HK40115884A - Compositions and methods of treating muscle atrophy and myotonic dystrophy - Google Patents

Description

Compositions and methods for treating muscle atrophy and myotonic dystrophy

This application is a divisional application of Chinese patent application No. 201880088816.0, filed on December 6, 2018, entitled "Composition and Method for Treating Amyotrophic Atrophy and Myotonic Dystrophy" (the corresponding PCT application was filed on December 6, 2018, with application number (PCT/US2018/064359).

sequence list

This application contains a sequence list, which has been electronically submitted in XML format and is incorporated herein by reference in its entirety. The XML copy created on June 13, 2024, is named 45532-722_7113_SL.xml and is 170,577 bytes in size.

Background Technology

Gene repression via RNA-induced gene silencing offers several levels of control: transcriptional inactivation, small interfering RNA (siRNA)-induced mRNA degradation, and siRNA-induced transcriptional attenuation. In some cases, RNA interference (RNAi) provides durable effects on multiple cell divisions. Therefore, RNAi represents a viable approach for drug target validation, gene function analysis, pathway analysis, and disease treatment.

Summary of the Invention

In some embodiments, this document discloses polynucleotide molecules and pharmaceutical compositions for regulating genes (or atrogenes) associated with muscle atrophy. In some embodiments, this document also describes methods for treating muscle atrophy using the polynucleotide molecules or polynucleotide conjugates disclosed herein.

In some embodiments, the present document discloses a molecule of formula (I): _AX1 - _BX2 -C (Formula I) wherein A is a binding moiety; B is a polynucleotide that hybridizes to the target sequence of the atrogene; C is a polymer; and _X1 and _X2 are each independently selected from bonded or non-polymerized linkers; wherein the polynucleotide comprises at least one 2' modified nucleotide, at least one modified internucleotide linker, or at least one reverse debasement moiety; and wherein A and C are not linked to the same end of B. In some embodiments, the atrogene comprises a differentially regulated (e.g., upregulated or downregulated) gene within the IGF1-Akt-FoxO pathway, glucocorticoid-GR pathway, PGC1α-FoxO pathway, TNFα-NFκB pathway, or myostatin-ActRIIb-Smad2/3 pathway. In some embodiments, the atrogene encodes an E3 ligase. In some embodiments, the atrogene encodes a forkhead box transcription factor. In some embodiments, the atrogene includes the atrogin-1 gene (FBXCTCCAACATCAAGGAAGATGGCATTTCTAG gacO32) (SEQ ID NO:881), the MuRF1 gene (TRIM63), FOXO1, FOXO3, or MSTN. In some embodiments, the atrogene includes DMPK. In some embodiments, B consists of a polynucleotide that hybridizes to the target sequence of the atrogene. In some embodiments, C consists of a polymer. In some embodiments, the at least one 2'-modified nucleotide comprises a nucleotide modified with 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA). In some embodiments, the at least one 2'-modified nucleotide comprises locked nucleic acid (LNA) or vinyl nucleic acid (ENA). In some embodiments, the linkage between the at least one modified nucleotide comprises a phosphate thioate linkage or a phosphate dithioate linkage. In some embodiments, the at least one reverse debasement moiety is at at least one end. In some embodiments, the polynucleotide comprises a single strand that hybridizes to the target sequence of the atrogene. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridizing with the first polynucleotide to form a double-stranded polynucleotide molecule, wherein the first or second polynucleotide also hybridizes with a target sequence of atrogene. In some embodiments, the second polynucleotide comprises at least one modification. In some embodiments, the first and second polynucleotides are RNA molecules. In some embodiments, the polynucleotide hybridizes with at least 8 consecutive bases of the target sequence of atrogene. In some embodiments, the polynucleotide comprises a sequence complementary to at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the sequences shown in SEQ ID NO:190-303 and 532-642. In some embodiments, the polynucleotide is about 8 to about 50 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 30 nucleotides in length. In some embodiments, the first polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequences shown in SEQ ID NO:304-417, 418-531, 643-753, 754-864, and 28-189. In some embodiments, the second polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequences shown in SEQ ID NO:304-417, 418-531, 643-753, 754-864, and 28-189. In some embodiments, _X1 and _X2 are independently _C1 - _C6 alkyl groups. In some embodiments, _X1 and _X2 are independently homo- or hetero-bifunctional linkers, optionally conjugated to a _C1 - _C6 alkyl group. In some embodiments, A is an antibody or a binding fragment thereof. In some embodiments, A includes a humanized antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a monovalent Fab', a bivalent Fab2, a single-chain variable fragment (scFv), a biantibody, a microantibody, a nanobody, a single-domain antibody (sdAb), or a camelid antibody or a binding fragment thereof. In some embodiments, A is an anti-transferrin receptor antibody or a binding fragment thereof. In some embodiments, C is polyethylene glycol. In some embodiments, _AX1 is conjugated to the 5' end of B, and _X2 -C is conjugated to the 3' end of B. In some embodiments, _X2 -C is conjugated to the 5' end of B, and _AX1 is conjugated to the 3' end of B. In some embodiments, A is directly conjugated to _X1 . In some embodiments, C is directly conjugated to _X2 . In some embodiments, B is directly conjugated to _X1 and _X2 . In some embodiments, the molecule further comprises D. In some embodiments, D is conjugated to C or to A. In some embodiments, D is an endosome-dissolved polymer.

