1285-nal 1295, nal 1305-nal 1315, nal 1125-nal 1135, nal 1155-nal 1165, nal 1225-nal 1235, nal 1235-nal 1245, nal 1275-nal 1285, nal 1285-nal 1295, nal 1305-nal 1315, nal 1315-nal 1325, nal 1335-nal 1345, nal 1345-nal 1355, nal 1525-nal 1535, nal 1535-nal 1545, nal 1605-nal 1615, nal 1615-nal 1625, nal 1625-nal 1635.

[0064] In some embodiments, the sequence of the oligonucleotide is at least 50% complementary to a target, sequence described herein. In some embodiments, the sequence of the oligonucleotide is at least 60% complementary to a target sequence described herein. In some embodiments, the sequence of the oligonucleotide is at least 70% complementary to a target sequence described herein. In some embodiments, the sequence of the oligonucleotide is at least 80% complementary to a target sequence described herein. In some embodiments, the sequence of the oligonucleotide is at least 90% complementary to a target sequence described herein. In some embodiments, the sequence of the oligonucleotide is at least 95% complementary to a target sequence described herein. In some embodiments, the sequence of the oligonucleotide is at least 99% complementary to a target sequence described herein. In some instances, the sequence of the oligonucleotide is 100% complementary to a target sequence described herein.

[0065] In some embodiments, the sequence of the oligonucleotide has 5 or less mismatches to a target sequence described herein. In some embodiments, the sequence of the oligonucleotide has 4 or less mismatches to a target sequence described herein. In some instances, the sequence of the oligonucleotide has 3 or less mismatches to a target sequence described herein. In some cases, the sequence of the oligonucleotide has 2 or less mismatches to a target sequence described herein. In some cases, the sequence of the oligonucleotide has 1 or less mismatches to a target sequence described herein. In some embodiments, a group of oligonucleotides among all the oligonucleotides potentially binds to the target sequence of BCL11A are selected to generate an oligonucleotide library. In certain embodiments, such selection process is conducted in silico via one or more steps of eliminating less desirable oligonucleotides from candidates. For example, in some embodiments, the selection process comprises an elimination step of one or more oligonucleotide that has single nucleotide polymorphism (SNP) and / or MEF < -5. Alternatively and / or additionally, in some embodiments, the selection process comprises an elimination step of one or more oligonucleotide with 0 and 1 mismatch (MM) in the human transcriptome (such that only hits allowed are BCL11A). Alternatively and / or additionally, in some embodiments, the selection process comprises an elimination step of one or more oligonucleotide with 0 MM in the human intragenic regions (such that only hits allowed are BCLl lA pseudogenes). Alternatively and / or additionally, in some embodiments, the selection process comprises an elimination step of one or more oligonucleotide predicted viability < 60. Alternatively and / or additionally, such selection process comprises carrying forward one or more oligonucleotide predicted viability >60 Alternatively and / or additionally, in some embodiments, the selection process comprises an elimination step of one or more oligonucleotide with a match to a seed region of known miRNAs 1-1000. Alternatively and / or additionally, in some embodiments, the selection process comprises an elimination step of one or more oligonucleotide with %GC content 75 and above. Alternatively and / or additionally, in some embodiments, the selection process comprises a selection step of 8 or less predicted off-target hits with 2 MM. In some embodiments, for the region 295-1132 (nal 295-1132), 12 or less predicted off -target hits with 2 MM is allowed. In some embodiments, selection process is conducted in silico via one or more consecutive steps of eliminating less desirable oligonucleotides from candidates. For example, in some embodiments, selection process begins with collecting candidate oligonucleotides to generate a library From the library, the first eliminating step comprises eliminating one or more oligonucleotide that has single nucleotide polymorphism (SNP) and / or MEF < -5. Then, the second eliminating step comprises eliminating one or more oligonucleotide with 0 and 1 MM in the human transcriptome (such that only hits allowed are BCLHA). Then, the third eliminating step comprises eliminating one or more oligonucleotide with 0 MM in the human intragenic regions (such that only hits allowed are BCL11A pseudogenes). Then, the next step is carrying forward only or one or more oligonucleotide with predicted viability >60. Next, the eliminating step comprises eliminating one or more oligonucleotide with a match to a seed region of known miRNAs 1-1000. Then, the eliminating step continues with eliminating one or more oligonucleotide with %GC content 75 and above Then, the final selection process comprises with 8 or less predicted off-target hits with 2 MM, except for the region 295-1132, for which up to 12 hits are allowed.

[0066] In some embodiments, the specificity of the oligonucleotide that hybridizes to a target sequence described herein is a 95%, 98%, 99%, 99.5% or 100% sequence complementarity of the oligonucleotide to a target sequence. In some instances, the hybridization is a high stringent hybridization condition.

[0067] In some embodiments, the oligonucleotide has reduced off-target effect. In some instances, "off-target" or "off-target effects" refer to any instance in which a nucleic acid polymer directed against a given target causes an unintended effect by interacting either directly or indirectly with another mRNA sequence, a DNA sequence or a cellular protein or other moiety. In some instances, an "off-target effect" occurs when there is a simultaneous degradation of other transcripts due to partial homology or complementarity between that other transcript and the sense and / or antisense strand of the oligonucleotide.

[0068] In some embodiments, the oligonucleotide comprises natural or synthetic or artificial nucleotide analogues or bases. In some cases, the oligonucleotide comprises combinations of DNA, RNA and / or nucleotide analogues. In some instances, the synthetic or artificial nucleotide analogues or bases comprise modifications at one or more of ribose moiety, phosphate moiety, nucleoside moiety, or a combination thereof.

[0069] In some embodiments, nucleotide analogues or artifi cial nucleotide base comprise a nucleic acid with a modification at a 2' hydroxyl group of the ribose moiety. In some instances, the modification includes an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. Exemplary alkyl moiety includes, but is not limited to, halogens, sulfurs, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary'), amides, ethers, esters, alcohols and oxygen. In some instances, the alkyl moiety further comprises a modification. In some instances, the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso, group, a. nitrile group, a heterocycle (e., imidazole, hydrazino or hydroxyl amino) group, an isocyanate or cyanate group, or a sulfur containing group (e.g., sulfoxide, sulfone, sulfide, and disulfide). In some instances, the alkyl moiety further comprises a hetero substitution. In some instances, the carbon of the heterocyclic group is substituted by a nitrogen, oxygen or sulfur. In some instances, the heterocyclic substitution includes but is not limited to, morpholino, imidazole, and pyrrolidino. In some instances, the modification at the 2' hydroxyl group is a 2'-O-methyl modification or a 2'-O-methoxy ethyl (2’-0-M0E) modification. In some cases, the 2'-O-methyl modification adds a. methyl group to the 2' hydroxyl group of the ribose moiety whereas the 2'0-methoxyethyl modification adds a methoxy ethyl group to the 2' hydroxyl group of the ribose moiety. Exemplary chemical structures of a 2'-0-methyl modification of an adenosine molecule and 20-methoxy ethyl modification of a uridine are illustrated below.

[0070]

[0071] ne

[0072] In some instances, the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification in which an extended amine group comprising a propyl linker binds the amine group to the 2' oxygen. In some instances, this modification neutralizes the phosphate derived overall negative charge of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar and thereby improves cellular uptake properties due to its zwitterionic properties. An exemplary’ chemical structure of a 2'-O-aminopropyl nucleoside phosphoramidite is illustrated below.

[0073]

[0074] 2’-0-aminopropyl nucleoside phosphorami di te

[0075] In some instances, the modification at the2'hydroxyl group is a locked or bridged ribose modification (e.g., locked nucleic acid or LNA) in which the oxygen molecule bound at the2'carbon is linked to the 4' carbon by a methylene group, thus forming a 2'-C,4'-C-oxy-methylene-linked bicyclic ribonucleotide monomer. Exemplary representations of the chemical structure of LNA are illustrated below. The representation shown to the left highli ghts the chemical connectivity of an LNA monomer. The representation shown to the right highlights the locked 3’-endo (3E) conformation of the furanose ring of an LNA monomer.

[0076]

[0077] LNA (Locked Nucleic Acids)

[0078] In some instances, the modification at the 2' hydroxyl group comprises ethylene nucleic acids (ENA) such as for example 2'-4'-ethylene-bridged nucleic acid, which locks the sugar conformation into a Cf -endo sugar puckering conformation. ENA are part of the bridged nucleic acids class of modified nucleic acids that also comprises LNA. Exemplary chemical structures of the ENA and bridged nucleic acids are illustrated below. In some embodiments, additional modifications at the2'hydroxyl group include 2'-deoxy, 2'-deoxy-2'-fluoro, 2 0-ami nopropyl (2’-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-0-DMAP), 2'-O-dimethyl aminoethyloxy ethyl (2'-0~MAE0E), or 2‘-O-N-methylacetamido (2'-0-NMA).

[0079] In some embodiments, nucleotide analogues comprise modified bases such as, but not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyl adenine, 6-methylguanine, N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5-(2-amino) propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2, 2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2 -thiouridine, other thio bases such as 2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any O-and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxy hydroxy alkyl nucleotides, carboxy alkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moieties, in some cases are or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4'-thioribose, and other sugars, heterocycles, or carbocycles The term nucleotide also includes what are known in the art as universal bases. By way of example, universal bases include but are not limited to 3 -nitropyrrole, 5~nitroindole, or nebularine.

[0080] In some embodiments, nucleotide analogues further comprise morpholinos, peptide nucleic acids (PNAs), methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, 1', 5'-anhydrohexitol nucleic acids (HNAs), or a combination thereof. Morpholino or phosphorodianiidate morpholino oligo (PMO) comprises synthetic molecules whose structure mimics natural nucleic acid structure but deviates from the normal sugar and phosphate structures. In some instances, the five member ribose ring is substituted with a six member morpholino ring containing four carbons, one nitrogen and one oxygen In some cases, the ribose monomers are linked by a phosphordiamidate group instead of a phosphate group. In such cases, the backbone alterations remove all positive and negative charges making morpholinos neutral molecules capable of crossing cellular membranes without the aid of cellular delivery agents such as those used by charged oligonucleotides.

[0081]

[0082] Morpholino

[0083] In some embodiments, peptide nucleic acid (PNA) does not contain sugar ring or phosphate linkage and the bases are attached and appropriately spaced by oligoglycine-like molecules, therefore, eliminating a backbone charge.

[0084]

[0085] A

[0086] In some embodiments, one or more modifications optionally occur at the internucleotide linkage. In some instances, modified internucleotide linkage include, but is not limited to, phosphorothioates, phosphorodithioates, methylphosphonates, 5'-alkylenephosphonates, 5'-methylphosphonate, 3'-alkylene phosphonates, borontrifluoridates, borano phosphate esters and selenophosphates of 3’-5' linkage or 2-5' linkage, phosphotriesters, thionoalkylphosphotriesters, hydrogen phosphonate linkages, alkyl phosphonates, alkylphosphonothioates, arylphosphonothi oates, phosphoroselenoates, phosphorodi sei enoates, phosphinates, phosphoramidates, 3 -alkylphosphoramidates, aminoalkylphosphoramidates, thionophosphorami dates, phosphoropiperazidates, phosphoroanilothioates, phosphoroanili dates, ketones, sulfones, sulfonamides, carbonates, carbamates, methylenehydrazos, methylenedimethylhydrazos, formacetals, thioform acetals, oximes, methyleneiminos, methylenemethyliminos, thioamidat.es, linkages with riboacetyl groups, aminoethyl glycine, silyl or siloxane linkages, alkyl or cycloalkyl linkages with or without heteroatoms of, for example, 1 to 10 carbons that are saturated or unsaturated and / or substituted and / or contain heteroatoms, linkages with morpholino structures, amides, polyamides wherein the bases are attached to the aza nitrogens of the backbone directly or indirectly, and combinations thereof.

[0087] Phosphorothioate antisense oligonucleotides (PS ASO) are antisense oligonucleotides comprising a phosphorothioate linkage. An exemplary PS ASO is illustrated below.

[0088]

[0089] In some instances, the modification is a methyl or thiol modification such as methylphosphonate or thiolphosphonate modification. Exemplary thiolphosphonate nucleotide (left) and methylphosphonate nucleotide (right) are illustrated below.

[0090]

[0091] In some instances, a modified nucleotide includes, but is not limited to, 2’-fluoro N3-P5'-phosphoramidites illustrated as:

[0092]

[0093] In some instances, a modified nucleotide includes, but is not limited to, hexitol nucleic acid (or 6, 5'-anhydrohexitol nucleic acids (HNA)) illustrated as:

[0094]

[0095] HNA

[0096] In some embodiments, one or more modifications further optionally include modifications of the ribose moiety, phosphate backbone and the nucleoside, or modifications of the nucleotide analogues at the 3' or the 5' terminus. For example, the 3’ terminus optionally include a 3' cationic group, or by inverting the nucleoside at the 3'-terminus with a 3’-3' linkage. In another alternative, the 3'-terminus is optionally conjugated with an aminoalkyl group, e.g., a 3' C5-aminoalkyl dT. In an additional alternative, the 3'-terminus is optionally conjugated with an abasic site, e g,, with an apurinic or apyrimidinic site. In some instances, the 5'-terminus is conjugated with an aminoalkyl group, e.g., a 5'-O-alkylaminosubstituent In some cases, the 5'-terminus is conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site.

[0097] In some embodiments, the oligonucleotide comprises one or more of the artificial nucleotide analogues described herein In some instances, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of the artificial nucleotide analogues described herein In some embodiments, the artificial nucleotide analogues include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'--deoxy-2' -fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2’-O-dimethylaminoethyl oxy ethyl (2'-O-DMAfiOE), or 2'-O-N-methylacetamido (2'-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiol phosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or a combination thereof. In some instances, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of the artificial nucleotide analogues selected from 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2‘-deoxy, 2‘-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2-0-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methyl phosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or a combination thereof. In some instances, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of 2'-O-methyl modified nucleotides. In some instances, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides. In some instances, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of thiolphosphonate nucleotides.

[0098] In some instances, the oligonucleotide comprises at least one of: from about 5% to about 100% modification, from about 10% to about 100% modification, from about 20% to about 100% modification, from about 30% to about 100% modification, from about 40% to about 100% modification, from about 50% to about 100% modification, from about 60% to about 100% modification, from about 70% to about 100% modification, from about 80% to about 100% modification, and from about 90% to about 100% modification.

[0099] In some cases, the oligonucleotide comprises at least one of: from about 10% to about 90% modification, from about 20% to about 90% modification, from about 30% to about 90% modification, from about 40% to about 90% modification, from about 50% to about 90% modification, from about 60% to about 90% modification, from about 70% to about 90% modification, and from about 80% to about 100% modification.

[0100] In some cases, the oligonucleotide comprises at least one of: from about 10% to about 80% modification, from about 20% to about 80% modification, from about 30% to about 80% modification, from about 40% to about 80% modification, from about 50% to about 80% modification, from about 60% to about 80% modification, and from about 70% to about 80% modification.

[0101] In some instances, the oligonucleotide comprises at least one of: from about 10% to about 70% modification, from about 20% to about 70% modification, from about 30% to about 70% modification, from about 40% to about 70% modification, from about 50% to about 70% modification, and from about 60% to about 70% modification.

[0102] In some instances, the oligonucleotide comprises at least one of: from about 10% to about 60% modification, from about 20% to about 60% modification, from about 30% to about 60% modification, from about 40% to about 60% modification, and from about 50% to about 60% modification.

[0103] In some cases, the oligonucleotide comprises at least one of: from about 10% to about 50% modification, from about 20% to about 50% modification, from about 30% to about 50% modification, and from about 40% to about 50% modification.

[0104] In some cases, the oligonucleotide comprises at least one of: from about 10% to about 40% modification, from about 20% to about 40% modification, and from about 30% to about 40% modification.

[0105] In some cases, the oligonucleotide comprises at least one of: from about 10% to about 30% modification, and from about 20% to about 30% modification.

[0106] In some cases, the oligonucleotide comprises from about 10% to about 20% modification. In some cases, the oligonucleotide comprises from about 15% to about 90%, from about 20% to about 80%, from about 30% to about 70%, or from about 40% to about 60% modifications.

[0107] In additional cases, the oligonucleotide comprises at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modification.

[0108] In some embodiments, the oligonucleotide comprises at least about I, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modifications. In some instances, the oligonucleotide comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modified nucleotides

[0109] In some instances, from about 5 to about 100% of the oligonucleotide comprise the artificial nucleotide analogues described herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the oligonucleotide comprise the artificial nucleotide analogues described herein. In some instances, about 5% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 10% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 15% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 20% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 25% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 30% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 35% of the oligonucleotide comprises the artificial nucleotide analogues described herein In some instances, about 40% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 45% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 50% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 55% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 60% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 65% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 70% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 75% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 80% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 85% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about. 90% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 95% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 96% of the oligonucleotide comprises the artificial nucleotide analogues described herein In some instances, about 97% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 98% of the oligonucleotide comprises the artificial nucleotide analogues described herein. In some instances, about 99% of the oligonucleotide comprises the artificial nucleotide analogues described herein In some instances, about 100% of the nucleic acid molecule comprises the artificial nucleotide analogues described herein. In some embodiments, the artificial nucleotide analogues include 2!-O-methyl, 2'-O-methoxyethyl (2‘-O-MOE), 2‘-O-aminopropyl, 2‘-deoxy, 2'-deoxy-2'-fluoro, 2‘-O-aminopropyl (2!-O-AP), 2'-O-dimethylami noethyl (2-O-DMAOE), 2'-()-dimethy 1 aminopropyl (2-0-DMAP), 2'-O-dimethylaminoethyloxy ethyl (2'-0-DMAE0E), or 2'-O-N-methylacetamido (2-0-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiol phosphonate nucleotides, 2'~fluoro N3-P5'-phosphoramidites, or a combination thereof.

[0110] In some embodiments, the oligonucleotide comprises from about 1 to about 25 modifications in which the modification comprises an artificial nucleotide analogues described herein. In some embodiments, the oligonucleotide comprises about 1 modification in which the modification comprises an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 2 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 3 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 4 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 5 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 6 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 7 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 8 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 9 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 10 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 11 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 12 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 13 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 14 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 15 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 16 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 17 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 18 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 19 modifications in which the modifications comprise an artificial nucleotide analogue described herein In some embodiments, the oligonucleotide comprises about 20 modifications in ’which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 21 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 22 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 23 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 24 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the oligonucleotide comprises about 25 modifications in which the modifications comprise an artificial nucleotide analogue described herein.

[0111] In some embodiments, an oligonucleotide is assembled from two separate polynucleotides wherein one polynucleotide comprises the sense strand and the second polynucleotide comprises the antisense strand of the oligonucleotide. In other embodiments, the sense strand is connected to the antisense strand via a linker molecule, which in some instances is a polynucleotide linker or a non-nucleotide linker. In some embodiments, an oligonucleotide comprises a sense strand and antisense strand, wherein pyrimidine nucleotides in the sense strand comprises 2'-O-methylpyrimidine nucleotides and purine nucleotides in the sense strand comprise 2'-deoxy purine nucleotides In some embodiments, an oligonucleotide comprises a sense strand and antisense strand, wherein pyrimidine nucleotides present in the sense strand comprise 2'-deoxy-2'-fluoro pyrimidine nucleotides and wherein purine nucleotides present in the sense strand comprise 2'-deoxy purine nucleotides.

[0112] In some embodiments, an oligonucleotide comprises a sense strand and antisense strand, wherein the pyrimidine nucleotides when present in said antisense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides and the purine nucleotides when present in said antisense strand are 2'-O-methyl purine nucleotides.

[0113] In some embodiments, an oligonucleotide comprises a sense strand and antisense strand, wherein the pyrimidine nucleotides when present in said antisense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides and wherein the purine nucleotides when present in said antisense strand comprise 2 '-deoxy -purine nucleotides.

[0114] In some embodiments, an oligonucleotide comprises a sense strand and antisense strand, and at least one of sense strand and antisense strands has a plurality of (e.g, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, etc.) 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides. In some embodiments, where at least two out of the plurality of 2'-O-methyl or 2'-deoxy-2' -fluoro modified nucleotides are consecutive nucleotides. In some embodiments, where consecutive 2‘-O-methyl or 2’-deoxy-2'-fluoro modified nucleotides are located at the 5'-end of the sense strand and / or the antisense strand. In some embodiments, where consecutive 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides are located at the 3' -end of the sense strand and / or the antisense strand In some embodiments, the sense strand of oligonucleotide includes at least four, at least five, at least six consecutive 2'-O-methyl modified nucleotides at its 5' end and / or 3' end, or both Optionally, in such embodiments, the sense strand of oligonucleotide includes at least one, at least two, at least three, at least four 2'-deoxy-2'-fluoro modified nucleotides at the 3' end of the at least four, at least five, at least six consecutive 2'-O-methyl modified nucleotides at the polynucleotides’ 5’ end, or at the 5' end of the at least four, at least five, at least six consecutive 2'-O-methyl modified nucleotides at polynucleotides' 3' end Also optionally, such at least two, at least three, at least four 2'-deoxy-2’-fluoro modified nucleotides are consecutive nucleotides.