In some embodiments, this document discloses a polynucleotide conjugate comprising a binding moiety conjugated to a polynucleotide that hybridizes to a target sequence of an atrogene; wherein the polynucleotide optionally comprises at least one 2' modified nucleotide, at least one modified internucleotide linker, or at least one reverse debasement moiety; and wherein the polynucleotide conjugate mediates RNA interference against the atrogene, thereby treating muscle atrophy in a subject. In some embodiments, the atrogene comprises a differentially regulated (e.g., upregulated or downregulated) gene within the IGF1-Akt-FoxO pathway, glucocorticoid-GR pathway, PGC1α-FoxO pathway, TNFα-NFκB pathway, or myostatin-ActRIIb-Smad2/3 pathway. In some embodiments, the atrogene encodes an E3 ligase. In some embodiments, the atrogene encodes a forkhead box transcription factor. In some embodiments, the atrogene comprises a ligand of the TGF-beta (transforming growth factor-beta) protein superfamily. In some embodiments, the atrogene comprises DMPK. In some embodiments, the binding moiety is an antibody or a binding fragment thereof. In some embodiments, the binding moiety includes a humanized antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a monovalent Fab', a bivalent Fab2, a single-stranded variable fragment (scFv), a biantibody, a microantibody, a nanobody, a single-domain antibody (sdAb), or a camelid antibody or a binding fragment thereof. In some embodiments, the binding moiety is an anti-transferrin receptor antibody or a binding fragment thereof. In some embodiments, the binding moiety is cholesterol. In some embodiments, the polynucleotide comprises a single strand that hybridizes to the target sequence of atrogene. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide that hybridizes to the first polynucleotide to form a double-stranded polynucleotide molecule, wherein the first or second polynucleotide also hybridizes to the target sequence of atrogene. In some embodiments, the second polynucleotide comprises at least one modification. In some embodiments, the first and second polynucleotides are RNA molecules. In some embodiments, the polynucleotide hybridizes to at least eight consecutive bases of the target sequence of atrogene. In some embodiments, the polynucleotide comprises a sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% complementary to the sequences shown in SEQ ID NO: 190-303 and 532-642. In some embodiments, the polynucleotide is about 8 to about 50 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 30 nucleotides in length. In some embodiments, the first polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequences shown in SEQ ID NO: 304-417, 418-531, 643-753, 754-864, and 28-189. In some embodiments, the second polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequences shown in SEQ ID NO: 142-255, 256-369, 481-591, 592-702, and 3407-14222. In some embodiments, the polynucleotide conjugate optionally comprises a linker that links the binding moiety to the polynucleotide. In some embodiments, the polynucleotide conjugate further comprises a polymer, which is optionally indirectly conjugated to the polynucleotide via an additional linker. In some embodiments, the linker and the additional linker are each independently a bonded or non-polymerized linker. In some embodiments, the polynucleotide conjugate comprises a molecule of formula (I): _AX1 - _BX2 -C (Formula I), wherein A is an antibody or a binding fragment thereof; B is a polynucleotide that hybridizes to the target sequence of atrogene; C is a polymer; and _X1 and _X2 are each independently selected from bonded or non-polymerized linkers; wherein the polynucleotide comprises at least one 2' modified nucleotide, at least one modified internucleotide linker, or at least one reverse debasement moiety; and wherein A and C are not linked to the same end of B. In some embodiments, the at least one 2'-modified nucleotide comprises a nucleotide modified with 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA). In some embodiments, the at least one 2'-modified nucleotide comprises locked nucleic acid (LNA) or vinyl nucleic acid (ENA). In some embodiments, the linkage between the at least one modified nucleotide comprises a phosphate thioate linkage or a diphosphate thioate linkage. In some embodiments, the at least one reverse debasement moiety is at at least one end. In some embodiments, the muscle atrophy is diabetes-related muscle atrophy. In some embodiments, the muscle atrophy is cancer cachexia-related muscle atrophy. In some embodiments, the muscle atrophy is associated with insulin deficiency. In some embodiments, the muscle atrophy is associated with chronic renal failure. In some embodiments, the muscle atrophy is associated with congestive heart failure. In some embodiments, the muscle atrophy is associated with chronic respiratory diseases. In some embodiments, the muscle atrophy is associated with chronic infections. In some embodiments, the muscle atrophy is associated with fasting. In some embodiments, the muscle atrophy is associated with denervation. In some embodiments, the muscle atrophy is associated with sarcopenia, glucocorticoid therapy, stroke, and/or heart attack. In some cases, type 1 myotonic dystrophy (DM1) is associated with the expansion of the CTG repeat sequence in the 3'UTR of the DMPK gene.

In some embodiments, this document discloses a pharmaceutical composition comprising: the molecules described above or polynucleic acid conjugates described above; and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated as a nanoparticle formulation. In some embodiments, the pharmaceutical composition is formulated for parenteral, oral, intranasal, buccal, rectal, or transdermal administration.

In some embodiments, this document discloses a method for treating muscular atrophy or myotonic dystrophy in a subject of need, comprising: administering to the subject a therapeutically effective amount of a polynucleotide molecular conjugate comprising a binding moiety conjugated to a polynucleotide, the polynucleotide hybridizing to a target sequence of an atrogene; wherein the polynucleotide optionally comprises at least one 2'-modified nucleotide, at least one modified internucleotide linker, or at least one reverse debasement moiety; and wherein the polynucleotide molecular conjugate mediates RNA interference against the atrogene, thereby treating the subject's muscular atrophy or myotonic dystrophy. In some embodiments, the muscular atrophy is diabetes-related. In some embodiments, the muscular atrophy is cancer cachexia-related. In some embodiments, the muscular atrophy is associated with insulin deficiency. In some embodiments, the muscular atrophy is associated with chronic renal failure. In some embodiments, the muscular atrophy is associated with congestive heart failure. In some embodiments, the muscular atrophy is associated with chronic respiratory disease. In some embodiments, the muscular atrophy is associated with chronic infection. In some embodiments, the muscular atrophy is associated with fasting. In some embodiments, the muscular atrophy is associated with denervation. In some embodiments, the muscle atrophy is associated with sarcopenia. In some embodiments, the myotonic dystrophy is DM1. In some embodiments, the atrogene includes differentially regulated (e.g., upregulated or downregulated) genes within the IGF1-Akt-FoxO pathway, glucocorticoid-GR pathway, PGC1α-FoxO pathway, TNFα-NFκB pathway, or myostatin-ActRIIb-Smad2/3 pathway. In some embodiments, the atrogene encodes an E3 ligase. In some embodiments, the atrogene encodes a forkhead box transcription factor. In some embodiments, the atrogene includes the atrogin-1 gene (FBXO32), the MuRF1 gene (TRIM63), FOXO1, FOXO3, or MSTN. In some embodiments, the atrogene includes DMPK. In some embodiments, the polynucleotide conjugate comprises a molecule of formula (I): _AX1 - _BX2 -C (Formula I), wherein A is a binding moiety; B is a polynucleotide that hybridizes to the target sequence of the atrogene; C is a polymer; and _X1 and _X2 are each independently selected from bonded or non-polymerized linkers; wherein the polynucleotide comprises at least one 2'-modified nucleotide, at least one modified internucleotide linker, or at least one reverse debasement moiety; and wherein A and C are not linked to the same end of B. In some embodiments, B consists of a polynucleotide that hybridizes to the target sequence of the atrogene. In some embodiments, C consists of a polymer. In some embodiments, the at least one 2'-modified nucleotide comprises a nucleotide modified with 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA). In some embodiments, the at least one 2'-modified nucleotide comprises locked nucleic acid (LNA) or vinyl nucleic acid (ENA). In some embodiments, the linkage between the at least one modified nucleotide comprises a phosphate thioate linkage or a phosphate dithioate linkage. In some embodiments, the at least one reverse debasement moiety is at at least one end. In some embodiments, the polynucleotide comprises a single strand that hybridizes to the target sequence of the atrogene. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridizing with the first polynucleotide to form a double-stranded polynucleotide molecule, wherein the first or second polynucleotide also hybridizes with a target sequence of atrogene. In some embodiments, the second polynucleotide comprises at least one modification. In some embodiments, the first and second polynucleotides are RNA molecules. In some embodiments, the polynucleotide hybridizes with at least 8 consecutive bases of the target sequence of atrogene. In some embodiments, the polynucleotide comprises a sequence complementary to at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the sequences shown in SEQ ID NO:190-303 and 532-642. In some embodiments, the polynucleotide is about 8 to about 50 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 30 nucleotides in length. In some embodiments, the first polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequences shown in SEQ ID NO:304-417, 418-531, 643-753, 754-864, and 28-189. In some embodiments, the second polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequences shown in SEQ ID NO:304-417, 418-531, 643-753, 754-864, and 28-189. In some embodiments, _X1 and _X2 are independently _C1 - _C6 alkyl groups. In some embodiments, _X1 and _X2 are independently homo- or hetero-bifunctional linkers, optionally conjugated to a _C1 - _C6 alkyl group. In some embodiments, A is an antibody or a binding fragment thereof. In some embodiments, A includes a humanized antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a monovalent Fab', a bivalent Fab2, a single-chain variable fragment (scFv), a biantibody, a microantibody, a nanobody, a single-domain antibody (sdAb), or a camelid antibody or a binding fragment thereof. In some embodiments, A is an anti-transferrin receptor antibody or a binding fragment thereof. In some embodiments, C is polyethylene glycol. In some embodiments, _AX1 is conjugated to the 5' end of B, and _X2 -C is conjugated to the 3' end of B. In some embodiments, _X2 -C is conjugated to the 5' end of B, and _AX1 is conjugated to the 3' end of B. In some embodiments, A is directly conjugated to _X1 . In some embodiments, C is directly conjugated to _X2 . In some embodiments, B is directly conjugated to _X1 and _X2 . In some embodiments, the method further includes D. In some embodiments, D is conjugated to C or to A. In some embodiments, D is an endosome-soluble polymer. In some embodiments, the polynucleic acid conjugate is formulated for parenteral, oral, intranasal, buccal, rectal, or transdermal administration. In some embodiments, the subject is a human.

In some embodiments, this document discloses kits comprising the molecules described above or polynucleotide conjugates described above.

Attached Figure Description

The various aspects of this disclosure are set forth in detail in the appended claims. A better understanding of the features and advantages of this disclosure will be gained by referring to the following detailed description of illustrative embodiments utilizing the principles of this disclosure, and the accompanying drawings. This patent application document contains at least one color-drawn drawing. Upon request and payment of the necessary fees, the Patent Office will provide a copy of the published text of this patent application with color drawings.

Figure 1 shows an exemplary structure of a cholesterol-myosin siRNA conjugate.

Figure 2 shows the SAX HPLC chromatogram of TfR mAb-(Cys)-HPRT-PEG5k,DAR1.

Figure 3 shows the SEC HPLC chromatogram of TfR mAb-(Cys)-HPRT-PEG5k,DAR1.

Figure 4 shows the overlay of DAR1 and DAR2SAX HPLC chromatograms of the TfR1mAb-Cys-BisMal-siRNA conjugate.

Figure 5 shows the overlay of DAR1 and DAR2SEC HPLC chromatograms of the TfR1mAb-Cys-BisMal-siRNA conjugate.

Figure 6 shows the SEC chromatogram of CD71 Fab-Cys-HPRT-PEG5.

Figure 7 shows the SAX chromatogram of CD71 Fab-Cys-HPRT-PEG5.

Figure 8 shows the relative expression levels of Murf1 and atrogin-1 in C2C12 myoblasts and myotubes. C2C12 myoblasts and myotubes were generated as described in Example 4. mRNA levels were measured as described in Example 4.