[0115] In some embodiments, an oligonucleotide comprises a sense strand and antisense strand, and at least one of sense strand and antisense strands has 2'-O-methyl modified nucleotide located at the 5'-end of the sense strand and / or the antisense strand. In some embodiments, at least one of sense strand and antisense strands has 2'-O-methyl modified nucleotide located at the 3'-end of the sense strand and / or the antisense strand. In some embodiments, the 2'-O-methyl modified nucleotide located at the 5'-end of the sense strand and / or the antisense strand is a purine nucleotide. In some embodiments, the 2'-O-methyl modified nucleotide located at the 5'-end of the sense strand and / or the antisense strand is a pyridine nucleotide In some embodiments, an oligonucleotide comprises a sense strand and antisense strand, and the antisense strand has two or more consecutive 2'-deoxy-2’-fluoro modified nucleotides at 5'-end. In some embodiments, an oligonucleotide comprises a sense strand and antisense strand, and the antisense strand has two or more consecutive 2'-O-methyl modified nucleotides at 3'-end. In some embodiments, an oligonucleotide comprises a sense strand and antisense strand, and the antisense strand has at least 2, 3, 4, 5, 6, or 7 consecutive 2'-O-methyl modified nucleotides.

[0116] In some embodiments, an oligonucleotide comprises a sense strand and antisense strand, and the sense strand comprises a nucleic acid of 5'-nsnsnnnnNfNfNfnnnnnnnnsnsa-3' (lower case (n) = 2'-0-Me (methyl), Nf = 2'-F (fluoro); s = phosphorothioate backbone modification). In some embodiments., an oligonucleotide comprises a sense strand and antisense strand, and the antisense strand comprises a nucleic acid of 5'-UfsNfsnnnNfnnnnnnnNfnNfnnnsusu-3' (lower case (n) ~ 2'-O-Me (methyl), Nf = 2'-F (fluoro), s = phosphorothioate backbone modification). In some embodiments, an oligonucleotide comprises a sense strand and antisense strand, and the sense strand comprises a nucleic acid of 5'-nsnsnnnnNfNfNfnnnnnnnnsnsa-3' (lower case (n) = 2'-O-Me (methyl), Nf = 2'-F (fluoro); s = phosphorothioate backbone modification) and the antisense strand comprises a nucleic acid of 5'-UfsNfsnnnNfnnnnnnnNfnNfnnnsusu-3' (lower case (n) = 2'-0-Me (methyl), Nf 2'-F (fluoro); s phosphorothioate backbone modification).

[0117] In some embodiments, an oligonucleotide comprises a sense strand and antisense strand, wherein the sense strand includes a terminal cap moiety at the 5'-end, the 3'-end, or both of the 5' and 3' ends of the sense strand. In other embodiments, the terminal cap moiety is an inverted deoxy abasic moiety.

[0118] In some embodiments, an oligonucleotide comprises a sense strand and an antisense strand, wherein the antisense strand comprises a glyceryl modification at the 3' end of the antisense strand.

[0119] In some embodiments, an oligonucleotide comprises a sense strand and an antisense strand, in which the sense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2’-O-methyl, 2'-deoxy-2'-fluoro, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense strand; and in which the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the antisense strand. In other embodiments, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and / or antisense strand are chemically-modified with 2'-deoxy, 2’-O-methyl and / or 2'-deoxy-2'-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages and / or a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3' and 5'-ends, being present in the same or different strand.

[0120] In some embodiments, an oligonucleotide comprises a sense strand and an antisense strand, in which the sense strand comprises about 1 to about 25, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2' -deoxy, 2'-O-methyl, 2'-deoxy-2‘-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3 -end, the 5'-end, or both of the 3’ and 5'-ends of the sense strand, and in which the antisense strand comprises about 1 to about 25 or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the antisense strand. In other embodiments, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and / or antisense strand are chemically-modified with 2'-deoxy, 2'-O-methyl and / or 2'-deoxy-2' -fluoro nucleotides, with or without about 1 to about 25 or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate internucleotide linkages and / or a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3' and 5'-ends, being present in the same or different strand. In some embodiments, an oligonucleotide comprises a sense strand and an antisense strand, in which the antisense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate internucleotide linkages, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2' -fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides at the S'-end, the 5'-end, or both of the 3' and 5‘-ends of the sense strand and / or antisense strand, and optionally a terminal cap molecule at the 3'-end, the 5!-end, or both of the 3' and 5'-ends of the sense strand. In some embodiments, the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2' -deoxy, 2’-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (eg., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the antisense strand. In other embodiments, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more pyrimidine nucleotides of the sense and / or antisense strand are chemically-modified with 2‘-deoxy, 2'-O-methyl and / or 2'-deoxy-2'-fluoro nucleotides, with or without one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and / or a terminal cap molecule at the 3‘-end, the 5'-end, or both of the 3' and 5'-ends, being present in the same or different strand.

[0121] In some embodiments, an oligonucleotide comprises a sense strand and an antisense strand, in which the antisense strand comprises about 1 to about 25 or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate intemucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2‘-deoxy, 2-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense strand, and the antisense strand comprises about 1 to about 25 or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate intemucleotide linkages, and / or one or more (e g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the antisense strand. In other embodiments, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and / or antisense strand are chemically-modified with 2'-deoxy, 2-O-methyl and / or 2'-deoxy-2'-fluoro nucleotides, with or without about 1 to about 5, for example about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and / or a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3' and 5'-ends, being present in the same or different strand In some embodiments, an oligonucleotide described herein is a chemically-modified short interfering nucleic acid molecule having about 1 to about 25, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate internucleotide linkages in each strand of the oligonucleotide. In some embodiments, an oligonucleotide comprises a sense strand and an antisense strand, and the antisense strand comprises a phosphate backbone modification at the 3' end of the antisense strand. Alternatively and / or additionally, an oligonucleotide comprises a sense strand and an antisense strand, and the sense strand comprises a phosphate backbone modification at the 5' end of the antisense strand. In some instances, the phosphate backbone modification is a phosphorothioate. In some embodiments, the sense or antisense strand has three consecutive nucleosides that are coupled via two phosphorothioate backbone.

[0122] In another embodiment, an oligonucleotide described herein comprises 2'-5' intemucleotide linkages In some instances, the 2'-5' intemucleotide linkage(s) is at the 3'-end, the 5'-end, or both of the 3' and 5’-ends of one or both sequence strands. In addition instances, the 2'-5' intemucleotide linkage(s) is present at various other positions within one or both sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every intemucleotide linkage of a pyrimidine nucleotide in one or both strands of the oligonucleotide comprise a 2'-5' internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a purine nucleotide in one or both strands of the oligonucleotide comprise a 2'-5' intemucleotide linkage.

[0123] In some embodiments, an oligonucleotide is a single stranded oligonucleotide that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the oligonucleotide comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present, in the nucleic acid are 2'-deoxy-2' -fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2’-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the nucleic acid are 2'-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-deoxy purine nucleotides), and a terminal cap modification. that is optionally present at the 3'~end, the 5'-end, or both of the 3' and 5'-ends of the antisense sequence, the oligonucleotide optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2'-deoxynucleotides at the 3'-end of the oligonucleotide, wherein the terminal nucleotides further comprise one or more (eg., 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and wherein the oligonucleotide optionally further comprises a terminal phosphate group, such as a 5'-terminal phosphate group.

[0124] In some cases, one or more of the artificial nucleotide analogues described herein are resistant toward nucleases such as for example ribonuclease such as RNase H, deoxyribonuclease such as DNase, or exonuclease such as 5'-3' exonuclease and 3 -5' exonuclease when compared to natural oligonucleotides. In some instances, artificial nucleotide analogues comprising 2-0-methyl,2’-0-m ethoxy ethyl (2'-O-M0E), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-aminopropyl (2'-0-AP), 2'-O-dimethylaminoethyl (2-0-DMA0E), 2'-0-dimethyl aminopropyl (2'-0-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-0-DMAE0E), or 2'-O-N-methylacetamido (2-0-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidites, or combinations thereof are resistant toward nucleases such as for example ribonuclease such as RNase H, deoxyribonuclease such as DNase, or exonuclease such as 5'-3' exonuclease and 3!-5‘ exonuclease. In some instances, 2'-0-methyl modified oligonucleotide is nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some instances, 2'0-m ethoxy ethyl (2'-0-M0E) modified oligonucleotide is nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some instances, 2'-O-aminopropyl modified oligonucleotide is nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some instances, 2' -deoxy modified oligonucleotide is nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistance) In some instances, 2'-deoxy-2'-fluoro modified oligonucleotide is nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5’ exonuclease resistance). In some instances, 2'-O-aminopropyl (2'-O-AP) modified oligonucleotide is nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some instances, 2'-O-dimethylarninoethyl (2'-0-DMA0E) modified oligonucleotide is nuclease resistance (e.g, RNase H, DNase, 5'-3’ exonuclease or 3'-5!exonuclease resistance). In some instances, 2'-O-dimethylaminopropyl (2'-0-DMAP) modified oligonucleotide is nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some instances, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified oligonucleotide is nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some instances, 2'-O-N-methylacetamido (2-0-NMA) modified oligonucleotide is nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some instances, LNA modified oligonucleotide is nuclease resistance (e.g, RNase H, DNase, 5'-3' exonuclease or 3 -5' exonuclease resistance). In some instances, ENA modified oligonucleotide is nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some instances, HNA modified oligonucleotide is nuclease resistance (e.g, RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistance).

[0125] In some instances, morpholines is nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease or 3 -5' exonuclease resistance). In some instances, PNA modified oligonucleotide is resistant to nucleases (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some instances, methylphosphonate nucleotides modified oligonucleotide is nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistance).

[0126] In some instances, thiolphosphonate nucleotides modified oligonucleotide is nuclease resistance (e.g., RNase H, DNase, 5'-3‘ exonuclease or 3'-5' exonuclease resistance). In some instances, oligonucleotide comprising 2'-fluoro N3-P5'-phosphoramidites is nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistance). In some instances, the 5' conjugates described herein inhibit 5'-3' exonucleolytic cleavage. In some instances, the 3' conjugates described herein inhibit 3'-5' exonucleolytic cleavage.

[0127] In some embodiments, one or more of the artificial nucleotide analogues described herein have increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. The one or more of the artificial nucleotide analogues comprising 2'-O-methyl, 2'-O-methoxy ethyl (2-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2-0-DMA0E), 2'-O-dimethylaminopropyl (2-0-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-0-DMAE0E), or 2'-O-N-methylacetamido (2'-0-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, or 2'-fluoro N3-P5'-phosphoramidites have increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide In some instances, 2'-O-methyl modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some instances, 2'-O-m ethoxy ethyl (2-O-MOE) modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some instances, 2’-O-aminopropyl modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some instances, 2'-deoxy modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some instances, 2'-deoxy-2’-fluoro modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some instances, 2'-O-aminopropyl (2-O-AP) modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some instances, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some instances, 2'-O-dimethylaminopropyl (2-O-DMAP) modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some instances, 2'-O-dimethylaminoethyloxy ethyl (2'-O-DMAEOE) modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent, natural oligonucleotide. In some instances, 2'-O-N-methylacetamido (2'-O-NMA) modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some instances, LNA modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some instances, ENA modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some instances, PNA modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some instances, HNA modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some instances, morpholino modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some instances, methylphosphonate nucleotides modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some instances, thiolphosphonate nucleotides modified oligonucleotide has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some instances, oligonucleotide comprising 2'-fluoro N3-P5'-phosphoramidites has increased binding affinity toward their mRNA target relative to an equivalent natural oligonucleotide. In some cases, the increased affinity is illustrated with a lower Kd, a higher melt temperature (Tm), or a combination thereof

[0128] In some embodiments, an oligonucleotide described herein is a chi rally pure (or stereo pure) oligonucleotide, or an oligonucleotide comprising a single enantiomer. In some instances, the oligonucleotide comprises L-nucleotide. In some instances, the oligonucleotide comprises D-nucleotides. In some instance, an oligonucleotide composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its mirror enantiomer. In some cases, an oligonucleotide composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic mixture. In some instances, the oligonucleotide is an oligonucleotide described in: U. S. Patent Publication Nos. 2014 / 194610 and 2015 / 211006; and PCT Publication No: WO2015107425.

[0129] In some embodiments, an oligonucleotide described herein is further modified to include an aptamer conjugating moiety. In some instances, the aptamer conjugating moiety is a DNA aptamer conjugating moiety. In some instances, the aptamer conjugating moiety is Alphamer (Centauri Therapeutics), which comprises an aptamer portion that recognizes a specific cellsurface target and a portion that presents a specific epitopes for attaching to circulating antibodies. In some instance, an oligonucleotide described herein is further modified to include an aptamer conjugating moiety as described in U. S. Patent Nos: 8,604,184, 8,591,910, and 7,850,975.

[0130] In additional embodiments, an oligonucleotide described herein is modified to increase its stability. In some embodiment, the oligonucleotide is RNA (e.g., siRNA). In some instances, the oligonucleotide is modified by one or more of the modifications described above to increase its stability. In some cases, the oligonucleotide is modified at the2'hydroxyl position, such as by 2'-O-methyl, 2' -O-m ethoxy ethyl (2-O-MOE), 2'-O-aminopropyI, 2'-deoxy, 2'-deoxy-2’-fluoro, 2'-O-aminopropyl (2'-0 -AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O"dimethy laminoethyl oxy ethyl (2' -O-DMAEOE), or 2'-O-N -methyl acetamido (2-O-NMA) modification or by a locked or bridged ribose conformation (e.g., LNA or ENA), In some cases, the oligonucleotide is modified by 2-O-methyl and / or 2-O-methoxyethyl ribose. In some cases, the oligonucleotide also includes morpholinos, P As, HNA, methylphosphonate nucleotides, thiolphosphonate nucleotides, and / or 2!~fluoro N3-P5'-phospboratnidites to increase its stability. In some instances, the oligonucleotide is a chirally pure (or stereo pure) oligonucleotide. In some instances, the chirally pure (or stereo pure) oligonucleotide is modified to increase its stability. Suitable modifications to the RNA to increase stability for delivery will be apparent to the skilled person. In some instances, the oligonucleotide is a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In some instances, the oligonucleotide is assembled from two separate polynucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (e.g., each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 19, 20, 21, 22, 23, or more base pairs); the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof Alternatively, the oligonucleotide is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the oligonucleotide are linked by means of a nucleic acid based or non-nucleic acid-based linker(s)

[0131] In some cases, the oligonucleotide is a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In other cases, the oligonucleotide is a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide is processed either in vivo or in vitro to generate an active oligonucleotide capable of mediating RNAi. In additional cases, the oligonucleotide also comprises a single-stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such oligonucleotide does not require the presence within the oligonucleotide of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide further comprises a terminal phosphate group, such as a 5'-phosphate (see, e.g., Martinez et ah, 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or 5', 3 '-di phosphate.

[0132] In some instances, an asymmetric hairpin is a linear oligonucleotide comprising an antisense region, a loop portion that comprises nucleotides or non-nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complimentary nucleotides to base pair with the antisense region and form a duplex with loop. For example, an asymmetric hairpin oligonucleotide comprises an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (eg., about 19 to about 22 nucleotides) and a loop region comprising about 4 to about 8 nucleotides, and a sense region having about 3 to about 18 nucleotides that are complementary to the antisense region. In some cases, the asymmetric hairpin oligonucleotide also comprises a 5'~terminal phosphate group that is chemically modified. In additional cases, the loop portion of the asymmetric hairpin oligonucleotide comprises nucleotides, non-nucleotides, linker molecules, or conjugate molecules.

[0133] In some embodiments, an asymmetric duplex is an oligonucleotide having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complimentary nucleotides to base pair with the antisense region and form a duplex. For example, an asymmetric duplex oligonucleotide comprises an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g., about 19 to about 22 nucleotides) and a sense region having about 3 to about 18 nucleotides that are complementary to the antisense region.

[0134] In some cases, a universal base refers to nucleotide base analogs that form base pairs with each of the natural DNA / RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3 -nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see, e.g, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447). Oligonucleotide Synthesis

[0135] In some embodiments, an oligonucleotide described herein is constructed using chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. For example, an oligonucleotide is chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the oligonucleotide and target nucleic acids. Exemplary methods include those described in: U. S. Patent Nos. 5,142,047; 5,185,444; 5,889,136; 6,008,400; and 6,111,086; PCT Publication No. W02009099942; or European Publication No 1579015. Additional exemplary methods include those described in: Griffey et al., "2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides," J. Med. Chem. 39(26):5100-5109 (1997)); Obika, et al. "Synthesis of 2'-0,4'-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering". Tetrahedron Letters 38 (50): 8735 (1997); Koizumi, M. "ENA oligonucleotides as therapeutics". Current opinion in molecular therapeutics 8 (2): 144-149 (2006), and Abramova et al., "Novel oligonucleotide analogues based on morpholino nucleoside subunits-antisense technologies: new chemical possibilities," Indian Journal of Chemistry 48B: 1721-1726 (2009) Alternatively, the oligonucleotide is produced biologically-using an expression vector into which an oligonucleotide has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted oligonucleotide will be of an antisense orientation to a target oligonucleotide of interest ).

[0136] In some embodiments, an oligonucleotide is synthesized via a tandem synthesis methodology, wherein both strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate fragments or strands that hybridize and permit purification of the duplex

[0137] In some instances, an oligonucleotide is also assembled from two distinct nucleic acid strands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the molecule.

[0138] Additional modification methods for incorporating, for example, sugar, base and phosphate modifications include: Eckstein et al., International Publication PCT No. WO 92 / 07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman etal. International Publication PCT No. WO 93 / 15187; Sproat, U S. Pat. No. 5,334,711 and Beigelman et al., 1995, J Biol Cbem., 270, 25702; Beigelman et al., International PCT publication No. WO 97 / 26270; Beigelman et al., U. S. Pat. No.

[0139] 5,716,824; Usman et al., U. S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No WO 98 / 13526; Thompson et al., U. S. Ser. No. 60 / 082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett, 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Anna. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010. Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and / or phosphate modifications and the like into nucleic acid molecules without modulating catalysis.

[0140] In some instances, while chemical modification of the oligonucleotide intemucleotide linkages with phosphorothioate, phosphorodithioate, and / or 5'-methylphosphonate linkages improves stability, excessive modifications sometimes cause toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages in some cases is minimized. In such cases, the reduction in the concentration of these linkages lowers toxicity, increases efficacy and higher specificity of these molecules.

[0141] Oligonucleotide Conj ugates

[0142] In some embodiments, an oligonucleotide (B) is further conjugated to a polypeptide (A) for delivery to a site of interest In some instances, at least one polypeptide A is conjugated to at least one B. In some instances, the at least one polypeptide A is conjugated to the at least one B to form an A-B conjugate. In some embodiments, at least one A is conjugated to the 5' terminus of B, the 3' terminus of B, an internal site on B, or in any combinations thereof. In some instances, the at least one polypeptide A is conjugated to at least two B. In some instances, the at least one polypeptide A is conjugated to at least 2, 3, 4, 5, 6, 7, 8, or more B.

[0143] In some cases, an oligonucleotide is conjugated to a polypeptide (A) and optionally a polymeric moiety (C) In some embodiments, at. least one polypeptide A is conjugated at one terminus of at least one B while at least one C is conjugated at the opposite terminus of the at least one B to form an A-B-C conjugate. In some instances, at least one polypeptide A is conjugated at one terminus of the at least one B while at least one of C is conjugated at an internal site on the at least one B. In some instances, at least one polypeptide A is conjugated directly to the at least one C. In some instances, the at least one B is conjugated indirectly to the at least one polypeptide A via the at least one C to form an A-C-B conjugate In some instances, at least one B and / or at least one C are conjugated to at least one polypeptide A. In some instances, the at least one B is conjugated at a terminus (e.g., a 5' terminus or a 3' terminus) to the at least one polypeptide A or are conjugated via an internal site to the at least one polypeptide A. In some cases, the at least one C is conjugated either directly to the at least one polypeptide A or indirectly via the at least one B. If indirectly via the at least one B, the at least one C is conjugated either at the same terminus as the at least one polypeptide A on B, at opposing terminus from the at least one polypeptide A, or independently at an internal site. In some instances, at least one additional polypeptide A is further conjugated to the at least one polypeptide A, to B, or to C.

[0144] Binding Moiety

[0145] In some embodiments, the binding moiety A is a polypeptide. In some instances, the polypeptide is an antibody or its fragment thereof. In some cases, the fragment is a binding fragment. In some instances, the antibody or antigen binding fragment thereof comprises a humanized antibody or antigen binding fragment thereof, murine antibody or antigen binding fragment thereof, chimeric antibody or antigen binding fragment thereof, monoclonal antibody or antigen binding fragment thereof a binding fragment having a light chain domain and a heavy chain domain, a binding fragment having two light chain domains and two heavy chain domains, a binding fragment having two or more light chain domains and heavy chain domains, monovalent Fab', divalent Fab'2, F(ab')2fragments, single-chain variable fragment (scFv), bis-scFv, (scFv)2, diabody, minibody, nanobody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or antigen binding fragment thereof, bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof.