Figure 9A illustrates an in vivo study design to evaluate the ability of an exemplary conjugate to mediate the downregulation of myostatin (MSTN) mRNA in skeletal muscle.

Figure 9B shows siRNA-mediated mRNA knockdown of mouse MSTN in the mouse gastrocnemius muscle.

Figure 10A illustrates an in vivo study design to evaluate the ability of an exemplary conjugate to mediate the downregulation of myostatin (MSTN) mRNA in skeletal muscle.

Figure 10B shows the tissue concentration-time curve up to 1008 h after administration of the exemplary formula (I) molecule.

Figure 10C shows siRNA-mediated mRNA knockdown of mouse MSTN in the mouse gastrocnemius muscle.

Figure 10D shows the reduction in plasma mRNA protein after siRNA-mediated mRNA knockdown of mouse MSTN in the mouse gastrocnemius muscle.

Figure 10E shows the changes in muscle size in the mouse gastrocnemius muscle after siRNA-mediated mRNA knockdown of mouse MSTN.

Figure 10F shows the Welch two-tailed unpaired t-test for Figure 10E.

Figure 11A illustrates an exemplary in vivo study design.

Figure 11B shows tissue accumulation of siRNA in the gastrocnemius muscle of mice after a single intravenous administration of the exemplary formula (I) molecule at the indicated dose.

Figure 11C shows siRNA-mediated mRNA knockdown of mouse MSTN in the mouse gastrocnemius muscle.

Figure 12A illustrates an exemplary in vivo study design.

Figure 12B shows the accumulation of siRNA in various muscle tissues.

Figure 12C shows siRNA-mediated mRNA knockdown of mouse MSTN in the gastrocnemius and myocardium of mice.

Figure 12D shows the RISC load of the MSTN guide chain in the mouse gastrocnemius muscle.

Figure 13A illustrates an exemplary in vivo study design.

Figure 13B shows siRNA-mediated mRNA knockdown of mouse MSTN in the gastrocnemius, quadriceps, triceps, and heart muscles of mice.

Figure 13C shows plasma myostatin levels.

Figure 13D shows the accumulation of siRNA in different tissue types: gastrocnemius, triceps, quadriceps, and cardiac tissue.

Figure 13E shows the RISC load of the MSTN guide chain in the mouse gastrocnemius muscle.

Figure 13F shows the changes in muscle area.

Figure 13G shows the Welch two-tailed unpaired t-test of Figure 13F.

Figure 14A illustrates an exemplary in vivo study design.

Figure 14B shows HPRT mRNA expression in the gastrocnemius muscle induced by the exemplary conjugate described herein.

Figure 14C shows the SSB mRNA expression in the gastrocnemius muscle induced by the exemplary conjugate described herein.

Figure 14D shows HPRT mRNA expression in cardiac tissue induced by the exemplary conjugates described herein.

Figure 14E shows the expression of SSB mRNA in cardiac tissue induced by the exemplary conjugates described herein.

Figure 14F shows the accumulation of siRNA in the gastrocnemius muscle.

Figure 15A illustrates an exemplary in vivo study design.

Figure 15B shows the downregulation of Atrogin-1 in the gastrocnemius muscle.

Figure 15C shows the downregulation of Atrogin-1 in cardiac tissue.

Figure 16A illustrates an exemplary in vivo study design.

Figure 16B shows the downregulation of MuRF-1 in the gastrocnemius muscle.

Figure 16C shows the downregulation of MuRF-1 in cardiac tissue.

Figure 17 shows the siRNAs transfected into mouse C2C12 myoblasts in vitro. All four DMPK siRNAs evaluated showed DMPK mRNA knockdown, while the negative control siRNA did not. The dashed line is the three-parameter curve fitted by nonlinear regression.

Figures 18A-18F show in vivo results demonstrating a stable dose response to DMPK mRNA knockdown 7 days after a single intravenous administration of the DMPK siRNA-antibody conjugate. Figure 18A: Gastrocnemius muscle; Figure 18B: Tibialis anterior muscle; Figure 18C: Quadriceps muscle; Figure 18D: Diaphragm muscle; Figure 18E: Heart; Figure 18F: Liver.

Figures 19A-19L show the exemplary antibody-nucleic acid conjugates described herein.

Figure 19M shows the antibody cartoon used in Figures 19A-19L.

Figures 20A and 20B illustrate the exemplary 2l-mer double strand used in Example 20. Figure 20A shows a representative structure of the siRNA transit strand with a C6- _NH2 stalk at the 5' end and a C6-S-NEM at the 3' end. Figure 20B shows a representative structure of the 2l-mer double strand with 19 complementary bases and a 3' dinucleotide overhang.

Figures 21A-21B illustrate the second exemplary 2l-polymer double strand used in Example 20. Figure 21A shows a representative structure of the siRNA transit strand with a 5' conjoined handle. Figure 21B shows a representative structure of a blunt-ended double strand with 19 complementary bases and a 3' dinucleotide overhang.

Figure 22 shows an illustrative in vivo study design.

Figure 23 shows the time course of Atrogin-1 mRNA downregulation in gastrocnemius muscle mediated by a TfR1 antibody siRNA conjugate following a single intravenous dose of 3 mg/kg.

Figure 24 shows the time course of Atrogin-1 mRNA downregulation in myocardium mediated by TfR1 antibody siRNA conjugate following a single intravenous dose of 3 mg/kg.

Figure 25 shows an illustrative in vivo study design.

Figure 26 shows the downregulation of MuRF1 mRNA in the gastrocnemius muscle 96 hours after intravenous delivery at the dose indicated.

Figure 27 shows the downregulation of MuRF1 mRNA in the myocardium 96 hours after intravenous delivery at the dose shown, mediated by the TfR1 antibody siRNA conjugate.

Figure 28 shows the time course of MuRF1 and Atrogin-1 mRNA downregulation in the gastrocnemius muscle mediated by TfR1 antibody siRNA conjugate (intravenous delivery of 3 mg/kg siRNA) in the absence and presence of dexamethasone-induced muscle atrophy.

Figure 29 shows the time course of MuRF1 and Agtrogin1 mRNA downregulation in the myocardium mediated by TfR1 antibody siRNA conjugate (intravenous delivery of 3 mg/kg siRNA) in the absence and presence of dexamethasone-induced myocardial atrophy.

Figure 30 shows the time course of gastrocnemius muscle weight changes mediated by TfR1 antibody siRNA conjugate (intravenous delivery of 3 mg/kg siRNA) in the absence and presence of muscle atrophy.

Figure 31 shows the time course of siRNA tissue concentrations in the gastrocnemius and myocardium mediated by TfR1 antibody siRNA conjugates (delivered intravenously at 3 mg/kg siRNA) in the absence and presence of muscle atrophy.

Figure 32 shows an illustrative in vivo study design.

Figure 33 shows the downregulation of Atrogin-1 mRNA in the gastrocnemius muscle 10 days after TfR1 antibody-siRNA conjugate, relative to the measured concentration of siRNA in the tissue, in the absence and presence of dexamethasone-induced atrophy (starting on day 7).

Figure 34 shows the relative Atrogin-1 mRNA levels in the gastrocnemius muscle of the scrambled control group in the absence of dexamethasone (groups 10 and 13, and groups 11 and 14) and in the presence of dexamethasone-induced atrophy (groups 12 and 15).

Figure 35 shows the relative RISC load of the Atrogin-1 guide chain in mouse gastrocnemius muscle after TfR1-mAb conjugate delivery in the absence and presence of dexamethasone-induced atrophy.

Figure 36 shows the time course of MSTN mRNA downregulation in gastrocnemius muscle following delivery of the TfR1 antibody siRNA conjugate, relative to the PBS control, in the absence of (solid line) and presence of (dashed line) dexamethasone-induced atrophy (starting on day 7).

Figure 37 shows the rate of leg muscle growth in the gastrocnemius muscle after delivery of the TfR1-mAb conjugate in the absence and presence of dexamethasone-induced atrophy.

Figure 38 shows an illustrative in vivo study design.

Figure 39A shows that a single treatment with 4.5 mg/kg (siRNA) of Atrogin-1 siRNA or MuRF1 siRNA, or a single dose of a combination of the two siRNAs, resulted in downregulation of up to 75% of each target in the gastrocnemius muscle.

Figure 39B shows that, up to 37 days after ASC administration, the mRNA knockdown of the two targets in the gastrocnemius muscle remained at 75% throughout the leg.

Figure 39C shows the changes in muscle area.

Figure 39D shows the change in the weight of the gastrocnemius muscle.

Figure 39E shows the percentage reduction in muscle atrophy caused by treatment in terms of leg muscle area. Statistical analysis was performed using Welch’s T-test to compare the treatment group with the disordered siRNA control group.

Figure 39F shows the percentage reduction in muscle atrophy caused by treatment, in terms of gastrocnemius muscle weight.

Detailed Implementation

Muscle atrophy refers to the loss of muscle mass or progressive weakening and degeneration of muscles such as skeletal or voluntary muscles that control movement, cardiac muscle, and smooth muscle. Various pathophysiological conditions, including disuse, starvation, cancer, diabetes, and renal failure, or glucocorticoid treatment, can lead to muscle atrophy and decreased strength. The phenotypic effects of muscle atrophy are caused by a variety of molecular events, including inhibition of muscle protein synthesis, increased muscle protein turnover, abnormal regulation of satellite cell differentiation, and abnormal transformation of muscle fiber types.

Extensive research has established that muscle atrophy is an active process controlled by specific signaling pathways and transcriptional programs. Exemplary pathways involved in this process include, but are not limited to, the IGF1-Akt-FoxO pathway, the glucocorticoid-GR pathway, the PGC1α-FoxO pathway, the TNFα-NFκB pathway, and the myostatin-ActRIIb-Smad2/3 pathway.

In some cases, therapeutic interventions targeting the mechanisms regulating muscle atrophy focus on IGF1-Akt, TNFα-NfκB, and myostatin. While IGF1 analogs have shown efficacy in treating muscle atrophy, the IGF1-Akt pathway's involvement in promoting tumorigenesis and hypertrophy hinders these therapies. Modulation of the Akt-mTOR pathway using β-adrenergic agonists also involves similar risks. Inhibition of myostatin by using soluble ActRIIB or ligands that block ActRIIb antibodies can prevent and reverse skeletal muscle loss and prolong the survival of tumor-bearing animals. However, the mechanism of myostatin blockade in preventing muscle atrophy remains uncertain, as neither dominant-negative ActRIIb expression nor Smad2/3 knockdown prevented muscle loss after denervation (Satori et al., "Smad2 and 3 transcription factors control muscle mass in adulthood", Am J Physiol Cell Physiol 296:C1248-C1257, 2009).

Comparison of gene expression in different models of muscle atrophy (including diabetes, cancer cachexia, chronic renal failure, fasting, and denervation) has led to the identification of atrophy-associated genes, termed atrogenes (Sacheck et al., “Rapid disuse and denervation atrophy involve transcriptional changes similar to those of muscle wasting during systemic diseases”, The FASEB Journal, 21(1):140-155, 2007), which are typically upregulated or downregulated in atrophied muscle. Genes strongly upregulated under atrophic conditions include muscle-specific ubiquitin (E3) ligases (e.g., atrogin-1, MuRF1), forkhead box transcription factors, and proteins mediating stress responses. In some cases, many of these effector proteins are difficult to modulate using conventional drugs.