[0146] In some embodiments, the binding moiety A is a bispecific antibody or antigen binding fragment thereof. In some instances, the bispecific antibody is a trifunctional antibody or a bispecific mini -antibody. In some cases, the bispecific antibody is a tri function al antibody. In some instances, the trifunctional antibody is a full-length monoclonal antibody comprising binding sites for two different antigens.

[0147] In some cases, the bispecific antibody is a bispecific mini-antibody. In some instances, the bispecific mini-antibody comprises divalent Fab'2, F(ab)'3 fragments, bis-scFv, (scFv)2, diabody, minibody, triabody, tetrabody or a bi-specific T-cell engager (BiTE). In some embodiments, the bi-specific T-cell engager is a fusion protein that contains two single-chain variable fragments (scFvs) in which the two scFvs target epitopes of two different antigens. In some embodiments, the binding moiety A is a bispecific mini-antibody. In some instances, A is a bispecific Fabi. In some instances, A is a bispecific F(ab)'s fragment. In some cases, A is a bispecific bis-scFv. In some cases, A is a bispecific (scFv)2.. In some embodiments, A is a bi specific diabody. In some embodiments, A is a bispecific minibody. In some embodiments, A is a bispecific triabody. In other embodiments, A is a bispecific tetrabody. In other embodiments, A is a bi-specific T-cell engager (BiTE).

[0148] In some embodiments, the binding moiety A is a trispecific antibody. In some instances, the trispecific antibody comprises F(ab)'3 fragments or a triabody. In some instances, A is a trispecific F(ab')3 fragment. In some cases, A is a triabody. In some embodiments, A is a trispecific antibody as described in Dimas, et al., "Development of a trispecific antibody designed to simultaneously and efficiently target three different antigens on tumor cells," Mol. Pharmaceutics, 12(9): 3490-3501 (2015).

[0149] In some embodiments, the binding moiety A is an antibody or antigen binding fragment thereof that recognizes a cell surface protein. In some instances, the binding moiety A is an antibody or antigen binding fragment thereof that recognizes a cell surface protein on a muscle cell. In some cases, the binding moiety. A is an antibody or antigen binding fragment thereof that recognizes a cell surface protein.

[0150] In certain embodiments, the compositions and methods disclosed herein generally relate to compositions, methods, therapies and regimens that are useful for conditioning a subject's tissues for engraftment or transplant (e.g., hematopoietic stem cell transplant). In particular, such compositions and methods selectively target a marker (e.g., a cell surface marker such as the CD45 or CD 117 receptor) and facilitate the intracellular delivery of an oligonucleotide to one or more cells (e.g., CD45+ or CD117+ cells) of the target tissue, for example, hematopoietic stem cells (HSCs) and / or progenitor cells in the bone marrow tissue of a subject. By selectively targeting cells expressing a selected marker (e.g., CD45 or CD117), the compositions and methods disclosed herein are able to exert their effect on those targeted cells (e.g., downregulation of BCL11A, thereby achieving increased fetal hemoglobin expression in a subject), while sparing, minimizing, and in certain instances eliminating, adverse effects on non-targeted cells and tissues. For example, in certain instances, the compositions and methods disclosed herein selectively inhibit BCL11 Ain the endogenous stem cell niche of a target tissue (e.g., bone marrow tissue), however, in contrast to traditional conditioning regimens (e.g., the reduced conditioning regimen for sickle cell anemia disclosed by Bolanos-Meade, et al., Blood (2012), 120(22): 4286), such compositions and methods do not induce life-threatening neutropenia, thrombocytopenia and / or anemia in the subject.

[0151] The CD45 receptor is a unique and ubiquitous membrane glycoprotein that is expressed on almost all hematopoietic cells. Similarly, CD117 is a cytokine receptor that is expressed on the surface of hematopoietic stem cells, progenitor cells, as well as other cell types. The compositions and methods disclosed herein are based in-part upon the discovery that certain markers (e.g., cell surface markers such as CD45 and CD117) have internalizing properties that may be exploited to facilitate the intracellular delivery of an oligonucleotide to the cells of a target tissue and thereby induce cell death. Accordingly, in certain embodiments the antibodies or antigen binding fragments thereof and compositions disclosed herein are characterized as being internalizing and thus can cause or otherwise facilitate the intracellular delivery of one or more oligonucleotides to cells of the target tissue that express a targeted marker (e.g., a targeted cell surface marker).

[0152] In certain aspects, the inventions disclosed herein contemplate the selection of one or more markers (e.g., a cell surface marker) to facilitate the selective targeting of the antibodies or antigen binding fragments thereof to the cells of a target tissue As used herein, the term "selectively" means that the antibody or antigen binding fragment thereof preferentially or discriminatorily recognizes and / or binds to a marker or a fragment or epitope of such marker (e.g., a cell surface marker). Exemplary antibodies or antigen binding fragments thereof that selectively recognize and / or bind a cell surface marker (e g., CD45, CD117 and CD34) and that may be used in accordance with the present inventions include, clone 104, clone 30F11, clone ACK2, clone 2B8, clone 3C11, clone MEM-28, clone HI30, clone 581 and clone 4H11. In certain aspects, the antibody or antigen binding fragment thereof comprises an antibody that selectively recognizes and / or binds to the CD34 marker (e.g., clone 581 or clone 4H11). In certain aspects, the antibody or antigen binding fragment thereof comprises an antibody that, selectively recognizes and / or binds to the CD45 marker (e.g., clone MEM-28 or clone HI30). In certain aspects, the antibody or antigen binding fragment thereof is an antibody selected from the group consisting of clone 1.243, clone TS2 / 4, clone TS1 / 18, clone 581, clone 4H11, clone A2A9 / 6, clone CD43-10G7, clone BHPT-1, clone orb 12060, clone 2D1, clone CC2C6, clone TS2 / 9, clone CY1G4, clone OKT9, clone CD84.1.21, clone VIM.3b, clone A3C6E2, clone EMK08, clone TMP4, clone KPL-l, clone 3a6, clone HD83 and clone MEM-216. By selectively targeting the cells of the target tissues, the methods and compositions disclosed herein may reduce, limit or otherwise avoid toxicities that have historically plagued traditional conditioning regimens and that result in life-threatening complications.

[0153] As used herein, the term "marker" generally refers to any protein, receptor, antigen, carbohydrates, lipids or other moieties that may be located or expressed on the surface of the cells of the target tissue and that can be used to discriminate a cell population. In particular, such markers may be used to selectively target the antibodies or antigen binding fragments thereof that comprise the oligonucleotide compositions disclosed herein to the cells of the target tissue. While certain embodiments disclosed herein contemplate the selective targeting of a cell using, for example the CD34, CD45 and / or CD 117 markers, the inventions are not limited to those markers. Rather, the present inventions contemplate the selection and use of any markers (e.g., cell surface markers) that may be useful or suitable for selectively targeting a cell population, inclusive of any yet to be discovered markers. Optionally, the selected marker is selectively expressed on the surface of the target cell population, thereby facilitating the selective or discriminatory targeting of such cell population using the antibodies or antigen binding fragments thereof disclosed herein. For example, in certain aspects, the selected marker is expressed on hematopoietic stem cells or progenitor cells. Exemplary markers may be selected from the group of markers consisting of HLA-DR, CD11a, CD 18, CD34, CD41 / 61, CD43, CD45, CD49d (VLA-4), CD49f (VLA-6), CD51, CD58, CD71, CD84, CD90, CD97, CD117 (c-kit), CD133, CD134, CD 162, CD166, CD 184 (CXCR4), CD205 and CD361. In certain embodiments, the selected marker is only expressed on the targeted cell population (e.g., the target HSC population), thereby limiting or avoiding the "off-target" effects that have limited the utility of traditional conditioning regimens.

[0154] In certain embodiments, the marker is a receptor. Exemplary human receptors that may be used or selected as markers in accordance with the inventions disclosed herein may be selected from the group of markers consisting of CD1.3, CD33, CD34, CD44, CD45, CD49d: VLA-4, CD49f: VLA-6, CD59, CD84: CD 150 family, CD90: Thyl, CD93, CD105: Endoglin, CD117: cKit / SCF receptor, CD123. IL-3R, CD126: IL-6R, CD133, CD135: Flt.3 receptor, CD166: ALCAM, CD184: CXCR4, Prominin 2, Erythropoietin R, Endothelial Cell-Selective Adhesion Molecule, CD244, Tiel, Tie2, MPL, G-CSFR or CSF3R, IL-1R, gpl30, Leukemia inhibitory factor Receptor, oncostatin M receptor, Ernbigin and 1L-18R. In certain aspects, exemplary markers that are expressed on human hematopoietic stem cells, that may be targeted and to which the antibodies or antigen binding fragments thereof that comprise the oligonucleotide selectively bind may be selected from the group consisting of CD7, C-Dwl2, CD 13, CD15, CD 19, CD21, CD22, CD29, CD30, CD33, CD34, CD36, CD38, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD48, CD49b, CD49d, CD49e, CD49f, CD50, CD53, CD55, CD64a, CD68, CD71, CD72, CD73, CD81, CD82, CD85A, CD85K, CD90, CD99, CD 104, CD105, CD 109, CD110, CD111, CD112, CD114, CD115, CD117, CD123, CD124, CD126, CD127, CD130, CD131, CD133, CD135, CD138, CD151, CD157, CD 162, CD 164, CD 168, CD 172a, CD173, CD 174, CD175, CD175s, CD176, CD183, CD191, CD200, CD201, CD205, CD217, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD235a, CD235b, CD236, CD236R, CD238, CD240, CD242, CD243, CD277, CD292, CDw293, CD295, CD298, CD309, CD318, CD324. CD325, CD338, CD344, CD349, and CD350.

[0155] Antibodies of the present invention can be raised against an appropriate marker or antigen, such as, for example, isolated and / or recombinant mammalian CD34, CD45, or CD117: cKit / SCF receptor or portions or epitopes thereof.

[0156] Typically, such antibodies are raised by immunizing an animal (e.g., a rabbit, rat, mouse, donkey, etc.) by multiple subcutaneous or intraperitoneal injections of the relevant antigen (e.g., CD34, CD45, or CD117: cKit / SCF receptor) optionally conjugated to keyhole limpet hemocyanin (KLH), serum albumin, other immunogenic carrier, diluted in sterile saline and combined with an adjuvant (e g., Complete or Incomplete Freund's Adjuvant) to form a stable emulsion. The polyclonal antibody is then recovered from blood or ascites of the immunized animal. Collected blood is clotted, and the serum decanted, clarified by centrifugation, and assayed for antibody titer. The polyclonal antibodies can be purified from serum or ascites according to standard methods in the art including affinity chromatography, ion-exchange chromatography, gel electrophoresis, dialysis, etc. Polyclonal antiserum can also be rendered monospecific using standard procedures (see, e.g, Agaton et al., "Selective Enrichment of Monospecific Polyclonal Antibodies for Anti body -Based Proteomics Efforts," J Chromatography A 1043(l):33-40 (2004), which is hereby incorporated by reference in its entirety).

[0157] In some embodiments, monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity," Nature 256:495-7 (1975), which is hereby incorporated by reference in its entirety. Using the hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized to elicit the production by lymphocytes of antibodies that will specifically bind to an immunizing antigen. Alternatively, lymphocytes can be immunized in vitro. Following immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells that can then be selected away from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies directed specifically against for example, a cell surface marker such as CD34, CD45, or CD117: cKit / SCF receptor, as determined by immunoprecipitation, immunoblotting, or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) can then be propagated either in vitro culture using standard methods (James Goding, Monoclonal Antibodies: Principles and Practice (1986) which is hereby incorporated by reference in its entirety) or in vivo as ascites tumors in an animal. The monoclonal antibodies can then be purified from the culture medium or ascites fluid as described for polyclonal antibodies above.

[0158] In some embodiments, the monoclonal antibody against a cell surface marker or antigen, such as CD34, CD45, or CD 117: cKit / SCF receptor, is a humanized antibody. In certain embodiments, the monoclonal antibody against a cell surface marker or antigen, such as HLA-DR, CD11a, CD18, CD34, CD41 / 61, CD43, CD45, CD47, CD58, CD71, CD84, CD97, CD117, CD133, CD162, CD 166, CD205 and / or CD361, is a humanized antibody. Humanized antibodies are antibodies that contain minimal sequences from non-human (e.g., murine) antibodies within the variable regions Such antibodies are used therapeutically to reduce antigenicity and human anti-mouse antibody responses when administered to a human subject. In practice, humanized antibodies are typically human antibodies with minimum to no non-human sequences. A human antibody is an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human.

[0159] In some embodiments, the antibodies or antigen binding fragments thereof disclosed herein binds to one or more markers selected from the group consisting of CD13, CD33, CD34, CD44, CD45, CD49d: VLA-4, CD49f: VLA-6, CD59, CD84: CD150 family, CD90: Thyl, CD93, CD105: Endoglin, CD! 17: cKit / SCF receptor, CD123: IL-3R, CD126: IL-6R, CD133, CD135: Flt3 receptor, CD166: ALCAM, CD184: CXCR4, Prominin 2, Erythropoietin R, Endothelial Cell-Selective Adhesion Molecule, CD244, Tiel, Tie2, MPL, G-CSFR or CSF3R, IL-1R, gpl3O, Leukemia inhibitory factor Receptor, oncostatin M receptor, Embigin and IL-18R. In certain embodiments, the antibody or antigen binding fragment thereof disclosed herein binds to one or more markers selected from the group consisting of HLA-DR, CDlla, CD 18, CD34, CD41 / 61, CD43, CD45, CD47, CD58, CD71, CD84, CD97, CD117, CD133, CD 162, CD 166, CD205 and CD361.

[0160] In some embodiments, the antibodies or antigen binding fragments thereof disclosed herein bind to two or more markers selected from the group consisting of CD 13, CD33, CD34, CD44, CD45, CD49d: VLA-4, CD49f: VLA-6, CD59, CD84: CD150 family, CD90: Thy 1, CD93, CD 105: Endoglin, CD 117: cKit / SCF receptor, CD 123: IL-3R, CD 126: IL-6R, CD133, CD135: Flt3 receptor, CD166: ALCAM, CD184: CXCR4, Prominin 2, Erythropoietin R, Endothelial Cell-Selective Adhesion Molecule, CD244, Tiel, Tie2, MPL, G-CSFR or CSF3R, IL-1R, gpl30, Leukemia inhibitory factor Receptor, oncostatin M receptor, Embigin and IL-18R. In certain embodiments, the antibody or antigen binding fragment thereof disclosed herein binds to two or more markers selected from the group consisting of HLA-DR, CDlla, CD 18, CD34, CD41 / 61, CD43, CD45, CD47, CD58, CD71, CD84, CD97, CD117, CD133, CD162, CD166, CD205 and CD361.

[0161] In certain embodiments, the antibody or antigen binding fragment thereof disclosed herein binds to two or more markers expressed on human hematopoietic stem cells and selected from the group consisting of CD7, CDw12, CD13, CD15, CD 19, CD21, CD22, CD29, CD30, CD33, CD34, CD36, CD38, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD48, CD49b, CD49d, CD49e, CD49f, CD50, CD53, CD55, CD64a, CD68, CD71, CD72, CD73, CD81, CD82, CD85A, CD85K, CD90, CD99, CD 104, CD105, CD 109, CD110, CD111, CD112, CD 114, CD115, CD117, CD123, CD 124, CD 126, CD127, CD130, CD131, CD133, CD135, CD138, CD151, CD157, CD 162, CD 164, CD168, CD 172a, CD173, CD 174, CD175, CD 175s, CD 176, CD183, CD191, CD200, CD201, CD205, CD217, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD235a, CD235b, CD236, CD236R, CD238, CD240, CD242, CD243, CD277, CD292, CDw293, CD295, CD298, CD309, CD318, CD324, CD325, CD338, CD344, CD349, and CD350.

[0162] In some embodiments, the antibody or antigen binding fragment is an anti-CD71 (antitransferrin receptor) antibody. In some instances, the anti-CD71 antibody comprises a variable heavy chain (VH) region and a variable light chain (VL) region In some instances, the antibody or antigen binding fragment is as described in U. S. Patent No. 11,555,190.

[0163] In some embodiments, the VH region of the anti-CD71 antibody comprises HCDR1, HCDR2, and HCDR3 sequences selected from:

[0164] Table 1

[0165]

[0166] In some embodiments, the VL region of the anti-CD71 antibody comprises LCDR1, LCDR2, and LCDR3 sequences selected from:

[0167] Table 2

[0168]

[0169] In some embodiments, the VH region of the anti-CD71 antibody comprises a sequence selected from:

[0170] Table 3

[0171]

[0172] The underlined regions in Table 3 denote the respective HCDR1, HCDR2, or HCDR3 sequence. In some embodiments, the VL. region of the anti~CD71 antibody comprises a sequence selected from:

[0173] Table 4

[0174]

[0175] The underlined regions in Table 4 denote the respective LCDR1, LCDR2, or LCDR3 sequence.

[0176] In some embodiments, the anti-CD7l antibody comprises a VH region and a VL region as illustrated in Table 5:

[0177] Table 5

[0178]

[0179]

[0180] In some embodiments, an anti-CD71 antibody described herein is a full-length antibody, comprising a heavy chain (HC) and a light chain (LC). In some cases, the heavy chain (HC) comprises a sequence selected from:

[0181] Table 6

[0182]

[0183]

[0184]

[0185]

[0186]

[0187] The underlined regions in Table 6 denote the respective CDRs.

[0188] In some cases, the light chain (LC) of an anti-CD71 antibody comprises a sequence selected from:

[0189] Table 7

[0190]

[0191] The underlined regions in Table 7 denote the respective CDRs.

[0192] In some embodiments, antibodies disclosed herein are capable of binding CD71 and CD117: cKit / SCF receptor. In some embodiments, antibodies disclosed herein are capable of binding CD34 and CD117: cKit / SCF receptor. In some embodiments, antibodies disclosed herein are capable of binding CD45 and CD117: cKit / SCF receptor In some embodiments, antibodies disclosed herein are capable of binding CD34 and CD45.

[0193] In certain embodiments, cells which express a cell surface marker can be used as an immunogen or in a screen for antibody which binds the marker. In one embodiment, the antibody has specificity for the marker, epitope or a portion thereof. In those embodiments where the antibody or antigen binding fragment thereof is or comprises an antibody, upon identifying and selecting a marker that is expressed on the surface of the cells of the target tissue (e.g., CD71, CD117 or portions or epitopes thereof), an antibody may be raised against such marker using art-recognized techniques and methods.

[0194] In certain aspects, the antibody or antigen binding fragment thereof is or comprises a ligand For example, in certain embodiments the antibody or antigen binding fragment thereof is or comprises a ligand, such as stem cell factor, and that interacts or binds to a cell surface receptor, such as CD 117.

[0195] In certain embodiments, the antibody or antigen binding fragment thereof is used to deliver, or to facilitate the delivery of an oligonucleotide to the cells of a target tissue and, following the delivery of such oligonucleotide to the cells of the target tissue, such oligonucleotide is internalized by such cells and thereby exerts a gene inhibitory effect on such cells of the target tissue In certain embodiments, upon delivery of an antibody or antigen binding fragment thereof coupled to an oligonucleotide to the cells of a target tissue, both the antibody or antigen binding fragment thereof and oligonucleotide are co-localized to an intracellular compartment of one or more cells of the target tissue, thereby downregulating a target gene (e g, BCL11A) in such cells.

[0196] In certain aspects, the compositions disclosed herein (e.g., antibody-oligonucleotide conjugates) are internalized by a cell expressing a marker (e.g., a CD71, CD117, CD34, CD45, etc. cell surface marker) upon binding of such antibody or antigen binding fragment thereof to an epitope of the marker (e.g., epitope of CD71, CD117, CD34, or CD45).

[0197] In certain embodiments, the oligonucleotide may be directly coupled or otherwise bound to an antibody or antigen binding fragment thereof (e.g., an antibody that specifically or selectively binds CD71, CD117, CD34, or CD45). For example, the antibody or antigen binding fragment thereof is directly coupled to one or more oligonucleotides. As used herein, the terms "couple" and "coupling" broadly refer to any physical, biological or chemical linking or joining of two or more moieties or components together. Such a coupling may be direct or indirect. For example, disclosed herein are antibodies or antigen binding fragments thereof (e.g., bispecific antibodies or antigen binding fragments thereof) that may be directly or indirectly coupled to oligonucleotides The antibodies or antigen binding fragments thereof and the oligonucleotide are covalently or non-covalently coupled or linked to each other. Such a coupling may be direct or indirect. In some embodiments, the oligonucleotide disclosed herein may be indirectly coupled to an antibody. Such antibodies may be biotinylated and coupled to a streptavidin-toxin moiety. Alternatively, in certain embodiments, the oligonucleotide may be biotinylated, which may be indirectly coupled to an anti-CD71, anti-CD34, anti-CD45 or anti-CD117 antibody that may be bound to or labeled with one or more of streptavidin, avidin, neutravidin and any other variants thereof. In certain aspects, the antibodies disclosed herein are humanized.