Nucleic acid (e.g., RNAi) therapy is a highly selective and specific targeted therapy. However, in some cases, nucleic acid therapy is also hampered by poor intracellular uptake, limited blood stability, and nonspecific immune stimulation. To address these issues, various modifications to nucleic acid compositions have been explored, such as novel linkers for better stability and/or reduced toxicity, optimization of binding moieties for improved target specificity and/or target delivery, and nucleic acid polymer modifications for improved stability and/or reduced off-target effects.

In some embodiments, the arrangement or sequence of the different components constituting the nucleic acid composition further affects intracellular uptake, stability, toxicity, efficacy, and/or nonspecific immune stimulation. For example, if the nucleic acid component includes a binding moiety, a polymer, and a polynucleotide molecule (or polynucleotide), the order or arrangement of the binding moiety, polymer, and/or polynucleotide molecule (or polynucleotide) (e.g., binding moiety-polynucleotide molecule-polymer, binding moiety-polymer-polynucleotide molecule, or polymer-binding moiety-polynucleotide molecule) further affects intracellular uptake, stability, toxicity, efficacy, and/or nonspecific immune stimulation.

In some embodiments, the polynucleotide molecules and polynucleotide conjugates described herein include those for the treatment of myasthenia gravis or myotonic dystrophy. In some cases, the polynucleotide conjugates described herein enhance intracellular uptake, stability, and/or potency. In some cases, the polynucleotide conjugate comprises a molecule of formula (I): _AX1 - _BX2 -C. In some cases, the polynucleotide molecule hybridizes to one or more target sequences of atrogene.

Other implementation methods described herein include methods for treating muscle atrophy or myotonic dystrophy, which include administering the polynucleotide molecules or polynucleotide conjugates described herein to a subject.

Atrogene

Atrogenes, or atrophy-related genes, are genes that are upregulated or downregulated in atrophied muscle. In some cases, upregulated atrogenes include genes encoding ubiquitin ligases, forkhead box transcription factors, growth factors, deubiquitinating enzymes, or proteins involved in glucocorticoid-induced atrophy.

ubiquitin ligase

In some embodiments, the atrogene described herein encodes an E3 ubiquitin ligase. Exemplary E3 ubiquitin ligases include, but are not limited to, Atrogin-1/MAFbx, muscle ring finger 1 (MuRF1), TNF receptor adaptor protein 6 (TRAF6), F-Box protein 30 (Fbxo30), F-Box protein 40 (Fbxo40), neural progenitor cell expression downregulation protein 4 (Nedd4-1), and protein 32 containing a three-part motif (Trim32). Exemplary mitochondrial ubiquitin ligases include, but are not limited to, mitochondrial E3 ubiquitin protein ligase 1 (Mul1) and the C-terminus (CHIP) of Hsc70 interacting proteins.

In some implementations, the atrogene described herein encodes Atrogin-1, also known as the muscle atrophy F-box (MAFbx), a member of the F-box protein family. Atrogin-1/MAFbx is one of the four subunits of the ubiquitin ligase complex SKP1-cullin-F-box (SCF), which promotes the degradation of MyoD—a muscle transcription factor—and the eukaryotic translation initiation factor 3 subunit F (eIF3-f). Atrogin-1/MAFbx is encoded by FBXO32.

In some embodiments, the atrogene described herein encodes muscle ring finger 1 (MuRF1). MuRF1 is a member of the muscle-specific ring finger protein family and is found along with family members MuRF2 and MuRF3 at the M- and Z-line lattices of myofibrils. Furthermore, some studies have shown that MuRF1 interacts with and/or regulates the half-life of muscle structural proteins such as troponin I, myosin heavy chain, actin, myosin-binding protein C, and myosin light chains 1 and 2. MuRF1 is encoded by TRIM63.

In some embodiments, the atrogene described herein encodes TNF receptor adaptor protein 6 (TRAF6) (also known as interleukin-1 signaling transducer, cyclic finger protein 85, or RNF85). TRAF6 is a member of the E3 ligase, which mediates the conjugation of Lys63-linked polyubiquitin chains to target proteins. The Lys63-linked polyubiquitin chains emit an autophagy-dependent cargo recognition signal via the scaffold protein p62 (SQSTM1). TRAF6 is encoded by the TRAF6 gene.

In some embodiments, the atrogene described herein encodes F-Box protein 30 (Fbxo30) (also known as F-Box-only protein, helicase 18; muscle ubiquitin ligase of the SCF complex in atrophy-1; or MUSA1). Fbxo30 is a member of the SCF complex family of E3 ubiquitin ligases. In one study, it was proposed that Fbox30 is inhibited by the bone morphogenetic protein (BMP) pathway and upregulated under conditions of induced atrophy, subsequently undergoing autoubiquitination. Fbxo30 is encoded by the FBXO30 gene.

In some embodiments, the atrogene described herein encodes F-Box protein 40 (Fbxo40) (also known as F-Box protein 40 only or muscle disease-associated protein). Fbxo40 is the second member of the SCF complex family of E3 ubiquitin ligases that regulate anabolic signaling. In some cases, Fbxo40 ubiquitinates insulin receptor substrate 1 (a downstream effector of insulin receptor-mediated signaling) and influences its degradation. Fbxo40 is encoded by the FBXO40 gene.

In some implementations, the atrogene described herein encodes neural progenitor cell expression downregulated protein 4 (Nedd4-1), a HECT domain E3 ubiquitin ligase that has been shown to be upregulated in muscle cells during disuse. Nedd4-1 is encoded by the NEDD4 gene.

In some implementations, the atrogene described herein encodes a trimeric motif protein 32 (Trim32). Trim32 is a member of the E3 ubiquitin ligase family and is involved in the degradation of filaments such as actin, tropomyosin, troponin, α-actin, and desmin. Trim32 is encoded by the TRIM32 gene.

In some embodiments, the atrogene described herein encodes mitochondrial E3 ubiquitin ligase 1 (Mul1) (also known as mitochondrial anchoring protein ligase, ring finger protein 218, RNF218, MAPL, MULAN, and GIDE). Mul1 is involved in mitochondrial network remodeling and is upregulated by the FoxO transcription factor family under catabolic conditions (e.g., denervation or fasting), subsequently leading to mitochondrial fragmentation and removal via autophagy (mitochondrial phagocytosis). Furthermore, Mul1 ubiquitinates the mitochondrial fusion-promoting protein, mitochondrial fusion protein 2 (a GTPase involved in mitochondrial fusion), leading to its degradation. Mul1 is encoded by the MUL1 gene.

In some embodiments, the atrogene described herein encodes the carboxyl terminus (CHIP) of an Hsc70 interacting protein (also known as a STIP1 homolog and containing U-Box protein 1, STUB1, CFF-associated antigen KW-8, antigen NY-CO-7, SCAR16, SDCCAG7, or UBOX1). CHIP is a mitochondrial ubiquitin ligase that regulates the ubiquitination and lysosomal-dependent degradation of serine C (a muscle protein found in Z-lines). Z-lines, or Z-discs, are structures formed between adjacent sarcomeres, the basic units of muscle. Changes in the filamentin structure trigger the binding of the co-chaperone protein BAG3, a complex containing chaperone proteins Hsc70 and HspB8 with CHIP. Subsequently, the ubiquitination of BAG3 and filamentin by CHIP activates the autophagy system, leading to the degradation of filamentin C. CHIP is encoded by the STUB1 gene.

Forkhead box transcription factors

In some implementations, the atrogene described herein encodes a forkhead box transcription factor. Exemplary forkhead box transcription factors include, but are not limited to, isotypes of forkhead box protein O1 (FoxO1) and forkhead box protein O3 (FoxO3).

In some implementations, the atrogene described herein encodes forkhead box protein O1 (FoxO1) (also known as the forkhead homolog in rhabdomyosarcoma, FKHR, or FKH1). FoxO1 is involved in the regulation of gluconeogenesis and glycogenolysis via insulin signaling, and initiation of adipogenesis via preadipocytes. FoxO1 is encoded by the FOXO1 gene.

In some embodiments, the atrogene described herein encodes forkhead box protein O3 (FoxO3) (also known as forkhead-like 1, FKHRL1, or FOXO3A in rhabdomyosarcoma). FOXO3 is activated by the AMP-activated protein kinase AMPK, which in turn induces the expression of atrogin-1 and MuRF1. FoxO3 is encoded by the FOXO3 gene.

growth factors

In some implementations, the atrogene described herein encodes a growth factor. Exemplary growth factors include myostatin.

In some cases, the atrogene described herein encodes myostatin (Mstn), also known as growth/differentiation factor 8 (GDF-8). Myostatin is converted into an activator within the cell and stimulates muscle degradation and inhibits muscle synthesis by inhibiting Akt via phosphorylation/activation of Smad (small mothers against decapentaplegic). In some cases, myostatin has been found to be regulated by the Akt-FoxO signaling pathway. In other cases, myostatin has been shown to inhibit satellite cell differentiation, stimulate muscle degradation by inhibiting the Akt pathway, and inhibit muscle synthesis via the mTOR pathway.

Deubiquitinase

In some embodiments, the atrogene described herein encodes a deubiquitinizing enzyme. Exemplary deubiquitinizing enzymes include, but are not limited to, ubiquitin-specific peptidase 14 (USP14) and ubiquitin-specific peptidase 19 (USP19). In some cases, the atrogene described herein encodes USP14 (also known as deubiquitinase 14 or TGT). In other cases, the atrogene described herein encodes USP19 (also known as zinc finger MYND domain protein 9, deubiquitinase 19, or ZMYND9). USP14 is encoded by the USP14 gene. USP19 is encoded by the USP19 gene.

Other Atrogene

In some embodiments, the atrogene described herein encodes developmental and DNA damage response regulation 1 (Redd1), also known as DNA damage-inducible transcript 4 (DDIT4) and the HIF-1 response protein RTP801. Redd1 inhibits mTOR function and increases TSC1/2 activity by chelating 14-3-3. Furthermore, Redd1 reduces phosphorylation of 4E-BP1 and S6K1, which are involved in muscle protein synthesis. Redd1 is encoded by the DDIT4 gene.

In some embodiments, the atrogene described herein encodes cathepsin L2, also known as cathepsin V. Cathepsin L2 is a lysosomal cysteine protease. It is encoded by the CTSL2 gene.

In some implementations, the atrogene described herein encodes the TG interacting factor, or homeobox protein TGIF1. The TG interacting factor is a transcription factor that regulates signal transduction pathways involved in embryonic development. This protein is encoded by the TGIF gene.

In some implementations, the atrogene described herein encodes myocyte-producing protein, also known as myogenic factor 4. Myocyte-producing protein is a member of the muscle-specific basic-helix-loop-helix (bHLH) transcription factor MyoD family, which is involved in the coordination of skeletal muscle development and repair. Myocyte-producing protein is encoded by the MYOG gene.

In some implementations, the atrogene described herein encodes myotonic protein kinase (MT-PK), also known as myotonic dystrophy protein kinase (MDPK) or dystrophic myotonic protein kinase (DMK). MT-PK is a serine/threonine kinase that further interacts with members of the Rho family of GTPases. In humans, MT-PK is encoded by the DMPK gene.