[0198] In some embodiments, an effective amount of the oligonucleotide compositions disclosed herein achieves maximal stem cell depletion (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 99%, 99.5% or more depletion of hematopoietic or progenitor stem cells from the target tissues of the subject). In some embodiments, an effective amount of the compositions disclosed herein is determined on the basis of a subject's weight For example, in certain aspects, such an effective amount of the compositions disclosed herein is or comprises one or more doses of ranging between about 10-0.01 mg / kg In certain aspects, an effective amount of the compositions disclosed herein (e.g., an anti-CD71 -oligonucleotide or anti-CD117-oligonucleotide conjugate) is or comprises one or more doses of 4.0 mg / kg. In some aspects, an effective amount of the compositions disclosed herein is or comprises one or more doses of 3.0 mg / kg. In certain aspects, an effective amount of the compositions disclosed herein is or comprises one or more doses of 2.0 mg / kg. In some aspects, an effective amount of the compositions disclosed herein is or comprises one or more doses of 2.5 mg / kg. In certain aspects, an effective amount of the compositions disclosed herein is or comprises one or more doses of 2.0 mg / kg In certain embodiments, an effective amount of the compositions disclosed herein (e.g., an anti-CD71-oligonucleotide or anti-CD117-oligonucleotide conjugate) is or comprises one or more doses of 1.5 mg / kg.

[0199] In some embodiments, an antibody described herein comprises an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some instances, the anti-CD71 antibody comprises an IgG framework (e.g., IgGl, IgG2, IgG3, or IgG4). In some cases, the anti-CD71 antibody compri ses an IgG 1 framework. In some cases, the anti-CD71 antibody comprises an IgG2 (e g, an IgG2a or IgG2b) framework. In some cases, the anti-CD71 antibody comprises an IgG2a framework. In some cases, the anti-CD71 antibody comprises an IgG2b framework In some cases, the anti-CD71 antibody comprises an IgG3 framework. In some cases, the anti-CD71 antibody comprises an IgG4 framework.

[0200] In some cases, an antibody comprises one or more mutations in a framework region, e g., in the CHI domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some instances, the one or more mutations are to stabilize the antibody and / or to increase half-life. In some instances, the one or more mutations are to modulate Fc receptor interactions, to reduce or eliminate Fc effector functions such as FcyR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC) In additional instances, the one or more mutations are to modulate glycosylation. In some embodiments, the one or more mutations are located in the Fc region. In some instances, the Fc region comprises a mutation at residue position L234, L235, or a combination thereof. In some instances, the mutations comprise L234 and L235. In some instances, the mutations comprise L234A and L235A. In some cases, the residue positions are in reference to IgGl

[0201] In some instances, the Fc region comprises a mutation at residue position L234, L235, D265, N297, K322, L328, or P329, or a combination thereof. In some instances, the mutations comprise L234 and 1.235 in combination with a mutation at residue position K322. L328, or P329. In some cases, the Fc region comprises mutations at L234, L235, and K322. In some cases, the Fc region comprises mutations at L234, L235, and L328. In some cases, the Fc region comprises mutations at L234, L235, and P329. In some cases, the Fc region comprises mutations at D265 and N297. In some cases, the residue position is in reference to IgGl.

[0202] In some instances, the Fc region comprises L234A, L235A, D265A, N297G, K322G, L328R, or P329G, or a combination thereof. In some instances, the Fc region comprises L234A and L235Ain combination with K322G, L328R, orP329G. In some cases, theFc region comprises L234A, L235A, and K322G. In some cases, the Fc region comprises L234A, L235A, and L328R. In some cases, the Fc region comprises L234A, L235A, and P329G In some cases, the Fc region comprises D265A and N297G. In some cases, the residue position is in reference to IgGl.

[0203] In some instances, the Fc region comprises a mutation at residue position L235, L236, D265, N297, K322, L328, or P329, or a combination of the mutations. In some instances, the Fc region comprises mutations at L235 and L236. In some instances, the Fc region comprises mutations at L235 and L236 in combination with a mutation at residue position K322, L328, or P329. In some cases, the Fc region comprises mutations at L235, L236, and K322. In some cases, the Fc region comprises mutations at L235, L236, and L328. In some cases, the Fc region comprises mutations at L235, L236, and P329. In some cases, the Fc region comprises mutations at D265 and N297 In some cases, the residue position is in reference to IgG2b.

[0204] In some embodiments, the Fc region comprises L235A, L236A, D265A, N297G, K322G, L328R, or P329G, or a combination thereof. In some instances, the Fc region comprises L235A and L236A. In some instances, the Fc region comprises L235A and L236A in combination with K322G, L328R, or P329G. In some cases, the Fc region comprises L235A, L236A, and K322G. In some cases, the Fc region comprises L235A, L236A, and L328R. In some cases, the Fc region comprises L235 A, L236A, and P329G. In some cases, the Fc region comprises D265 A and N297G. In some cases, the residue position is in reference to IgG2b. In some embodiments, the Fc region comprises a mutation at residue position L233, L234, D264, N296, K321, L327, or P328, wherein the residues correspond to positions 233, 234, 264, 296, 321, 327, and 328 of SEQ ID NO: 303. In some instances, the Fc region comprises mutations at. L233 and L234. In some instances, the Fc region comprises mutations at L233 and L234 in combination with a mutation at residue position K321, L327, or P328 In some cases, the Fc region comprises mutations at L233, L234, and K321, In some cases, the Fc region comprises mutations at L233, L234, and L327. In some cases, the Fc region comprises mutations at L233, L234, and K321. In some cases, the Fc region comprises mutations at L233, L234, and P328. In some instances, the Fc region comprises mutations at D264 and N296. In some cases, equivalent positions to residue L233, L234. D264, N296, K321, L327, or P328 in an IgGl, IgG2, IgG3, or IgG4 framework are contemplated. In some cases, mutations to a residue that corresponds to residue L233, L234, D264, N296, K321, L327, or P328 of SEQ ID NO: 303 in an IgGl, IgG2, or IgG4 framework are also contemplated

[0205] In some embodiments, the Fc region comprises L233A, L234A, D264A, N296G, K321G, L327R, or P328G, wherein the residues correspond to positions 233, 234, 264, 296, 321, 327, and 328 of SEQ ID NO: 303. In some instances, the Fc region comprises L233A and L234A. In some instances, the Fc region comprises L233A and L234A in combination with K321G, L327R, or P328G. In some cases, the Fc region comprises L233A, L234A. and K321G. In some cases, the Fc region comprises L233A, L234A, and L327R. In some cases, the Fc region comprises L233A, L234A, and K321G. In some cases, the Fc region comprises L233A, L234A, and P328G. In some instances, the Fc region comprises D264A and N296G.

[0206] In some embodiments, the human IgG constant region is modified to alter antibodydependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), e.g., with an amino acid modification described in Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Moore et al., 2010 mAbs, 2(2): 181-189; Lazar etal., 2006 PNAS, 103(11): 4005-4010, Shields et al., 2001 JBC, 276( 9): 6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48: 152-164, Alegre et al, 1992 J Immunol, 148: 3461-3468; Reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1): 1-11.

[0207] In some embodiments, an antibody described herein is a full-length antibody, comprising a heavy chain (HC) and a light chain (LC). In certain embodiments, the full-length antibody is an anti-CD117 antibody. In some embodiments, the anti-CD117 antibody is a clone 2B8 anti-CD117 antibody or is a clone 104DT anti-CD117 antibody.

[0208] In some embodiments, an antibody described herein has an improved serum half-life compared to a reference anti -transferrin receptor antibody. In some instances, the improved serum half-life is at least 30 minutes, 1 hour, 1.5 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, or longer than reference anti transferrin receptor antibody.

[0209] In some embodiments, the binding moiety A is conjugated to an oligonucleotide (B) non-specifically. In some instances, the binding moiety A is conjugated to an oligonucleotide (B) via a lysine residue or a cysteine residue, in a non-site specific manner. In some instances, the binding moiety A is conjugated to an oligonucleotide (B) via a lysine residue (e.g., lysine residue present in the binding moiety A) in a non-site specific manner In some cases, the binding moiety A is conjugated to an oligonucleotide (B) via a cysteine residue (e.g., cysteine residue present in the binding moiety A) in a non-site specific manner.

[0210] In some embodiments, the binding moiety A is conjugated to an oligonucleotide (B) in a site-specific manner In some instances, the binding moiety A is conjugated to an oligonucleotide (B) through a lysine residue, a cysteine residue, at the 5'-terminus, at the 3'-terminus, an unnatural amino acid, or an enzyme-modified or enzyme-catalyzed residue, via a site-specific manner. In some instances, the binding moiety A is conjugated to an oligonucleotide (B) through a lysine residue (e.g., lysine residue present in the binding moiety A) via a site-specific manner. In some instances, the binding moiety A is conjugated to an oligonucleotide (B) through a cysteine residue (e.g, cysteine residue present in the binding moiety A) via a site-specific manner. In some instances, the binding moiety A is conjugated to an oligonucleotide (B) at the 5'-terminus via a site-specific manner. In some instances, the binding moiety A is conjugated to an oligonucleotide (B) at the 3'-terminus via a site-specific manner. In some instances, the binding moiety A is conjugated to an oligonucleotide (B) through an unnatural amino acid via a site- specific manner. In some instances, the binding moiety A is conjugated to an oligonucleotide (B) through an enzyme-modified or enzyme-catalyzed residue via a site-specific manner.

[0211] In some embodiments, one or more oligonucleotide (B) is conjugated to a binding moiety A. In some instances, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more oligonucleotides are conjugated to one binding moiety A. In some instances, about 1 oligonucleotide is conjugated to one binding moiety A. In some instances, about 2 oligonucleotides are conjugated to one binding moiety A In some instances, about 3 oligonucleotides are conjugated to one binding moiety A. In some instances, about 4 oligonucleotides are conjugated to one binding moiety A. In some instances, about 5 oligonucleotides are conjugated to one binding moiety A In some instances, about 6 oligonucleotides are conjugated to one binding moiety A. In some instances, about 7 oligonucleotides are conjugated to one binding moiety A. In some instances, about 8 oligonucleotides are conjugated to one binding moiety A. In some instances, about 9 oligonucleotides are conjugated to one binding moiety A. In some instances, about 10 oligonucleotides are conjugated to one binding moiety A In some instances, about 11 oligonucleotides are conjugated to one binding moiety A In some instances, about 12 oligonucleotides are conjugated to one binding moiety A.

[0212] In some instances, about 13 oligonucleotides are conjugated to one binding moiety A In some instances, about 14 oligonucleotides are conjugated to one binding moiety A. In some instances, about 15 oligonucleotides are conjugated to one binding moiety A. In some instances, about 16 oligonucleotides are conjugated to one binding moiety A. In some cases, the one or more oli onucleotides are the same In other cases, the one or more oligonucleotides are different.

[0213] In some embodiments, the number of oligonucleotide (B) conjugated to a binding moiety A forms a ratio. In some instances, the ratio is referred to as a DAR (drug-to-antibody) ratio, in which the drug as referred to herein is the oligonucleotide (B). In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or greater. In some instances, the D AR ratio of the oligonucleotide (B) to binding moiety A is about 1 or greater. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 2 or greater. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 3 or greater. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 4 or greater. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 5 or greater. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 6 or greater. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 7 or greater. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 8 or greater. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 9 or greater. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 10 or greater In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 11 or greater. In some instances, the DAR ratio of the oli onucleotide (B) to binding moiety A is about 12 or greater. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 1. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 2. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 3. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 4. In some instances, the DAR ratio of the oli onucleotide (B) to binding moiety A is about 5 In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 6. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 7. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 8. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 9. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about. 10 In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 11. In some instances, the DAR ratio of the oli onucleotide (B) to binding moiety A is about 12. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 13. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 14 In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 15 In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is about 16.

[0214] In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is 1. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is 2. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is 4, In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is 6 In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is 8. In some instances, the DAR ratio of the oligonucleotide (B) to binding moiety A is 12.

[0215] In some instances, a conjugate comprising oligonucleotide (B) and binding moiety A has improved activity as compared to a conjugate comprising oligonucleotide (B) without a binding moiety A. In some instances, improved activity results in enhanced biologically relevant functions, e.g., improved stability, affinity, binding, functional activity, and efficacy in treatment or prevention of a disease state In some instances, the disease state is a result of one or more mutated exons of a gene. In some instances, the conjugate comprising oligonucleotide (B) and binding moiety A results in increased exon skipping of the one or more mutated exons as compared to the conjugate comprising oligonucleotide (B) without a binding moiety A. In some instances, exon skipping is increased by at least or about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% in the conjugate comprising oligonucleotide (B) and binding moiety A as compared to the conjugate comprising oligonucleotide (B) without a binding moiety A.

[0216] In some embodiments, an antibody or antigen binding fragment is further modified using conventional techniques known in the art, for example, by using amino acid deletion, insertion, substitution, addition, and / or by recombination and / or any other modification (e g, posttranslational and chemical modifications, such as glycosylation and phosphorylation) known in the art either alone or in combination In some instances, the modification further comprises a modification for modulating interaction with Fc receptors. In some instances, the one or more modifications include those described in, for example, International Publication No WO 97 / 34631, which discloses amino acid residues involved in the interaction between the Fc domain and the FcRn receptor. Methods for introducing such modifications in the nucleic acid sequence underlying the amino acid sequence of an antibody or antigen binding fragment is well known to the person skilled in the art.

[0217] In some instances, an antigen binding fragment further encompasses its derivatives and includes polypeptide sequences containing at least one CDR.

[0218] In some instances, the term "single-chain" as used herein means that the first and second domains of a bi-specific single chain construct are covalently linked, optionally in the form of a co-linear amino acid sequence encodable by a single nucleic acid molecule.

[0219] In some instances, a bi specific single chain antibody construct relates to a construct comprising two antibody derived binding domains. In such embodiments, bi-specific single chain antibody construct is tandem bi-scFv or diabody. In some instances, a scFv contains a VH and VL domain connected by a linker peptide. In some instances, linkers are of a length and sequence sufficient to ensure that each of the first and second domains can, independently from one another, retain their differential binding specificities.

[0220] In some embodiments, binding to or interacting with as used herein defines a binding / interaction of at least two antigen-interaction-sites with each other. In some instances, antigen-interaction-site defines a motif of a polypeptide that shows the capacity of specific interaction with a specific antigen or a specific group of antigens. In some cases, the binding / interaction is also understood to define a specific recognition. In such cases, specific recognition refers to that the antibody or its antigen binding fragment is capable of specifically interacting with and / or binding to at least two amino acids of each of a target molecule. For example, specific recognition relates to the specificity of the antibody molecule, or to its ability to discriminate between the specific regions of a target molecul e. In addi tional instances, the specific interaction of the antigen-interaction-site with its specific antigen results in an initiation of a signal, e g., due to the induction of a change of the conformation of the antigen, an oligomerization of the antigen, etc. In further embodiments, the binding is exemplified by the specificity of a "key -lock-principle". Thus in some instances, specific motifs in the amino acid sequence of the antigen-i nt eracti on-site and the antigen bind to each other as a result of their primary, secondary or tertiary structure as well as the result of secondary modifications of said structure. In such cases, the specific interaction of the antigen-interaction-site with its specific antigen results as well in a simple binding of the site to the antigen. In some instances, specific interaction further refers to a reduced cross-reactivity of the antibody or antigen binding fragment or a reduced off-target effect. For example, the antibody or antigen binding fragment that bind to the polypeptide / protein of interest but do not or do not essentially bind to any of the other polypeptides are considered as specific for the polypeptide / protein of interest. Examples for the specific interaction of an antigen-interaction-site with a specific antigen comprise the specificity of a ligand for its receptor, for example, the interaction of an antigenic determinant (epitope) with the antigenic binding site of an antibody.

[0221] Additional Binding Moielies

[0222] In some embodiments, the binding moiety is a plasma protein. In some instances, the plasma protein comprises albumin. In some instances, the binding moiety A is albumin. In some instances, albumin is conjugated by one or more of a conjugation chemistry described herein to an oligonucleotide. In some instances, albumin is conjugated by native ligation chemistry' to an oligonucleotide. In some instances, albumin is conjugated by lysine conjugation to an oligonucleotide.

[0223] In some instances, the binding moiety is a steroid. Exemplary' steroids include cholesterol, phospholipids, di-and triacylglycerols, fatty acids, hydrocarbons that, are saturated, unsaturated, comprise substitutions, or combinations thereof. In some instances, the steroid is cholesterol. In some instances, the binding moiety is cholesterol. In some instances, cholesterol is conjugated by one or more of a conjugation chemistry described herein to an oligonucleotide. In some instances, cholesterol is conjugated by native ligation chemistry to an oligonucleotide. In some instances, cholesterol is conjugated by lysine conjugation to an oligonucleotide.

[0224] In some instances, the binding moiety is a polymer, including but not limited to oligonucleotide aptamers that bind to specific surface markers on cells. In this instance the binding moiety is a nucleic acid that does not hybridize to a target gene or mRNA, but instead is capable of selectively binding to a cell surface marker similarly to an antibody binding to its specific epitope of a cell surface marker.

[0225] In some cases, the binding moiety is a peptide. In some cases, the peptide comprises between about 1 and about 3 kDa. In some cases, the peptide comprises between about 1.2 and about 2.8 kDa, about 1.5 and about 2.5 kDa, or about 1.5 and about 2 kDa. In some instances, the peptide is a bicyclic peptide. In some cases, the bicyclic peptide is a constrained bicyclic peptide. In some instances, the binding moiety is a bicyclic peptide (e.g, bicycles from Bicycle Therapeutics)

[0226] In additional cases, the binding moiety is a small molecule. In some instances, the small molecule is an antibody-recruiting small molecule. In some cases, the antibody-recruiting small molecule comprises a target-binding terminus and an antibody-binding terminus, in which the target-binding terminus is capable of recognizing and interacting with a cell surface receptor.

[0227] For example, in some instances, the target-binding terminus comprising a glutamate urea compound enables interaction with PSMA, thereby, enhances an antibody interaction with a cell that expresses PSMA. In some instances, a binding moiety is a small molecule described in Zhang et al, "A remote arene-binding site on prostate specific membrane antigen revealed by antibodyrecruiting small molecules," J Am Chem Soc. 132(36): 12711-12716 (2010); orMcEnaney, et al., "Antibody -recruiting molecules: an emerging paradigm for engaging immune function in treating human disease," ACS Chem Biol 7(7): 1139-1151 (2012).

[0228] Production of Antibodies or Antigen Binding Fragment Thereof

[0229] In some embodiments, polypeptides described herein (e.g., antibodies and antigen binding fragments) are produced using any method known in the art to be useful for the synthesis of polypeptides (e.g., antibodies), in particular, by chemical synthesis or by recombinant expression, and are optionally produced by recombinant expression techniques. In some instances, an antibody or antigen binding fragment thereof is expressed recombinantly, and the nucleic acid encoding the antibody or antigen binding fragment is assembled from chemically synthesized oligonucleotides (e g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0230] Alternatively, a nucleic acid molecule encoding an antibody is optionally generated from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence. In some instances, an antibody or its antigen binding is optionally generated by immunizing an animal, such as a rabbit, to generate polyclonal antibodies or, optionally, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp 77-96). Alternatively, a clone encoding at least the Fab portion of the antibody is optionally obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246: 1275- 1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (see, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

[0231] In some embodiments, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855, Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity are used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g., humanized antibodies.

[0232] In some embodiments, techniques described for the production of single chain antibodies (U. S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883, and Ward et al., 1989, Nature 334:544-54) are adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242: 1038-1041).

[0233] In some embodiments, an expression vector comprising the nucleotide sequence of an antibody or the nucleotide sequence of an antibody is transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody

[0234] In specific embodiments, the expression of the antibody is regulated by a constitutive, an inducible or a tissue, specific promoter. In some embodiments, a variety of host-expression vector systems is utilized to express an antibody or its antigen binding fragment described herein Such host-expression systems represent vehicles by which the coding sequences of the antibody is produced and subsequently purified, but also represent cells that are, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody or its antigen binding fragment in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing an antibody or its antigen binding fragment coding sequences; yeast (e g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing an antibody or its antigen binding fragment coding sequences, insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an antibody or its antigen binding fragment coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an antibody or its antigen binding fragment coding sequences, or mammalian cell systems (e.g., COS, CHO, BH, 293, 293 T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e g., metallothionein promoter) or from mammalian viruses (e.g, the adenovirus late promoter: the vaccinia virus 7.5K promoter). For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some instances, cell lines that stably express an antibody are optionally engineered. Rather than using expression vectors that contain viral origins of replication, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells are then allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn are cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the antibody or its antigen binding fragments.

[0235] In some instances, a number of selection systems are used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes are employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance are used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G- 418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol.

[0236] 32:573-596, Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993,. Ann. Rev Biochem. 62:191-217; May, 1993, TIB TECH 11(5): 155-215) and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY, Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et. al., 1981, J Mol Biol 150:1).

[0237] In some instances, the expression levels of an antibody are increased by vector amplification (for a review, see Bebbington and Hentschel. The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing an antibody is amplifiable, an increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody, production of the antibody will also increase (Crouse et al., 1983, Mol Cell Biol. 3:257).

[0238] In some instances, any method known in the art for purification or analysis of an antibody or antibody conjugates is used, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Exemplary chromatography methods included, but are not limited to, strong anion exchange chromatography, hydrophobic interaction chrom tography, size exclusion chromatography, and fast protein liquid chromatography. Conjugation Chemistry

[0239] In some embodiments, an oligonucleotide B is conjugated to a binding moiety. In some embodiments, an oligonucleotide B is conjugated to a binding moiety in a formula A-X-B (X is a linker conjugating A and B). In some instances, the binding moiety comprises amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, as well as analogs or derivatives of all of these classes of substances. Additional examples of binding moiety also include steroids, such as cholesterol, phospholipids, di-and triacylglycerols, fatty acids, hydrocarbons (e.g., saturated, unsaturated, or contains substitutions), enzyme substrates, biotin, digoxigenin, and polysaccharides. In some instances, the binding moiety is an antibody or antigen binding fragment thereof. In some instances, the oligonucleotide is further conjugated to a polymer, and optionally an endosomolytic moiety

[0240] In some embodiments, the oligonucleotide is conjugated to the binding moiety by a chemical ligation process. In some instances, the oligonucleotide is conjugated to the binding moiety by a native ligation. In some instances, the conjugation is as described in: Dawson, et al. "Synthesis of proteins by native chemical ligation," Science 1994, 266, 776-779; Dawson, et al. "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives," J Am. Chem. Soc. 1997, 119, 4325-4329; Hackeng, et al. "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology.," Proc. Natl Acad. Sci. USA 1999, 96, 10068-10073, or Wu, et al. "Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol," Angew. Chem Int. Ed. 2006, 45, 4116-4125. In some instances, the conjugation is as described in U. S. Patent No. 8,936,910. In some embodiments, the oligonucleotide is conjugated to the binding moiety either site-specifically or non-specifically via native ligation chemistry.

[0241] In some instances, the oligonucleotide is conjugated to the binding moiety by a site-directed method utilizing a "traceless" coupling technology (Philochem). In some instances, the "traceless" coupling technology utilizes an N-terminal 1,2-aminothiol group on the binding moiety which is then conjugate with an oligonucleotide containing an aldehyde group (see, Casi et al, "Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery," JACS 134(13): 5887-5892 (2012)) In some instances, the oligonucleotide is conjugated to the binding moiety by a site-directed method utilizing an unnatural amino acid incorporated into the binding moiety.

[0242] In some instances, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some instances, the keto group of pAcPhe is selectively coupled to an alkoxy-amine derivatized conjugating moiety to form an oxime bond (see, Axup et al., "Synthesi s of site-specific antibodydrug conjugates using unnatural amino acids," PNAS 109(40): 16101-16106 (2012)). In some instances, the oligonucleotide is conjugated to the binding moiety by a site-directed method utilizing an enzyme-catalyzed process. In some instances, the site-directed method utilizes SMART ag™ technology (Catalent, Inc.). In some instances, the SMART ag™ technology comprises generation of a formylglycine (FGly) residue from cysteine by formylglycine-generating enzyme (FGE) through an oxidation process under the presence of an aldehyde tag and the subsequent conjugation of FGly to an alkylhydrazine-functionalized oligonucleotide via hydrazino-Pictet-Spengler (HIPS) ligation (see, Wu et al., "Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag," PNAS 106(9): 3000-3005 (2009); Agarwal, et al., "A Pictet-Spengler ligation for protein chemical modification," PNAS 110(1): 46-51 (2013))

[0243] In some instances, the enzyme-catalyzed process comprises microbial transglutaminase (mTG). In some cases, the oligonucleotide is conjugated to the binding moiety utilizing a microbial transglutaminase-catalyzed process. In some instances, mTG catalyzes the formation of a covalent bond between the amide side chain of a glutamine within the recognition sequence and a primary amine of a functionalized oligonucleotide. In some instances, mTG is produced from Streptomyces mobarensis. (see, Strop et al., "Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates," Chemistry and Biology 20(2) 161-167 (2013)).

[0244] In some instances, the oligonucleotide is conjugated to the binding moiety by a method as described in PCT Publication No. W02014 / 140317, which utilizes a sequence-specific transpeptidase.

[0245] In some instances, the oligonucleotide is conjugated to the binding moiety by a method as described in U. S. Patent Publication Nos 2015 / 0105539 and 2015 / 0105540. Polymer Conjugating Moiety

[0246] In some embodiments, a polymer moiety C is further conjugated to an oligonucleotide described herein, a binding moiety' described herein, or in combinations thereof. In some instances, a polymer moiety C is conjugated an oligonucleotide in a formula A-X1-B-X2-C (X1, X2 as two linkers conjugating A and B, B and C, respectively). In some cases, a polymer moiety C is conjugated to a binding moiety. In other cases, a polymer moiety C is conjugated to an oligonucleotide -binding moiety' molecule. In additional cases, a polymer moiety C is conjugated, as illustrated supra.

[0247] In some instances, the polymer moiety C is a natural or synthetic polymer, consisting of long chains of branched or unbranched monomers, and / or cross-linked network of monomers in two or three dimensions. In some instances, the polymer moiety C includes a polysaccharide, lignin, rubber, or polyalkylene oxide (e.g., polyethylene glycol). In some instances, the at least, one polymer moiety C includes, but is not limited to, alpha-, omega-dihydroxylpolyethyleneglycol, biodegradable lactone-based polymer, e.g., polyacrylic acid, polylactide acid (PL A), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin, polyamide, polycyanoacrylate, polyimide, polyethylene terephthalate (also known as poly(ethylene terephthalate), PET, PETG, or PETE), polytetramethylene glycol (PTG), or polyurethane as well as mixtures thereof. As used herein, a mixture refers to the use of different polymers within the same compound as well as in reference to block copolymers. In some cases, block copolymers are polymers wherein at least one section of a polymer is build up from monomers of another polymer. In some instances, the polymer moiety C comprises polyalkylene oxide. In some instances, the polymer moiety C comprises PEG In some instances, the polymer moiety C comprises polyethylene imide (PEI) or hydroxy ethyl starch (HES). In some instances, C is a PEG moiety. In some instances, the PEG moiety is conjugated at the 5' terminus of the oligonucleotide while the binding moiety is conjugated at the 3' terminus of the oligonucleotide. In some instances, the PEG moiety is conjugated at the 3’ terminus of the oligonucleotide while the binding moiety is conjugated at the 5' terminus of the oligonucleotide. In some instances, the PEG moiety is conjugated to an internal site of the oligonucleotide. In some instances, the PEG moiety, the binding moiety, or a combination thereof, are conjugated to an internal site of the oligonucleotide. In some instances, the conjugation is a direct conjugation. In some instances, the conjugation is via native ligation In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some instances, poly disperse material comprises disperse distribution of different molecular weight of the material, characterized by mean weight (weight average) size and dispersity. In some instances, the monodisperse PEG comprises one size of molecules. In some embodiments, C is poly or monodispersed polyalkylene oxide (e.g., PEG) and the indicated molecular weight represents an average of the molecular weight of the polyalkylene oxide, e.g., PEG, molecules.

[0248] In some embodiments, the molecular weight of the polyalkylene oxide (e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.

[0249] In some embodiments, C is polyalkylene oxide (e g., PEG) and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da In some embodiments, C is PEG and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some instances, the molecular weight of C is about 200 Da

[0250] In some instances, the molecular weight of C is about 300 Da. In some instances, the molecular weight of C is about 400 Da. In some instances, the molecular weight of C is about 500 Da. In some instances, the molecular weight of C is about 600 Da. In some instances, the molecular weight of C is about 700 Da. In some instances, the molecular weight of C is about 800 Da. In some instances, the molecular weight of C is about 900 Da. In some instances, the molecular weight of C is about 1000 Da In some instances, the molecular weight of C is about 1100 Da.

[0251] In some instances, the molecular weight of C is about 1200 Da. In some instances, the molecular weight of C is about 1300 Da. In some instances, the molecular weight of C is about 1400 Da In some instances, the molecular weight of C is about 1450 Da In some instances, the molecular weight of C is about 1500 Da. In some instances, the molecular weight of C is about 1600 Da. In some instances, the molecular weight of C is about 1700 Da. In some instances, the molecular weight, of C is about 1800 Da. In some instances, the molecular weight of C is about 1900 Da. In some instances, the molecular weight of C is about 2000 Da. In some instances, the molecular weight of C is about 2100 Da. In some instances, the molecular weight of C is about 2200 Da. In some instances, the molecular weight of C is about 2300 Da. In some instances, the molecular weight of C is about 2400 Da. In some instances, the molecular weight, of C is about 2500 Da. In some instances, the molecular weight of C is about 2600 Da. In some instances, the molecular weight of C is about 2700 Da. In some instances, the molecular weight of C is about 2800 Da. In some instances, the molecular weight of C is about. 2900 Da. In some instances, the molecular weight of C is about 3000 Da. In some instances, the molecular weight of C is about 3250 Da. In some instances, the molecular weight of C is about 3350 Da. In some instances, the molecular weight of C is about 3500 Da. In some instances, the molecular weight of C is about 3750 Da. In some instances, the molecular weight of C is about 4000 Da, In some instances, the molecular weight of C is about 4250 Da. In some instances, the molecular weight of C is about 4500 Da. In some instances, the molecular weight of C is about 4600 Da. In some instances, the molecular weight of C is about 4750 Da. In some instances, the molecular weight of C is about 5000 Da. In some instances, the molecular weight of C is about 5500 Da. In some instances, the molecular weight of C is about 6000 Da. In some instances, the molecular weight of C is about 6500 Da. In some instances, the molecular weight of C is about 7000 Da. In some instances, the molecular weight of C is about 7500 Da. In some instances, the molecular weight of C is about 8000 Da. In some instances, the molecular weight of C is about 10,000 Da. In some instances, the molecular weight of C is about 12,000 Da. In some instances, the molecular weight of C is about 20,000 Da. In some instances, the molecular weight of C is about 35,000 Da. In some instances, the molecular weight, of C is about 40,000 Da. In some instances, the molecular weight of C is about 50,000 Da. In some instances, the molecular weight of C is about 60,000 Da. In some instances, the molecular weight of C is about 100,000 Da.

[0252] In some embodiments, the polyalkylene oxide (e.g., PEG) comprises discrete ethylene oxide units (e.g., four to about 48 ethylene oxide units). In some instances, the polyalkylene oxide comprising the discrete ethylene oxide units is a linear chain. In other cases, the poly alkylene oxide comprising the discrete ethylene oxide units is a branched chain.

[0253] In some instances, the polymer moiety C is a polyalkylene oxide (e.g., PEG) comprising discrete ethylene oxide units. In some cases, the polymer moiety C comprises between about 4 and about 48 ethylene oxide units. In some cases, the polymer moiety C comprises about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, or about 48 ethylene oxide units.

[0254] In some instances, the polymer moiety C is a discrete PEG comprising, e.g., between about 4 and about 48 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, or about 48 ethylene oxide units In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 4 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 5 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 6 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 7 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 8 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 9 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g, about 10 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about I I ethylene oxide units In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 12 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 13 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 14 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 15 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 16 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 17 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 18 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 19 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 20 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 21 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 22 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 23 ethylene oxide units In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 24 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g, about 25 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 26 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 27 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 28 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 29 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 30 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 31 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 32 ethylene oxide units In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 33 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 34 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 35 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 36 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 37 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g, about 38 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 39 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, eg., about 40 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 41 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 42 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 43 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 44 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 45 ethylene oxide units In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 46 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 47 ethylene oxide units. In some cases, the polymer moiety C is a discrete PEG comprising, e.g., about 48 ethylene oxide units

[0255] In some cases, the polymer moiety C is dPEG® (Quanta Biodesign Ltd). In some embodiments, the polymer moiety C comprises a cationic mucic acid-based polymer (cMAP). In some instances, cMAP comprises one or more subunit of at least one repeating subunit, and the subunit structure is represented as Formula (V):

[0256]

[0257] Formula V

[0258] wherein m is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, optionally 4-6 or 5; and n is independently at each occurrence 1, 2, 3, 4, or 5. In some embodiments, m and n are, for example, about 10.

[0259] In some instances, cMAP is further conjugated to a PEG moiety, generating a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer In some instances, the PEG moiety is in a range of from about 500 Da to about 50,000 Da. In some instances, the PEG moiety is in a range of from about 500 Da to about 1000 Da, greater than 1000 Da to about 5000 Da, greater than 5000 Da to about 10,000 Da, greater than 10,000 to about 25,000 Da, greater than 25,000 Da to about 50,000 Da, or any combination of two or more of these ranges In some instances, the polymer moiety C is cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some cases, the polymer moiety C is cMAP-PEG copolymer. In other cases, the polymer moiety C is an mPEG-cMAP-PEGm triblock polymer. In additional cases, the polymer moiety C is a cMAP-PEG-cMAP triblock polymer.

[0260] In some embodiments, the polymer moiety C is conjugated to the oligonucleotide, the binding moiety, and optionally to the endosomolytic moiety as illustrated supra. Endosomolytic or Cell Membrane Penetration Moiety

[0261] In some embodiments, a molecule of Formula (I): A-X₁-B-X₂-C, further comprises an additional conjugating moiety. In some instances, the additional conjugating moiety is an endosomolytic moiety and / or a cell membrane penetration moiety. In some cases, the endosomolytic moiety is a cellular compartmental release component, such as a compound capable of releasing from any of the cellular compartments known in the art, such as the endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubule, peroxisome, or other vesicular bodies with the cell. In some cases, the endosomolytic moiety comprises an endosomolytic polypeptide, an endosomolytic polymer, an endosomolytic lipid, or an endosomolytic small molecule In some cases, the endosomolytic moiety comprises an endosomolytic polypeptide. In other cases, the endosomolytic moiety comprises an endosomolytic polymer. In some cases, the cell membrane penetration moiety comprises a cell penetrating peptide (CPP). In other cases, the cell membrane penetration moiety comprises a cell penetrating lipid. In other cases, the cell membrane penetration moiety comprises a cell penetrating small molecule.

[0262] In some embodiments, the endosomolytic moiety comprises a sequence as illustrated in Table 8. In some instances, the endosomolytic moiety is as described in U. S. Patent No.

[0263] 11,555, 190.

[0264] Table 8

[0265]

[0266]

[0267] In some embodiments, a cell penetrating polypeptide of the disclosure comprises positively charged short peptides with 5-30 amino acids. In some embodiments, a cell penetrating polypeptide comprises arginine or lysine rich amino acid sequences. In some embodiments, a cell penetrating polypeptide includes any polypeptide or combination thereof listed in Table 9.

[0268] Table 9

[0269]

[0270] Endosomolytic and Cell Membrane Penetration Polypeptides

[0271] In some embodiments, a molecule of Formula (I): A-X₁-B-X₂-C, is further conjugated with an endosomolytic polypeptide. In some cases, the endosomolytic polypeptide is a pH-dependent membrane active peptide. In some cases, the endosomolytic polypeptide is an amphipathic polypeptide. In additional cases, the endosomolytic polypeptide is a peptidomimetic. In some instances, the endosomolytic polypeptide comprises INF, melittin, meucin, or their respective derivatives thereof. In some instances, the endosomolytic polypeptide comprises INF or its derivatives thereof. In other cases, the endosomolytic polypeptide comprises melittin or its derivatives thereof. In additional cases, the endosomolytic polypeptide comprises meucin or its derivatives thereof.

[0272] In some cases, the endosomolytic moiety comprises a BakBH3 polypeptide which induces apoptosis through antagonization of suppressor targets such as Bcl-2 and / or BC1-XL. In some instances, the endosomolytic moiety comprises a Bak BH3 polypeptide described in Albarran, et al., "Efficient intracellular delivery of a pro-apoptotic peptide with a pH-responsive carrier," Reactive & Functional Polymers 71: 261-265 (2011).

[0273] In some instances, the endosomolytic moiety comprises a polypeptide (e.g, a cellpenetrating polypeptide) as described in PCT Publication Nos WO2013 / 166155 or WO2015 / 069587.

[0274] Endosomolytic Lipids

[0275] In some embodiments, the endosomolytic moiety is a lipid (e.g., a fusogenic lipid). In some embodiments, a molecule of Formula (I): A-X₁-B-X₂-C, is further conjugated with an endosomolytic lipid (e.g., fusogenic lipid). Exemplary fusogenic lipids include 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k-DMA) and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (XTC). In some instances, an endosomolytic moiety is a lipid (e.g., a fusogenic lipid) described in PCT Publication No. WO09 / 126,933.

[0276] Endosomolytic Small Molecules

[0277] In some embodiments, the endosomolytic moiety is a small molecule. In some embodiments, a molecule of Formula (I): A-X₁-B-X₂-C, is further conjugated with an endosomolytic small molecule. Exemplary small molecules suitable as endosomolytic moieties include, but are not limited to, quinine, chloroquine, hydroxychloroquines, amodiaquins (carnoquines), amopyroquines, primaquines, mefloquines, nivaquines, halofantrines, quinone imines, or a combination thereof. In some instances, quinoline endosomolytic moieties include. but are not limited to, 7-chloro-4-(4-diethylamino-l-methylbutyl-amino)quinoline (chloroquine); 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-l-methylbutyl-amino)quinoline (hydroxychloroquine); 7-fluoro-4-(4-diethylamino-l-methylbutyl-amino)quinoline; 4-(4-diethylamino-l-methylbutylamino)quinoline; 7-hydroxy-4-(4-diethyl-amino-1-methylbutylamino)quinoline; 7-chloro-4-(4-diethylamino-1-butylamino)quinoline (desmethylchloroquine), 7-fluoro-4-(4-diethylamino-1-butylamino)quinoline; 4-(4-di ethyl-amino-l-butylamino)quinoline; 7-hydroxy-4-(4-diethylamino-l-butylamino)quinoline; 7-chloro-4-(l-carboxy-4-diethylamino-l-butylamino)quinoline; 7-fluoro-4-(l -carboxy -4-di ethyl-amino- 1 -butyl aminojquinoline; 4-(l-carboxy-4-diethylamino-l-butylamino) quinoline, 7-hydroxy-4-(l-carboxy-4-diethylamino-l-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline; 7-fluoro-4-(1-carboxy-4-diethyl-amino-1-methylbutylamino)quinoline; 4-(l-carboxy-4-diethylamino-l-methyl butyl amino)quinoline; 7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline; 7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-l-methylbutylamino)quinoline; 4-(4-ethyl-(2-hydroxy-ethyl)-amino-1-methylbutylamino)quinoline, 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-l-methylbutylamino)quinoline;

[0278] hydroxy chloroquine phosphate; 7-chl oro-4-(4-ethyl-(2-hydroxyethyl-l)-amino-l-butylamino)quinoline (desmethylhydroxychloroquine); 7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-l-butylamino)quinoline; 4-(4-ethyl -(2 -hydroxy ethyl)-amino-l-butylamino)quinoline; 7-hydroxy -4-(4-ethyl-(2-hydroxyethyl)-amino-l-butylamino) quinoline; 7-chloro-4-(l-carboxy-4-ethyl-(2-hydroxyethyl)-aminO”l-butylamino)quinoline; 7-fluoro-4-(l-carboxy-4-ethyl-(2-hydroxy ethyl )-amino-l-butylamino)quinoline; 4-(l-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1 -butylamino)quinoline; 7-hydroxy-4-(l-carboxy-4-ethyl-(2-hydroxyethyl)-amino- 1 -butylamino)quinoline; 7-chloro-4-(l-carboxy-4-ethyl-(2-hydroxyethyl)-amino-l-methylbutylamino)quinoline; 7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 8-[(4-aminopentyl)amino-6-methoxy dihydrochloride quinoline; 1-acetyl-l,2,3,4-tetrahydroquinoline; 8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride; 1-butyryl-1,2,3,4-tetrahydroquinoline; 3-chloro-4-(4-hydroxy-alpha,alpha'-bis(2-methyl-l-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethyl-amino)-l-methylbutyl-amino]-6-methoxyquinoline; 3-fluoro-4-(4-hydroxy-alpha,alpha'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethylamino)-l-methylbutyl-amino]-6-methoxy quinoline; 4-(4-hydroxy-alpha, alpha' -bis(2-methyl-l-pyrrolidinyl)-2,5-xylidinoquinoline; 4-[(4-diethylamino)-l-methylbutyl-amino]-6-methoxy quinoline; 3,4-dihydro-1-(2H)-quinolinecarboxaldehyde, 1,1'-pentamethylene diquinolinium diiodide; 8-quinolinol sulfate and amino, aldehyde, carboxylic, hydroxyl, halogen, keto, sulfhydryl and vinyl derivatives or analogs thereof. In some instances, an endosomolytic moiety is a small molecule described in Naisbitt et al. (1997, J Pharmacol Exp Therapy 280:884-893) and in U. S. Patent No. 5,736,557

[0279] Cell Penetrating Polypeptide (CPP)

[0280] In some embodiments, a cell penetrating polypeptide comprises positively charged short peptides with 5-30 amino acids. In some embodiments, a cell penetrating polypeptide comprises arginine or lysine rich amino acid sequences.

[0281] In some embodiments, a linker described herein is a cleavable linker or a non-cleavable linker. In some instances, the linker is a cleavable linker In other instances, the linker is a non-cleavable linker.

[0282] In some cases, the linker is a non-polymeric linker. A non-polymeric linker refers to a linker that does not contain a repeating unit of monomers generated by a polymerization process Exemplary non-polymeric linkers include, but are not limited to, C₁-C₅ alkyl group (e.g., a C₅, C₄, C₃, C₂, or C₁ alkyl group), homobifunctional cross linkers, heterobifunctional cross linkers, peptide linkers, traceless linkers, self-immolative linkers, maleimide-based linkers, or combinations thereof. In some cases, the non-polymeric linker comprises a Ci-C& alkyl group (e.g., a Cs, C4, C3, C2, or Ci alkyl group), a homobifunctional cross linker, a heterobifunctional cross linker, a peptide linker, a traceless linker, a self-immolative linker, a maleimide-based linker, or a combination thereof. In additional cases, the non-polymeric linker does not comprise more than two of the same type of linkers, e.g., more than two homobifunctional cross linkers, or more than two peptide linkers. In further cases, the non-polymeric linker optionally comprises one or more reactive functional groups. In some instances, the non-polymeric linker does not encompass a polymer that is described above. In some instances, the non-polymeric linker does not encompass a polymer encompassed by the polymer moiety C. In some cases, the non-poly meric linker does not encompass a polyalkylene oxide (e.g., PEG). In some cases, the non-polymeric linker does not encompass a PEG.

[0283] In some instances, the linker comprises a homobifunctional linker. Exemplary homobifunctional linkers include, but are not limited to, Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3’3'-dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS³), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG), N, N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'~dinitrophenylsulfone (DFDNPS), bis-[P-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid di hydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, a,a'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N, N'-ethylene-bis(iodoacetamide), or N, N'-hexamethylene-bis(iodoacetamide).

[0284] In some embodiments, the linker comprises a heterobifunctional linker. Exemplary heterobifunctional linker include, but are not limited to, amine-reactive and sulfhydryl cross linkers such as N-succinimidyl 3 -(2-pyri dyl dithi o)propi onate (sPDP), long-chain N-succinimidyl-3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[a-methyl-a-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-l -carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1 -carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxy succinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs), N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB), sulfosuccinimidyl(4-iodoacteyl)ami nobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB),sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(y-maleimidobutyiyloxy)succinimide ester (GMBs), N-(y-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-l -carboxylate (sIAC), succinimidyl 6-((((4-iodoacetyl)amino)methyl)cyclohexane-l-carbonyl)amino) hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl -reactive and sulfhydryl-reactive cross-linkers such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-l-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive and photoreactive cross-linkers such as N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-l,3'-dithiopropi onate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4‘-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl -6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o~nitrobenzarnido)-ethyl-l,3'-dithiopropi onate (sAND), N-succinimidyl-4(4-azidophenyl)l,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfo-sPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-l,3'-dithiopropi onate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive cross-linkers such as 1-(p-Azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-mal eimide carbonyl -reactive and photoreactive cross-linkers such as p-azidobenzoyl hydrazide (ABH), carboxylate-reactive and photoreactive cross-linkers such as 4-(p-azidosalicylamido)butylamine (sBA), and arginine-reactive and photoreactive cross-linkers such as p-azidophenyl glyoxal (APG).

[0285] In some instances, the linker comprises a reactive functional group In some cases, the reactive functional group comprises a nucleophilic group that is reactive to an electrophilic group present on a binding moiety. Exemplary electrophilic groups include carbonyl groups — such as aldehyde, ketone, carboxylic acid, ester, amide, enone, acyl halide or acid anhydride. In some embodiments, the reactive functional group is aldehyde. Exemplary nucleophilic groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0286] In some embodiments, the linker comprises a maleimide group. In some instances, the maleimide group is also referred to as a maleimide spacer. In some instances, the maleimide group further encompasses a caproic acid, forming maleimidocaproyl (me). In some cases, the linker comprises maleimidocaproyl (me). In some cases, the linker is maleimidocaproyl (me).

[0287] In other instances, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-l -carboxylate (sulfo-sMCC) described above.

[0288] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some instances, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis of tiosuccinimide ring hydrolysis, thereby eliminating maleimide from undergoing an elimination reaction through a retro-Michael reaction. In some instances, the self-stabilizing maleimide is a maleimide group described in Lyon et al., "Self-hydrolyzing mal eimides improve the stability and pharmacological properties of antibody-drug conjugates," Nat. Biotechnol. 32(10): 1059-1062 (2014). In some instances, the linker comprises a self-stabilizing maleimide. In some instances, the linker is a selfstabilizing mal eimi de.

[0289] In some embodiments, the linker comprises a peptide moiety. In some instances, the peptide moiety comprises at least 2, 3, 4, 5, or 6 more amino acid residues. In some instances, the peptide moiety comprises at most 2, 3, 4, 5, 6, 7. or 8 amino acid residues. In some instances, the peptide moiety comprises about 2, about 3, about 4, about 5, or about 6 amino acid residues. In some instances, the peptide moiety is a cleavable peptide moiety (e.g., either enzymatically or chemically) In some instances, the peptide moiety is a non-cleavable peptide moiety In some instances, the peptide moiety comprises Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some instances, the linker comprises a peptide moiety such as: Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala- Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some cases, the linker comprises Val-Cit. In some cases, the linker is Val-Cit.

[0290] In some embodiments, the linker comprises a benzoic acid group, or its derivatives thereof. In some instances, the benzoic acid group or its derivatives thereof comprise paraaminobenzoic acid (PABA). In some instances, the benzoic acid group or its derivatives thereof comprise gamma-aminobutyric acid (GABA).

[0291] In some embodiments, the linker comprises one or more of a maleimide group, a peptide moiety, and / or a benzoic acid group, in any combination. In some embodiments, the linker comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group.

[0292] In some instances, the maleimide group is maleimidocaproyl (me). In some instances, the peptide group is val-cit. In some instances, the benzoic acid group is PABA. In some instances, the linker comprises a mc-val-cit group. In some cases, the linker comprises a val-cit-PAB A group In additional cases, the linker comprises a mc-val-cit-PABA group.

[0293] In some embodiments, the linker is a self-immolative linker or a self-elimination linker. In some cases, the linker is a self-immolative linker. In other cases, the linker is a self-elimination linker (e.g., a cyclization self-elimination linker). In some instances, the linker comprises a linker described in U S Patent No 9,089,614 or PCT Publication No. WO2015038426.

[0294] In some embodiments, the linker is a dendritic type linker. In some instances, the dendritic type linker comprises a branching, multifunctional linker moiety. In some instances, the dendritic type linker is used to increase the molar ratio of polynucleotide B to the binding moiety A. In some instances, the dendritic type linker comprises PAMAM dendrimers.

[0295] In some embodiments, the linker is a traceless linker or a linker in which after cleavage does not leave behind a linker moiety (e.g., an atom or a linker group) to a binding moiety A, a polynucleotide B, a polymer C, or an endosomolytic moiety D. Exemplary traceless linkers include, but are not limited to, germanium linkers, silicium linkers, sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers, boron linkers, chromium linkers, or phenylhydrazide linker In some cases, the linker is a traceless aryl-triazene linker as described in Hejesen, et al., "A traceless aryl-triazene linker for DNA-directed chemistry," Org Biomol Chem 11(15): 2493-2497 (2013). In some instances, the linker is a traceless linker described in Blaney, et al., "Traceless solid-phase organic synthesis," Chem. Rev. 102: 2607-2024 (2.002). In some instances, a linker is a traceless linker as described in U. S. Patent No. 6,821,783. In some instances, the linker is a linker described in U. S. Patent Nos. 6,884,869; 7,498,298; 8,288,352; 8,609,105; or 8,697,688; U. S. Patent Publication Nos. 2014 / 0127239; 2013 / 028919; 2014 / 286970; 2013 / 0309256; 2015 / 037360; or 2014 / 0294851; or PCT Publication Nos WO2015057699; W02014080251; WO2014197854; W02014145090; or WO2014177042.

[0296] In some embodiments, Xi and X2 are each independently a bond or a non-polymeric linker. In some instances, Xi and X2 are each independently a bond. In some cases, Xi and X2 are each independently a non-polymeric linker.

[0297] In some instances, Xi is a bond or a non-polymeric linker. In some instances, Xi is a bond In some instances, Xi is a non-polymeric linker. In some instances, the linker is a C1-C6 alkyl group. In some cases, Xi is a C1-C6 alkyl group, such as for example, a C, C4, C3, C2, or Ci alkyl group. In some cases, the C1-C6 alkyl group is an unsubstituted C1-C6 alkyl group As used in the context of a linker, and in particular in the context of Xi, alkyl means a saturated straight or branched hydrocarbon radical containing up to six carbon atoms. In some instances, Xi includes a homobifunctional linker or a heterobifunctional linker described supra. In some cases, Xi includes a heterobifunctional linker. In some cases, Xi includes sMCC, In other instances, Xi includes a heterobifunctional linker optionally conjugated to a Ci-Cs alkyl group.

[0298] In other instances, Xi includes sMCC optionally conjugated to a C1-C6 alkyl group. In additional instances, Xi does not include a homobifunctional linker or a heterobifunctional linker described supra.

[0299] In some instances, X2 is a bond or a linker. In some instances, X2 is a bond. In other cases, X2 is a linker. In additional cases, X2 is a non-polymeric linker. In some embodiments, X2 is a C1-C6 alkyl group. In some instances, X2 is a homobifunctional linker or a heterobifunctional linker described supra. In some instances, X2 is a homobifunctional linker described supra. In some instances, X2 is a heterobifunctional linker described supra. In some instances, X2 comprises a maleimide group, such as maleimidocaproyl (me) or a self-stabilizing maleimide group described above. Tn some instances, X2 comprises a peptide moiety, such as Val-Cit. In some instances, X2 comprises a benzoic acid group, such as PABA, In additional instances, X2 comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In additional instances, X2 comprises a me group. In additional instances, X2 comprises a mc-val-cit group. In additional instances, X2 comprises a val-cit-PABA group. In additional instances, X2 comprises a mc-val-cit-PABA group. Methods of Use

[0300] A hemoglobinopathy is a disease or disorder characterized by one or more mutation(s) in the genome that results in abnormal structure of one or more of the globin chains of the hemoglobin molecule. Exemplary hemoglobinopathies include hemolytic anemia, sickle cell disease, and thalassemia. Sickle cell disease is characterized by the presence of abnormal, sickle-shaped hemoglobins, which can result in severe infections, severe pain, stroke, and an increased risk of death. Subjects having sickle cell disease can be identified, e.g., using one or more of a complete blood count, a blood film, hemoglobin electrophoresis, and genetic testing. Thalassemias are a group of autosomal recessive diseases characterized by a reduction in the amount of hemoglobin produced. Symptoms include iron overload, infection, bone deformities, enlarged spleen, and cardiac disease. The subgroups of thalassemias include alpha-thalassemia, beta-thalassemia, and delta thalassemia. Additional hemoglobinopathies such as hemophilia A and hemophilia B may be treated using any of the particles, vectors, virions, expression systems, compositions, and host cells described herein.

[0301] In some embodiments, described herein is a method of treating a hemoglobinopathy in a subject, which comprises providing oligonucleotide described herein and administering to the subject a therapeutically effective amount of an oligonucleotide described herein or aoligonucleotide antibody -oligonucleotide conjugate described herein to reduces a quantity of the mRNA transcript of human BCL11A In some instances, the hemoglobinopathy is Sickle Cell Disease (SCD). The nucleic acid moiety mediates RNA interference against the human BCL11A as to modulating fetal hemoglobin expression in a subject. In some embodiments, expression of one or more marker genes that are affected by BCL11A expression is also altered or modulated (e.g., decreased) by the decreased expression of human BCL11A.

[0302] In some embodiments, described herein is a method of treating a hemoglobinopathy in a subject, which comprises providing an siRNA antibody conjugate described herein and administering to the subject a therapeutically effective amount of the siRNA antibody conjugate described herein and reducing the levels of mRNA transcript of human BCL11 A in said subject.

[0303] In some instances, the hemoglobinopathy is SCD. The siRNA antibody conjugate mediates RNA interference against the human BCL 1 1 A mRNA as to treat hemoglobinopathy in the subject, which comprises administering to the subject a therapeutically effective amount of the siRNA antibody conjugate described herein and reducing the levels of mRNA transcript of human BCLI 1 A in said subject

[0304] In some embodiments, described herein is a method of treating hemoglobinopathyin a subject, which comprises providing a BCL11A siRNA antibody conjugate (BC-L11A siRNAconjugate or BCL I I A -AOC) described herein and administering to the subject a therapeutically effective amount of the BCL1 IA siRNA antibody conjugate described herein and reducing the levels of mRNA transcript of human BCLI 1 A in said subject. In some instances, the hemoglobinopathy is SCD. The BCLI 1 A siRNA antibody conjugate mediates RNA interference against the human BCL11A mRNA as to treat hemoglobinopathy in the subject, which comprises administering to the subject a therapeutically effective amount of the BCLI 1 A siRNA antibody conjugate described herein and reducing the levels of mRNA transcript of human BCLI I A in said subject.

[0305] In some embodiments, described herein is a method of treating SCD in a subject, which comprises providing a BCLI 1 A siRNA antibody conjugate (BCLI 1 siRNA conjugate or BCLHA-AOC) described herein and administering to the subject a therapeutically effective amount of the BCL11A siRNA antibody conjugate described herein and reducing the levels of mRNA transcript of human BCLI 1 A in said subject. The BCLI 1 A siRNA antibody conjugate mediates RNA interference against the human BCLI 1 A mRNA as to treat SCD in the subject, which comprises administering to the subject a therapeutically effective amount of the BCLI 1 A siRNA-conjugate described herein and reducing the levels of mRNA transcript of human BCLI 1 A in said subject In some embodiments, expression levels of one or more marker genes that are affected by BCL I 1 A expression are also altered or modulated by the decreased expression levels of human BCL 11 A.

[0306] In some embodiments, provided herein is a method of alleviating symptoms in a subject with SCD, which involves providing a BCLI 1 A siRNA -anti body conjugate (BCLI 1 A siRNA conjugate or BCLI 1 A AOC) described herein and administering to the subject a therapeutically effective amount of the si NA conjugate described herein by reducing the levels of mRNA transcript of human BCL I 1 A. In another embodiments, described herein is a method of allevi ating symptoms in a SCD patient, which comprises providing an siRNA conjugate described herein and administering to the SCD patient a therapeutically effective amount of the siRNA conjugate described herein by reducing the levels of mRNA transcript of human BCLIIA or reducing the levels of B CL I I A protein.

[0307] In some embodiments, described herein is a method of treating SCD in a subject, which involves providing an antisense oligonucleotide (ASO) antibody conjugate (ASO conjugate) described herein and administering to the subject a therapeutically effective amount of the ASO-antibody conjugate described herein and reducing the levels of mRNA transcript of human BCLIIA in said subject. The ASO-conjugate mediates RNA interference against the human BCLIIA mRNA as to treat SCD in the subject, which comprises administering to the subject a therapeutically effective amount of the ASO-antibody conjugate described herein and reducing the levels of mRNA transcript of human BCL11 A in said subject In some embodiments, expression levels of one or more marker genes that are affected by BCLIIA expression is also altered or modulated by the decreased expression levels of human BCLIIA.

[0308] Pharmaceutical Formulation

[0309] In some embodiments, the pharmaceutical formulations described herein are administered to a subject by multiple administration routes, including but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal administration routes. In some instances, the pharmaceutical composition describe herein is formulated for parenteral (e.g.,, intravenous, subcutaneous, intramuscular, intra-arterial, intraperitoneal, intrathecal, intracerebral, intracerebroventricular, or intracranial) admini tration. In other instances, the pharmaceutical composition describe herein is formulated for oral administration. In still other instances, the pharmaceutical composition describe herein is formulated for intranasal administration.

[0310] In some embodiments, the pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate and controlled release formulations.

[0311] In some instances, the pharmaceutical formulation includes multiparticulate formulations. In some instances, the pharmaceutical formulation includes nanoparticle formulations. In some instances, nanoparticles comprise cMAP, cyclodextrin, or lipids. In some cases, nanoparticles comprise solid lipid nanoparticles, polymeric nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micellar solutions. Additional exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as with covalently attached metal chelates), nanofibers, nanohoms, nano-onions, nanorods, nanoropes and quantum dots. In some instances, a nanoparticle is a metal nanoparticle, e.g., a nanoparticle of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium, lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium, potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, and combinations, alloys or oxides thereof.

[0312] In some instances, a nanoparticle includes a core or a core and a shell, as in a core-shell nanoparticle

[0313] In some instances, a nanoparticle is further coated with molecules for attachment of functional elements (e.g., with one or more of an oligonucleotide or binding moiety described herein). In some instances, a coating comprises chondroitin sulfate, dextran sulfate, carboxymethyl dextran, alginic acid, pectin, carragheenan, fucoidan, agaropectin, porphyran, karaya gum, gellan gum, xanthan gum, hyaluronic acids, glucosamine, galactosamine, chitin (or chitosan), polyglutamic acid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, a-chymotrypsin, polylysine, polyarginine, histone, protamine, ovalbumin or dextrin or cyclodextrin. In some instances, a nanoparticle comprises a graphene-coated nanoparticle

[0314] In some cases, a nanoparticle has at least one dimension of less than about 500nm, 400nm, 300nm, 200nm, or lOOnm.

[0315] In some instances, the nanoparticle formulation comprises paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as with covalently attached metal chelates), nanofibers, nanohoms, nano-onions, nanorods, nanoropes or quantum dots. In some instances, an oligonucleotide or a binding moiety described herein is conjugated either directly or indirectly to the nanoparticle. In some instances, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more oligonucleotides or binding moieties described herein are conjugated either directly or indirectly to a nanoparticle.

[0316] In some embodiments, the pharmaceutical formulation comprises a delivery vector, e.g., a recombinant vector, the delivery of the oligonucleotide into cells. In some instances, the recombinant vector is DNA plasmid In other instances, the recombinant vector is a viral vector. Exemplary viral vectors include vectors derived from adeno-associated virus, retrovirus, adenovirus, or alphavirus. In some instances, the recombinant vectors capable of expressing the oligonucleotides provide stable expression in target cells In additional instances, viral vectors are used that provide for transient expression of oligonucleotides.

[0317] In some embodiments, the pharmaceutical formulation includes a carrier or carrier materials selected on the basis of compatibility with the composition disclosed herein, and the release profile properties of the desired dosage form. Exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, e.g.. Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N. Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0318] In some instances, the pharmaceutical formulation further includes pH adjusting agents or buffering agents which include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range. In some instances, the pharmaceutical formulation includes one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0319] In some instances, the pharmaceutical formulation further includes diluent which are used to stabilize compounds because they provide a more stable environment. Salts dissolved in buffered solutions (which also provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution. In certain instances, diluents increase bulk of the composition to facilitate compression or create sufficient bulk for homogenous blend for capsule filling. Such compounds include e.g., lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®; dibasic calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugar, such as Di-Pac® (Am star); mannitol, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents, confectioner’s sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate tri hydrate, dextrates; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

[0320] In some cases, the pharmaceutical formulation includes disintegration agents or disintegrants to facilitate the breakup or disintegration of a substance. The term "disintegrate" include both the dissolution and dispersion of the dosage form when contacted with gastrointestinal fluid. Examples of disintegration agents include a starch, e.g., a natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551 or Amijel®, or sodium starch glycolate such as Promogel® or Explotab®, a cellulose such as a wood product, methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel®PH102, Avicel® PH105, Elcema* P100, Eracocel®, Vivacel®, Ming Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or a crosslinked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linked croscarmellose, a cross-linked starch such as sodium starch glycolate, a cross-linked polymer such as crospovidone, a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate, a clay such as V eegum® HV (magnesium aluminum silicate), a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, a natural sponge, a surfactant, a resin such as a cation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination starch, and the like.

[0321] In some instances, the pharmaceutical formulation includes filling agents such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like

[0322] Lubricants and glidants are also optionally included in the pharmaceutical formulations described herein for preventing, reducing or inhibiting adhesion or friction of materials Exemplary lubricants include, e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a methoxypoly ethylene glycol such as Carbowax™, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil®, a starch such as corn starch, silicone oil, a surfactant, and the like.

[0323] Plasticizers include compounds used to soften the microencapsulation material or film coatings to make them less brittle. Suitable plasticizers include, e.g., polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethyl cellulose and triacetin. Plasticizers also function as dispersing agents or wetting agents

[0324] Solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxy ethyl pyrrolidone, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide and the like. Stabilizers include compounds such as any antioxidation agents, buffers, acids, preservatives and the like.

[0325] Suspending agents include compounds such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinyl pyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol, e.g., the polyethylene glycol has a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxy ethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like.

[0326] Surfactants include compounds such as sodium lauryl sulfate, sodium docusate. Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Plutonic® (BASF), and the like. Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil, and polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40 Sometimes, surfactants is included to enhance physical stability or for other purposes.

[0327] Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof.

[0328] Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts and the like.

[0329] Therapeutic Regimens In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic applications. In some embodiments, the pharmaceutical composition is administered once per day, twice per day, three times per day or more. The pharmaceutical composition is administered daily, every day, every alternate day, five days a week, once a week, every other week, two weeks per month, three weeks per month, once a month, twice a month, three times per month, once in two months, once in three months, once in four months, once in five months, once in six months or more The pharmaceutical composition is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.

[0330] In some embodiments, one or more pharmaceutical compositions are administered simultaneously, sequentially, or at an interval period of time. In some embodiments, one or more pharmaceutical compositions are administered simultaneously. In some cases, one or more pharmaceutical compositions are administered sequentially. In additional cases, one or more pharmaceutical compositions are administered at an interval period of time (e.g., the first administration of a first pharmaceutical composition is on day one followed by an interval of at least 1, 2, 3, 4, 5, or more days prior to the administration of at least a second pharmaceutical composition).

[0331] In some embodiments, two or more different pharmaceutical compositions are co administered. In some instances, the two or more different pharmaceutical compositions are co administered simultaneously. In some cases, the two or more different pharmaceutical compositions are co-administered sequentially without a gap of time between administrations.

[0332] In other cases, the two or more different pharmaceutical compositions are co-administered sequentially with a gap of about 0.5 hour, 1 hour, 2 hour, 3 hour, 12 hours, 1 day, 2 days, or more between administrations.

[0333] In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the composition is given continuously; alternatively, the dose of the composition being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). In some instances, the length of the drug holiday varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holi day is from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained.

[0334] In some embodiments, the amount of a given agent that correspond to such an amount varies depending upon factors such as the particular compound, the severity of the disease, the identity (e.g., weight) of the subject or host in need of treatment, but nevertheless is routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, and the subject or host being treated. In some instances, the desired dose is conveniently presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

[0335] The foregoing ranges are merely suggestive, as the number of variables in regard to an individual treatment regime is large, and considerable excursions from these recommended values are not uncommon. Such dosages is altered depending on a number of variables, not limited to the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0336] In some embodiments, toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio between LD50 and ED50, Compounds exhibiting high therapeutic indices are preferred The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for use in human. The dosage of such compounds lies optionally within a range of circulating concentrations that include the ED50 with minimal toxicity The dosage varies within this range depending upon the dosage form employed and the route of administration utilized. Kits / Article of Manufacture

[0337] Disclosed herein, in certain embodiments, are kits and articles of manufacture for use with one or more of the compositions and methods described herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic.

[0338] The articles of manufacture provided herein contain packaging materials Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

[0339] For example, the container(s) include target nucleic acid molecule described herein. Such kits optionally include an identifying description or label or instructions relating to its use in the methods described herein.

[0340] A kit typically includes labels listing contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[0341] In one embodiment, a label is on or associated with the container. In one embodiment, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.

[0342] In certain embodiments, the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein. The pack, for example, contains metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is also accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U S Food and Drug Administration for prescription drugs, or the approved product insert In one embodiment, compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0343] Certain Terminology

[0344] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and '’the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, use of the term "including" as well as other forms, such as "include", "includes," and "included," is not limiting.

[0345] As used herein, ranges and amounts can be expressed as "about" a particular value or range. About also includes the exact amount. Hence "about 5 pL" means "about 5 pL" and also "5 pL." Generally, the term "about" includes an amount that would be expected to be within experimental error.

[0346] The section headings used herein are for organizational purposes only and are not to be constmed as limiting the subject matter described

[0347] As used herein, the terms "individual(s)", "subject(s)" and "patient(s)" mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a nonhuman. None of the terms require or are limited to situations characterized by the supervision (e.g., constant or intermittent) of a health care worker (eg., a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly or a hospice worker).

[0348] The term "therapeutically effective amount" relates to an amount of an oligonucleotide antibody-oligonucleotide conjugate that is sufficient to provide a desired therapeutic effect in a mammalian subject. In some cases, the amount is single or multiple dose administration to a patient (such as a human) for treating, preventing, preventing the onset of, curing, delaying, reducing the severity of, ameliorating at least one symptom of a disorder or recurring disorder, or prolonging the survival of the patient beyond that expected in the absence of such treatment. Naturally, dosage levels of the particular oligonucleotide antibody-oligonucleotide conjugate employed to provide a therapeutically effective amount vary in dependence of the type of injury, the age, the weight, the gender, the medical condition of the subject, the severity of the condition, the route of administration, and the particular inhibitor employed. In some instances, therapeutically effective amounts of oligonucleotide antibody-oligonucleotide conjugate, as described herein, is estimated initially from cell culture and animal models. For example, IC50 values determined in cell culture methods optionally serve as a starting point in animal models, while IC50 values determined in animal models are optionally used to find a therapeutically effective dose in humans

[0349] The term antibody oligonucleotide conjugate (AOC) refers to an antibody conjugated to a nucleotide.

[0350] The term siRNA conjugate or siRNA antibody conjugate refers to an antibody conjugated to a siRNA.

[0351] The term BCL11A -AOC refers to an antibody conjugated to an siRNA hybridizing to a target sequence of the human BCL11A mRNA.

[0352] EXAMPLES

[0353] Example 1. siRNAs designed to Target Human Full-Length BCL11A mRNA Showed Robust HbF Induction in Contacted Primary Human CD34+ Cells Differentiated Along the Erythroid Lineage

[0354] Sequences of siRNAs potentially capable of RISC-mediated inhibition of BCL11A mRNA (NCBI_Gene: 53335; NM_022893), or a pre-determined region of the BCL11A gene, were collected to generate a starting set of BCL1 lA-targeting siRNAs, for lead siRNA screeni ng / evaluati on.

[0355] Table 10 shows selected siRNAs possessing no (0) mismatches to NM_022893, Table 11 shows selected siRNAs having three (3) nucleotide mismatches to NM_022893. Table 12 shows selected siRNAs having two (2) nucleotide mismatches to NM_022893. Table 13 shows selected siRNAs having one (1) nucleotide mismatches to NM 022893. Table 10: siRNAs matching NM_022893

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364] Table 11: siRNA having 3 nt mismatch to NM_022893

[0365]

[0366]

[0367]

[0368]

[0369] Table 12: siRNA having 2 nt mismatch to NM_022893

[0370]

[0371] Table 13: siRNA having 1 nt mismatch to NM_022893

[0372]

[0373] From the above set of candidate BCL11 A-targeting siRNAs, six BCL11 A-targeting siRNAs - WD12195609, WD12195617, WD12195621, WD12195625, WD12195629, and WD12195635 - were selected for initial evaluation of induction of HbF fold change, in CL)'-4 primary erythroid cells (HbF induction serving as a functionally and potentially therapeutically relevant proxy for BCL11A knockdown). Sequences of the six initially tested siRNAs are shown in Table 14, and evaluation of relative HbF induction in response to administration of these tested BCL11A-targeting siRNAs to primary human CD34+ cells differentiated along the erythroid lineage was performed using the materials and methods detailed in Example 2 below. HbF fold change results obtained for each respective siRNA are shown in Table 15.

[0374] Table 14: Tested BCL11A-Targeting siRNAs

[0375]

[0376]

[0377] Table 15: Observed HbF Fold Induction for Tested BCL11A-Targeting siRNAs

[0378]

[0379] Each of the tested BCL11A-targeting siRNAs exhibited robust and reproducible HbF induction, demonstrating the viability of these siRNAs for use in antibody oligonucleotide conjugates (AOCs).

[0380] Example 2. Custom BCL11A siRNA Screening in CD34+ Cells -- Materials and Methods Materials

[0381] ® TrueCut Cas9 Protein v2 (Thermo; A36499)

[0382] * Amaxa P3 Primary Cell 4D-Nucleofector X Kit L (Lonza, V4XP-3024)

[0383] ® BCL11A siRNA positive control (Thermo, s28682)

[0384] * Negative control siRNA (Invitrogen, 4390844)

[0385] « Custom siRNA (Sigma, Sales order 3035186500, Ref 50994, Anindita Basak)

[0386] * P3 Primary cell Nucleofector Solution LV set (Lonza Bioscience, V4LP3-22500) » Phase I and Phase II media for CD34+ primary erythroid cells (listed in below Table 16 and Table 17)

[0387] Table 16. Phase I Media:

[0388]

[0389]

[0390] Table 17. Phase II Media:

[0391]

[0392] Screening Methods - Cellular Assay

[0393] Day 0

[0394] 1. Thaw a vial of Human Mobilized Peripheral Blood CD34+ cells (StemCell Technologies, Cat#70060) into Phase I Media.

[0395] 2. Centrifuge cells at 300 x g for 5 minutes to pellet.

[0396] 3. Aspirate supernatant and resuspend pellet in ImL of Phase I Media.

[0397] 4. Count viable cells and adjust concentration to 1x104viable cells / mL.

[0398] 5. Seed cells in lOOmL Phase I medium in T-175 flask for initial expansion.

[0399] Day 3 6. Add 3X the current culture volume of pre- warmed Phase I media.

[0400] Day 6

[0401] 7. Add IX the current culture volume of pre-warmed Phase I media.

[0402] Day 7

[0403] 8. Centrifuge le06 viable cells per reaction at 300 x g for 5min.

[0404] 9. Wash by resuspending in PBS, centrifuge at 300 x g for 5min and aspirate off PBS.

[0405] 10. Resuspend cells in 97 pLP3 Nucleofector solution + 5% glycerol.

[0406] 11. Add 3 pL 1 pM siRNA working dilution and mix gently.

[0407] 12. Transfer 100 pL total reaction mixture into 4D-Nucleocuvette XL without bubbles.

[0408] 13. Electroporate each cuvette in Amaxa 4D-Nucleofector using program DG-137.

[0409] 14. Add 200 pL warm Phase I media to each cuvette and allow cells to recover lOmin at room temperature.

[0410] 15. Transfer 100 pL electroporated cells to one well of 6-well plate with 2.5mL pre-warmed Phase I media.

[0411] Day 10

[0412] 16. Count cells with Vi-CELL BLU cell viability analyzer.

[0413] 17. Centrifuge each condition at 300 x g for 5 minutes, aspirate supernatant, and resuspend pellet in Phase II media to a final density 4 x 105viable cells / mL.

[0414] Day 14

[0415] 18. Collect IxlO6viable cells per condition were centrifuged at 300 xg for 5 minutes.

[0416] 19. Aspirate media and lyse pellet in 100 pL nuclease-free water.

[0417] 20. Vortex briefly and snap freeze lysates on dry ice.

[0418] 21. Samples stored at -80°C until subsequent analysis by HPLC.

[0419] In screening for candidate lead BCL11A-targeting siRNAs, a further series of steps can be performed to eliminate BCL11A-targeting siRNAs that are insufficiently effective and / or exhibit off-target effects. Such additional steps are used to select one or more siRN As to be a component of a lead compound of the current disclosure, specifically by selecting an effective anti-BCL l 1 A siRNA that maximizes treatment potency and reduces or avoids side effects, whether alone or in the context of an AOC, or both.

[0420] Optimization of candidate siRNAs may include introduction of one or more modifications, e.g., use of 2'-fluoro nucleotides, 2’-O-methyl nucleotides, phosphorothioate modifications, vinyl phosphonate nucleotide(s), an inverted abasic moiety, an amine linker, etc., in the passenger strand, the guide strand, or both, of a disclosed siRNA.

[0421] Example 3. Conjugate synthesis

[0422] To create antibody oligonucleotide conjugates (AOCs), siRNA sequences identified in the above Examples are crosslinked to an antibody using standard methods. Steps of conjugate synthesis typically include: ( 1) activation of the antibody using a reducing agent like TCEP (tris(2-carboxyethyl)phosphine) or DTT (dithiothreitol); (2) modification of the oligonucleotides (siRNAs) to have a thiol group at the 5' or 3' end to be used for conjugation; (3) conjugate reaction combining the activated antibody and the modified oligonucleotide; and (4) purification to remove unreacted species.

[0423] Here, CD 117 (proto-onogene c-KIT) antibodies are conjugated with siRNA oligonucleotides targeting BCL 11 A. In some embodiments, the CD 117 antibody may be one that is commercially available Non-limiting examples of commercially available CD117 antibodies include those listed in Table 18 below The conjugate may further include a fluorophore.

[0424] Table 18: Commercially Available Antibodies

[0425]

[0426] Example 4. Expression profile of BCL11A in bone marrow

[0427] Bone marrow derived hematopoietic stem and progenitor cells from healthy and SCD patients will be evaluated for BCL11A expression using standard methods. Such methods may include immunohistochemistry using BCL11A antibodies, RNA sequencing (RNA-seq) and / or immunofluorescent microscopy.

[0428] It is predicted that BCL11A expression will be reduced in both healthy and SCD cells treated with BCL11 siRNA AOCs.

[0429] Example 5. Expression profile of fetal hemoglobin (HbF) in AOC-treated cells and / or subjects (Prophetic Example)

[0430] Next, bone marrow derived hematopoietic stem and progenitor cells from healthy and SCD patients are evaluated for fetal hemoglobin (HbF) using standard methods. Such methods may include immunohistochemistry using HbF antibodies, HPLC, immunofluorescent microscopy and / or flow cytometry.

[0431] It is predicted that fetal hemoglobin is increased in both healthy and SCD cells treated with BCL11A siRNA AOCs.

[0432] Example 6. FTX-6058 is expected to induce pancellular expression of fetal hemoglobin (HbF) After in vitro validation. Phase 1 and Phase lb studies are conducted. Healthy patients are recruited for treatment with FTX-6058 (which is a small molecular fetal hemoglobin inducer for SCD --- see, e.g., Blood, vol. 136, Supplement 1, 5 November 2020 (2020-11-05), US, pages 26 -27). FTX-6058 has the following structure:

[0433]

[0434] The target population is SCD patients who are at least 18 years old and who have had 2 to 10 SCD-related pain crises in the past 12 months prior to enrollment. For recruitment of SCD patients, it is expected that Phase 1 and / or Phase lb studies will include patients undergoing treatment. Standard of care (SOC) treatments for SCD patients that will not be considered grounds for exclusion include but are not limited to: narcotics, chemotherapy, vitamins, L-glutamine, blood transfusion, bone marrow transplantation, hydroxyurea, voxelotor, and / or crizanlizumab-tmca. It is expected that SCD patients undergoing SOC treatment will have been prescribed for at least 6 months and that the dose will be stable for at least 3 months. It is expected that initially SCD patients will be limited to patients having Sickle Cell Anemia (HbSS), Sickle Hemoglobin-C Disease (HbSC), with Sickle Beta-Thalasemia (either HbSP°-thalassemia or HbS[3 -thalassemia) being a near-term expansion opportunity.

[0435] It is expected that FTX-6058 will be administered via infusion treatment. Infusion may be administered intravenously, epidurally, subcutaneously, or intramuscularly. Treatment is expected to be every four weeks (Q4W), every six weeks (Q6W), or every twelve weeks (Q12W).

[0436] For Phase 1 and / or Phase lb studies, the primary endpoint will be greater than half of the treatment population achieve greater than 20% fetal hemoglobin. As a secondary endpoint, Phase 1 and / or Phase lb studies have a 50% reduction in median annual rate of vaso-occlusive crises (VOCs) that lead to healthcare visits. It is expected that the safety outcome of these studies shows that the drug is generally well tolerate and that any adverse events (AE) will be Grade 1 or 2 with neutropeni and / or anemi as potential adverse events.

[0437] For Phase 2 / 3 studies, the target patient population can include SCD patients who are at least two years old.

[0438] In summary, the identification and selection of BCL11A RNAi oligonucleotides, conjugation with an antibody (e.g., CD117), and administration to patients is expected to decrease BCL11A, thereby increasing the transcription of gamma globin and consequently increasing HbF levels.

[0439] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

[0440] One skilled in the art would readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the disclosure. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the disclosure, are defined by the scope of the claims. In addition, where features or aspects of the disclosure ar e described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

[0441] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including,” and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0442] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all exampl es, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-cl aimed element as essential to the practice of the disclosure.

[0443] Embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosed invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description.

[0444] The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present disclosure provides preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the ait, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the description and the appended claims.

[0445] It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present disclosure and the following claims. The present disclosure teaches one skilled in the art to test various combinations and / or substitutions of chemical modifications described herein toward generating conjugates possessing improved contrast, diagnostic and / or imaging activity. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying conjugates possessing improved contrast, diagnostic and / or imaging activity.

[0446] The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.

[0447] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby

[0448] Features described above as well as those claimed below may be combined in various ways without departing from the scope thereof. The following examples illustrate some possible, nonlimiting combinations:

[0449] (Al) An antibody-oligonucleotide conjugate including an antibody or antigen binding fragment thereof capable of binding a human hematopoietic stem cell marker conjugated to an oligonucleotide capable of hybridizing to a target sequence of a BCL11 A transcript and mediating antisense and / or RNA interference-mediated inhibition of the BCL11 A transcript.

[0450] (A2) For the antibody-oligonucleotide conjugate denoted as (Al), the human hematopoietic stem cell marker is selected from among: HLA-DR, CDlla, CD18, CD34, CD41 / 61, CD43, CD45, CD49d (VLA-4), CD49f (VLA-6), CD51, CD58, CD71, CD84, CD90, CD97, CD117 (c-kit), CD133, CD134, CD162, CD166, CD184 (CXCR4), CD205 and CD361.

[0451] (A3) For the antibody-oligonucleotide conjugate denoted as any one of (Al) through (A2), the human hematopoietic stem cell marker is CD117 or CD71.

[0452] (A4) For the antibody-oligonucleotide conjugate denoted as any one of (Al) through (A3), the oligonucleotide is a strand of a double-stranded nucleic acid, optionally of a small interfering RNA (siRNA), optionally where the oligonucleotide comprises one or more of the following siRNA sequences: SEQ ID NO: 23 and SEQ ID NO: 121; SEQ ID NO: 19 and SEQ ID NO: 117; SEQ ID NO: 25 and SEQ ID NO: 123; SEQ ID NO: 27 and SEQ ID NO: 125, SEQ ID NO: 29 and SEQ ID NO: 127; and SEQ ID NO: 32 and SEQ ID NO: 130.

[0453] (A5) For the antibody-oligonucleotide conjugate denoted as any one of (Al) through (A4), the antibody or antigen binding fragment thereof capable of binding the human hematopoietic stem cell marker includes: a variable heavy chain (VH) region including at least one sequence selected from Table 1 or Table 3; a variable light chain (VL) region including at least one sequence selected from Table 2 or Table 4; at least one sequence selected from Table 5, a heavy chain sequence selected from Table 6; and / or a light chain sequence selected from Table 7.

[0454] (Bl) An antibody-oligonucleotide conjugate including a cell penetrating peptide conjugated to an oligonucleotide capable of hybridizing to a target sequence of a BCL11A transcript and mediating antisense and / or RNA interference-mediated inhibition of the BCL11 A transcript.

[0455] (B2) For the antibody-oligonucleotide conjugate denoted as (Bl), the cell penetrating peptide includes a sequence selected from Table 9.

[0456] (B3) For the antibody-oligonucleotide conjugate denoted as any one of (Al) through (A5) and (Bl) through (B2), the antibody or binding fragment thereof includes a non-human antibody or antigen binding fragment thereof, a human antibody or antigen binding fragment thereof, a humanized antibody or antigen binding fragment thereof, chimeric antibody or antigen binding fragment thereof, monoclonal antibody or antigen binding fragment thereof monovalent Fab', divalent Faba, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or antigen binding fragment thereof.

[0457] (B4) For the antibody -oligonucleotide conjugate denoted as any one of (Al) through (A5) and (B 1) through (B3), the antibody or antigen binding fragment thereof is a clone 2B8 anti-CD117 antibody or antigen binding fragment thereof, a clone 104D2 anti-CD117 antibody or antigen binding fragment thereof, and / or is an anti-CD71 antibody or antigen binding fragment thereof.

[0458] (B5) For the antibody-oligonucleotide conjugate denoted as any one of (Al ) through (A5) and (B 1) through (B4), the nucleic acid molecule includes a sense strand and / or an antisense strand, and wherein the sense strand and / or the antisense strand each independently includes at least one 2' modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety.

[0459] (B6) For the antibody-oligonucleotide conjugate denoted as any one of (Al ) through (A5) and (B 1) through (B5), the polynucleotide hybridizes to at least 8 contiguous bases of the target sequence ofBCLHA.

[0460] (B7) For the antibody-oligonucleotide conjugate denoted as any one of (Al) through (A5) and (Bl) through (B6), the polynucleotide is from about 8 to about 50 nucleotides in length or from about 10 to about 30 nucleotides in length.

[0461] (B8) For the antibody-oligonucleotide conjugate denoted as any one of (Al) through (A5) and (B 1) through (B7), the nucleic acid molecule includes a sense strand and / or an antisense strand, and the sense strand includes a sequence of at least 12 nucleotides in length that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and / or at least 99% identical to a sequence selected from Table 10, Table 11, Table 12, Table 13, SEQ ID NOs: 1-98; SEQ IDNOs: 197-231; SEQ ID NOs: 267-277; SEQ ID NO: 289; and SEQ ID NOs: 291-580.

[0462] m (B9) For the antibody-oligonucleotide conjugate denoted as any one of (Al) through ( A5) and (B 1) through (B8), the nucleic acid molecule includes a sense strand and / or an antisense strand, and the antisense strand includes a sequence of at least 12 nucleotides in length that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and / or at least 99% identical to a sequence selected from Table 10, Table 11, Table 12, Table 13, SEQ ID NOs: 99-196, SEQ ID NOs: 232-266; SEQ ID NOs: 278-288; SEQ ID NO: 290, and SEQ ID NOs: 291-580.

[0463] (BIO) For the antibody-oligonucleotide conjugate denoted as any one of (Al) through (A5) and (Bl) through (B9), the oligonucleotide includes at least one 2' modified nucleotide, optionally further where the 2' modified nucleotide: includes 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2' -deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2-O-AP), 2'-O-dimethylaminoethyl (2-0-DMA0E), 2‘-O-dimethylaminopropyl (2‘-0-DMAP), 2'-O-di met yl aminoethyl oxyethyl (2'-0-DMAE0E), or 2-0-N-methylacetami do (2'-0-NM A) modified nucleotide: optionally wherein the oligonucleotide includes locked nucleic acid (LNA) or ethylene nucleic acid (ENA); or includes a combination thereof.

[0464] (Bll) For the antibody-oligonucleotide conjugate denoted as any one of (A l) through (A5) and (Bl) through (BIO), the at least one modified internucleotide linkage includes a phosphorothioate linkage or a phosphorodi thioate linkage.

[0465] (Bl 2) For the antibody-oligonucleotide conjugate denoted as any one of (Al) through (A5) and (Bl) through (Bl I), the oligonucleotide includes 3 or more 2’ modified nucleotides selected from 2'-O-methyl and 2 '-deoxy-2 '-fluoro.

[0466] (B13) For the antibody-oligonucleotide conjugate denoted as any one of (Al) through (A5) and (Bl) through (B12), the oli onucleotide includes a 5'-terminal vinylphosphonate modified nucleotide.

[0467] (Bl 4) For the antibody-oligonucleotide conjugate denoted as any one of (Al) through (A5) and (BI) through (B 13), the 2' modified nucleotide is 2'-O-methyl modified nucleotide, and 2'-O-methyl modified nucleotide is at the 5'-end of the sense strand and / or the antisense strand. (BI 5) For the antibody-oligonucleotide conjugate denoted as (Bl 4), the 2'-O-methyl modified nucleotide is a purine nucleotide

[0468] (Bl 6) For the antibody-oligonucleotide conjugate denoted as (B14), the 2'~O-methyl modified nucleotide is a pyrimidine nucleotide.

[0469] (B 17) For the antibody-oligonucleotide conjugate denoted as any one of (B 14) through (B 16), the sense and / or antisense strands include at least two, three, four consecutive the 2'-O-methyl modified nucleotides at the 5‘-end.

[0470] (Bl 8) For the antibody-oligonucleotide conjugate denoted as any one of (Al) through (A5) and (BI) through (B17), the antibody-oligonucleotide conjugate includes a linker connecting the antibody or antigen binding fragment thereof to the oligonucleotide.

[0471] (BI 9) For the antibody-oligonucleotide conjugate denoted as (Bl 8), the linker is C1-C6 alkyl linker.

[0472] (B20) For the antibody-oligonucleotide conjugate denoted as (Bl 8), the inker is a homobifunctional linker or heterobifunctional linker, and includes a maleimide group, a dipeptide moiety, a benzoic acid group, or its derivative thereof.

[0473] (B21) For the antibody-oligonucleotide conjugate denoted as (B18), the linker is a cleavable or non-c I eavabl e 1 i n ker.

[0474] (B22) For the antibody-oligonucleotide conjugate denoted as any one of (Al ) through (A5) and (BI) through (B21), the ratio between the nucleic acid molecule and the antibody or antigen binding fragment thereof is about 1:1, 2:1, 3:1, or 4:1.

[0475] (B23) For the antibody-oligonucleotide conjugate denoted as any one of (Al) through (A5) and (Bl) through (B22), the oligonucleotide is an RNA interference oligonucleotide that mediates inhibition of the BCL11 A transcript and upregulates fetal hemoglobin expression in a treated cell, tissue, or subject (B24) For the antibody-oligonucleotide conjugate denoted as (B23), the inhibition includes reducing expression of the BCL 11 A transcript by at least 50%, by at least 60%, or by at least 70% or more, as compared to a quantity of the BCL11 A transcript in an untreated cell.

[0476] (B25) For the antibody-oligonucleotide conjugate denoted as any one of (Al) through (A5) and (Bl) through (B24), the antibody-oligonucleotide conjugate includes a molecule of Formula (I):

[0477] A-X-B Formula I wherein,

[0478] A is the antibody or antigen binding fragment thereof,

[0479] B is the oligonucleotide that hybridizes to a target sequence of BCL11A; and

[0480] X is a bond or a non-polymeric linker; and wherein X is conjugated to a cysteine residue of A.

[0481] (Cl) An oligonucleotide conjugate including an endosomolytic moiety and / or a cell penetrating peptide conjugated to an oligonucleotide capable of hybridizing to a target sequence of a BCL11 A transcript and mediating antisense and / or RNA interference-mediated inhibition of the BCL11A transcript.

[0482] (C2) For the oligonucleotide conjugate denoted as (Cl), the endosomolytic moiety includes a sequence selected from Table 8.

[0483] (C3) For the oligonucleotide conjugate denoted as (Cl), the cell penetrating peptide includes a sequence selected from Table 9.

[0484] (DI) A pharmaceutical composition including: an antibody-oligonucleotide conjugate or oligonucleotide conjugate of any one of the compositions denoted as (Al) through (A5), (Bl) through (B25), and (Cl) through (C3); and a pharmaceutically acceptable excipient.

[0485] (D2) For the pharmaceutical composition denoted as (DI), the pharmaceutical composition is formulated as a nanoparticle formulation.

[0486] (D3) For the antibody-oligonucleotide conjugate denoted as (DI) or (D2), the pharmaceutical composition is formulated for parenteral, oral, intranasal, buccal, rectal, transderm al, intravenous, subcutaneous, or intrathecal administration. (El). A method for treating a hemoglobinopathy in a subject in need thereof, the method involving, providing an antibody-oligonucleotide conjugate or oligonucleotide conjugate of any one of (Al) through (A5), (Bl) through (B2.5), and (Cl) through (C3); and administering the antibody-oligonucleotide conjugate or oligonucleotide conjugate to the subject in need thereof to treat the hemoglobinopathy, where the antibody-oligonucleotide conjugate or oligonucleotide conjugate reduces BCL11A transcript levels in the subject.

[0487] (E2) For the method denoted as (El), the antibody-oligonucleotide conjugate or oligonucleotide conjugate mediates inhibition of the BCL11A transcript and upregulates fetal hemoglobin expression in the subject.

[0488] (E3) For the method denoted as (El) or (E2), the hemoglobinopathy is Sickle Cell Disease (SCD).

[0489] (Fl) Use of the antibody-oligonucleotide conjugate of any one of (Al) through (A5), (Bl) through (B25), and (Cl) through (C3) or the pharmaceutical composition of any one of (DI) through (D3), for treating a subject diagnosed with or suspected to have Sickle Cell Disease (SCD).

[0490] (Gl) Use of the antibody-oligonucleotide conjugate any one of (Al) through (A5), (Bl) through (B25), and (Cl) through (C3) or the pharmaceutical composition of any one of (DI) through (D3), in the manufacture of a medicament for treating a subject diagnosed with or suspected to have Sickle Cell Disease (SCD).

[0491] (Hl) A kit comprising an antibody-oligonucleotide conjugate of any one of (Al) through (A5), (Bl) through (B25), (Cl) through (C3), (DI) through (D3), (El) through (E3), (Fl), (Gl), (Hl), and (El).

[0492] (II). An oligonucleotide agent capable of inhibiting BCL11A mRNA and having one or more siRNA strand sequences selected from the group consisting of: SEQ ID NO: 23 and SEQ ID NO: 121; SEQ ID NO: 19 and SEQ ID NO: 117; SEQ ID NO: 25 and SEQ ID NO: 123; SEQ ID NO: 27 and SEQ ID NO: 125, SEQ ID NO: 29 and SEQ ID NO: 127; and SEQ ID NO: 32 and SEQ ID NO: 130.

[0493] (12). For the antibody-oligonucleotide conjugate, oligonucleotide conjugate, pharmaceutical composition, method, use, or oligonucleotide agent denoted as (Al) through (A5), (Bl) through (B25), (Cl) through (C3), (DI) through (D3), (El) through (E3), (Fl), (Gl) and (II), the antibody-oligonucleotide conjugate, oligonucleotide conjugate, pharmaceutical composition, method, use, or oligonucleotide agent of any one of (Al) through (A5), (Bl) through (B25), (C l) through (C3), (DI) through (D3), (El) through (E3), (Fl), (Gl) and (11), where HbF is induced at least two-fold in a target cell, tissue, or subject, optionally where HbF is induced at least three-fold in the target cell, tissue, or subject, optionally where HbF is induced at least four-fold in the target cell, tissue, or subject.

[0494] Although the invention has been described with reference to the embodiments disclosed herein, it is noted that equivalents may be employed and substitutions made herein without departing from the scope of the invention as recited in the claims.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. An antibody-oligonucleotide conjugate comprising an antibody or antigen binding fragment thereof capable of binding a human hematopoietic stem cell marker conjugated to an oligonucleotide capable of hybridizing to a target sequence of a BCL11 A transcript and mediating antisense and / or RNA interference-mediated inhibition of the BCL I 1 A transcript.

2. The antibody-oligonucleotide conjugate of claim 1, wherein the human hematopoietic stem cell marker is selected from the group consisting of HLA-DR, CD11a, CD 18, CD34, CD41 / 61, CD43, CD45, CD49d (VLA-4), CD49f (VLA-6), CD51, CD58, CD71, CD84, CD90, CD97, CD117 (c-kit), CD133, CD134, CD162, CD166, CD184 (CXCR4), CD205 and CD361.

3. The antibody-oligonucleotide conjugate of claim 1, wherein the human hematopoietic stem cell marker is CD 117 or CD71.

4. The antibody-oligonucleotide conjugate of claim 1, wherein the oligonucleotide is a strand of a double-stranded nucleic acid, optionally of a small interfering RNA (siRNA), optionally wherein the oligonucleotide comprises one or more sequences selected from the group consisting of siRNA sequences SEQ ID NO: 23 and SEQ ID NO: 121; SEQ ID NO: 19 and SEQ ID NO: 117; SEQ ID NO: 25 and SEQ ID NO: 123; SEQ ID NO: 27 and SEQ ID NO: 125; SEQ ID NO: 29 and SEQ ID NO: 127; and SEQ ID NO: 32 and SEQ ID NO: 130.

5. The antibody-oligonucleotide conjugate of claim 1, wherein the antibody or antigen binding fragment thereof capable of binding the human hematopoietic stem cell marker comprises:a variable heavy chain ( VH) region comprising at least one sequence selected from Table 1 or Table 3,a variable light chain (VL) region comprising at least one sequence selected from Table 2 or Table 4,at least one sequence selected from Table 5,a heavy chain sequence selected from Table 6, and / ora light chain sequence selected from Table 7.

6. An antibody-oligonucleotide conjugate comprising a cell penetrating peptide conjugated to an oligonucleotide capable of hybridizing to a target sequence of a BCL11 A transcript and mediating antisense and / or RNA interference-mediated inhibition of the BCL11 A transcript.

7. The antibody-oligonucleotide conjugate of claim 6, wherein the cell penetrating peptide comprises a sequence selected from Table 9.

8. The antibody-oligonucleotide conjugate of claim 1 or 7, wherein the antibody or binding fragment thereof comprises a non-human antibody or antigen binding fragment thereof, a human antibody or antigen binding fragment thereof, a humanized antibody or antigen binding fragment thereof, chimeric antibody or antigen binding fragment thereof, monoclonal antibody or antigen binding fragment thereof, monovalent Fab', divalent Fab;?, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or antigen binding fragment thereof.

9. The antibody-oligonucleotide conjugate of any one of claims 1-8, wherein the antibody or antigen binding fragment thereof is a clone 2B8 anti-CD117 antibody or antigen binding fragment thereof, a clone 104D2 anti-CD117 antibody or antigen binding fragment thereof, and / or is an anti- CD71 antibody or antigen binding fragment thereof.10 The antibody-oligonucleotide conjugate of any one of claims 1-9, wherein the nucleic acid molecule comprises a sense strand and / or an antisense strand, and wherein the sense strand and / or the antisense strand each independently comprises at least one 2' modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety11. The antibody-oligonucleotide conjugate of any one of claims 1-10, wherein the polynucleotide hybridizes to at least 8 contiguous bases of the target sequence of BCL11A.

12. The antibody-oligonucleotide conjugate of any one of claims 1-11, wherein the polynucleotide is from about 8 to about 50 nucleotides in length or from about 10 to about 30 nucleotides in length1.3 The antibody-oligonucleotide conjugate of any one of claims 1-12, wherein the nucleic acid molecule comprises a sense strand and / or an antisense strand, and the sense strand comprises a sequence of at least 12 nucleotides in length that is at least 90%, at least 95%, at least 96%, at least97%, at least 98%, and / or at least 99% identical to a sequence selected from Table 10, Table 11, Table 12, Table 13, SEQ ID NOs: 1-98; SEQ ID NOs: 197-231; SEQ ID NOs: 267-277; SEQ ID NO: 289; and SEQ ID NOs: 291-580.14 The antibody-oligonucleotide conjugate of any one of claims 1-13, wherein the nucleic acid molecule comprises a sense strand and / or an antisense strand, and the antisense strand comprises a sequence of at least 12 nucleotides in length that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and / or at. least. 99% identical to a sequence selected from Table 10, Table 11, Table 12, Table 13, SEQ ID NOs: 99-196; SEQ ID NOs: 232-266, SEQ ID NOs: 278-288; SEQ ID NO: 290; and SEQ ID NOs: 291-58015 The antibody-oligonucleotide conjugate of any one of claims 1-14, wherein the oligonucleotide comprises at least one 2' modified nucleotide, and further wherein the 2' modified nucleotide: comprises 2'-O-methyl, 2'-O-methoxyethyl (2-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-0-NM A) modified nucleotide, comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA); or comprises a combination thereof.

16. The antibody-oligonucleotide conjugate of any one of claims 1-15, wherein the at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage.

17. The antibody-oligonucleotide conjugate of any one of cl aims 1-16, wherein the oligonucleotide comprises 3 or more 2' modified nucleotides selected from 2'-O-methyl and 2 '-deoxy-2 '-fluoro.

18. The antibody-oligonucleotide conjugate of any one of claims 1-17, wherein the oligonucleotide comprises a 5'-terminal vinylphosphonate modified nucleotide.

19. The antibody-oligonucleotide conjugate of any one of claims 1-18, wherein the 2’ modified nucleotide is 2’-O-methyl modified nucleotide, and 2’-O-methyl modified nucleotide is at the 5’-end of the sense strand and / or the antisense strand.

20. The antibody-oligonucleotide conjugate of claim 19, wherein the 2'-O-methyl modified nucleotide is a purine nucleotide21. The antibody-oligonucleotide conjugate of claim 19, wherein the 2'-O-methyl modified nucleotide is a pyrimidine nucleotide.

22. The antibody-oligonucleotide conjugate of any one of claims 19-21, wherein the sense and / or antisense strands comprise at least two, three, four consecutive the 2’-O-methyl modified nucleotides at the 5'-end.

23. The antibody-oligonucleotide conjugate of any one of claims 1-22, wherein the antibody-oligonucleotide conjugate comprises a linker connecting the antibody or antigen binding fragment thereof to the oligonucleotide24. The antibody-oligonucleotide conjugate of claim 23, wherein the linker is C I-C6 alkyl linker.

25. The antibody-oligonucleotide conjugate of claim 23, wherein the linker is a homobifunctional linker or heterobifunctional linker, and comprises a maleimide group, a dipeptide moiety, a benzoic acid group, or its derivative thereof.26 The antibody-oligonucleotide conjugate of claim 23, wherein the linker is a cleavable or noncl eavable linker.

27. The antibody-oligonucleotide conjugate of any one of claims 1-26, wherein a ratio between the nucleic acid molecule and the antibody or antigen binding fragment thereof is about 1:1, 2:1, 3:1, or 4:1.

28. The antibody-oligonucleotide conjugate of any one of claims 1-26, wherein the oligonucleotide is an RNA interference oligonucleotide that mediates inhibition of the BCL11A transcript and upregulates fetal hemoglobin expression in a treated cell, tissue, or subject.29 The antibody-oligonucleotide conjugate of claim 28, wherein the inhibition comprises reducing expression of the BCL11A transcript by at least 50%, by at least 60%, or by at least 70% or more, as compared to a quantity of the BCL11 A transcript in an untreated cell.

30. The antibody-oligonucleotide conjugate of any one of claims 1-29, wherein the antibody-oligonucleotide conjugate comprises a molecule of Formula (I):A-X-B Formula I wherein,A is the antibody or antigen binding fragment thereof;B is the oligonucleotide that hybridizes to a target sequence of BCL11 A; andX is a bond or a non-polymeric linker; and wherein X is conjugated to a cysteine residue of A.

31. An oligonucleotide conjugate comprising an endosomolytic moiety and / or a cell penetrating peptide conjugated to an oligonucleotide capable of hybridizing to a target sequence of a BCL11A transcript and mediating antisense and / or RNA interference-mediated inhibition of the BCL11A transcript.

32. The oligonucleotide conjugate of claim 31, wherein the endosomolytic moiety comprises a sequence selected from Table 8.

33. The oligonucleotide conjugate of claim 31, wherein the cell penetrating peptide comprises a sequence selected from Table 9.

34. A pharmaceutical composition comprising: an antibody-oligonucleotide conjugate or oligonucleotide conjugate of any one of claims 1-33; and a pharmaceutically acceptable excipient35. The pharmaceutical composition of claim 34, wherein the pharmaceutical composition is formulated as a nanoparticle formulation.

36. The pharmaceutical composition of claim 34 or claim 35, wherein the pharmaceutical composition is formulated for parenteral, oral, intranasal, buccal, rectal, transdermal, intravenous, subcutaneous, or intrathecal administration.

37. A method for treating a. hemoglobinopathy in a subject in need thereof, comprising:providing an antibody-oligonucleotide conjugate or oligonucleotide conjugate of any one of claims 1-33; andadministering the antibody-oligonucleotide conjugate or oligonucleotide conjugate to the subject in need thereof to treat the hemoglobinopathy, wherein the antibody-oligonucleotide conjugate or oligonucleotide conjugate reduces BCL11 A transcript levels in the subject.

38. The method of claim 37, wherein the antibody-oligonucleotide conjugate or oligonucleotide conjugate mediates inhibition of the BCL11A transcript and upregulates fetal hemoglobin expression in the subject.

39. The method of claim 37 or claim 38, wherein the hemoglobinopathy is Sickle Cell Disease (SCD).

40. Use of the antibody-oligonucleotide conjugate of any one of claims 1-30 or the pharmaceutical composition of any one of claims 31-33 for treating a subject diagnosed with or suspected to have Sickle Cell Disease (SCD).

41. Use of the antibody-oligonucleotide conjugate of any one of claims 1-30, the oligonucleotide conjugate of any one of claims 31-33, or the pharmaceutical composition of any one of claims 33-35 in the manufacture of a medicament for treating in a subject diagnosed with or suspected to have Sickle Cell Disease (SCD).

42. A kit comprising an antibody-oligonucleotide conjugate of any one of claims 1-30, an oligonucleotide conjugate of any one of claims 31-33, or the pharmaceutical composition of any one of claims 34-36.

43. An oligonucleotide agent capable of inhibiting BCL11A mRNA and comprising one or more siRNA strand sequences selected from the group consisting of SEQ ID NO: 23 and SEQ ID NO: 121, SEQ ID NO: 19 and SEQ ID NO: 117; SEQ ID NO: 25 and SEQ ID NO: 123; SEQ ID NO: 27 and SEQ ID NO: 125; SEQ ID NO: 29 and SEQ ID NO: 127; and SEQ ID NO: 32 and SEQ ID NO: 130.

44. The antibody-oligonucleotide conjugate, oligonucleotide conjugate, pharmaceutical composition, method, use, or oligonucleotide agent of any one of claims 1-41 and 43, wherein HbF is induced at least two-fold in a target cell, tissue, or subject, optionally wherein HbF is induced atleast three-fold in the target cell, tissue, or subject, optionally wherein HbF is induced at least fourfold in the target cell, tissue, or subject.

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