In some implementations, the atrogene described herein encodes histone deacetylase 2—a member of the histone deacetylase family. Histone deacetylase 2 is encoded by the HDAC2 gene.

In some implementations, the atrogene described herein encodes histone deacetylase 3—another member of the histone deacetylase family. Histone deacetylase 3 is encoded by the HDAC3 gene.

In some embodiments, the atrogene described herein encodes metallothionein 1L—a member of the metallothionein family. Metallothioneins (MTs) are cysteine-rich, low-molecular-weight proteins that bind heavy metals, thereby providing protection against metal toxicity and/or oxidative stress. Metallothionein 1L is encoded by the MT1L gene.

In some implementations, the atrogene described herein encodes metallothionein 1B—the second member of the metallothionein family. Metallothionein 1B is encoded by the MT1B gene.

In some implementations, the atrogene described herein is the atrogene listed in Table 14.

Polynucleic acid molecules

In some embodiments, the polynucleotide molecule hybridizes to the target sequence of atrophy-associated genes (also known as atrogenes). In some cases, the polynucleotide molecule described herein hybridizes to the target sequence of ubiquitin ligases (e.g., E3 ubiquitin ligase or mitochondrial ubiquitin ligase). In some cases, the polynucleotide molecule described herein hybridizes to the target sequence of forkhead box transcription factors. In some cases, the polynucleotide molecule described herein hybridizes to the target sequence of growth factors. In some cases, the polynucleotide molecule described herein hybridizes to the target sequence of deubiquitinating enzymes.

In some embodiments, the polynucleotide molecules described herein hybridize with the target sequences of FBXO32, TRIM63, TRAF6, FBXO30, FBXO40, NEDD4, TRIM32, MUL1, STUB1, FOXO1, FOXO3, MSTN, USP14, USP19, DDIT4, CTSL2, TGIF, MYOG, HDAC2, HDAC3, MT1L, MT1B, or DMPK. In some cases, the polynucleotide molecules described herein hybridize with the target sequences of FBXO32, TRIM63, FOXO1, FOXO3, or MSTN. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of FBXO32. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of TRIM63. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of TRAF6. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of FBXO30. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of FBXO40. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of NEDD4. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of TRIM32. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of MUL1. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of STUB1. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of FOXO1. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of FOXO3. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of MSTN. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of USP14. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of USP19. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of DDIT4. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of CTSL2. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of TGIF. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of MYOG. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of HDAC2. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of HDAC3. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of MT1L. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of MT1B. In some cases, the polynucleotide molecules described herein hybridize with the target sequence of DMPK.

In some embodiments, the polynucleotide molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the target sequences shown in SEQ ID NO: 28-141 and 370-480. In some embodiments, the polynucleotide molecule comprises a sequence having at least 50% sequence identity with the target sequences shown in SEQ ID NO: 190-303 and 532-642. In some embodiments, the polynucleotide molecule comprises a sequence having at least 60% sequence identity with the target sequences shown in SEQ ID NO: 190-303 and 532-642. In some embodiments, the polynucleotide molecule comprises a sequence having at least 70% sequence identity with the target sequences shown in SEQ ID NO: 190-303 and 532-642. In some embodiments, the polynucleotide molecule comprises a sequence having at least 75% sequence identity with the target sequences shown in SEQ ID NO:190-303 and 532-642. In some embodiments, the polynucleotide molecule comprises a sequence having at least 80% sequence identity with the target sequences shown in SEQ ID NO:190-303 and 532-642. In some embodiments, the polynucleotide molecule comprises a sequence having at least 85% sequence identity with the target sequences shown in SEQ ID NO:190-303 and 532-642. In some embodiments, the polynucleotide molecule comprises a sequence having at least 90% sequence identity with the target sequences shown in SEQ ID NO:190-303 and 532-642. In some embodiments, the polynucleotide molecule comprises a sequence having at least 95% sequence identity with the target sequences shown in SEQ ID NO:190-303 and 532-642. In some embodiments, the polynucleotide molecule comprises a sequence having at least 96% sequence identity with the target sequences shown in SEQ ID NO:190-303 and 532-642. In some embodiments, the polynucleotide molecule comprises a sequence having at least 97% sequence identity with the target sequences shown in SEQ ID NO:190-303 and 532-642. In some embodiments, the polynucleotide molecule comprises a sequence having at least 98% sequence identity with the target sequences shown in SEQ ID NO:190-303 and 532-642. In some embodiments, the polynucleotide molecule comprises a sequence having at least 99% sequence identity with the target sequences shown in SEQ ID NO:190-303 and 532-642. In some embodiments, the polynucleotide molecule is composed of the target sequences shown in SEQ ID NO:190-303 and 532-642.

In some embodiments, the polynucleotide molecule comprises a first polynucleotide and a second polynucleotide. In some cases, 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 with the target sequences shown in SEQ ID NO: 304-417, 418-531, 643-753, 754-864, and 28-189. 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 with the target sequences shown in SEQ ID NO: 304-417, 418-531, 643-753, 754-864, and 28-189. In some embodiments, the polynucleotide molecule comprises a first polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the target sequences shown in SEQ ID NO:304-417, 643-753, and 28-108, and a second polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the target sequences shown in SEQ ID NO:418-531, 754-864, and 109-189.

In some embodiments, the polynucleotide molecule comprises a sense strand (e.g., a guest strand) and an antisense strand (e.g., a guide strand). In some cases, the sense strand (e.g., the guest 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 with the target sequences shown in SEQ ID NO: 304-417, 643-753, and 28-108. In some cases, the antisense strand (e.g., the guide strand) contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the target sequences shown in SEQ ID NO:418-531, 754-864, and 109-189.

In some embodiments, the polynucleotide molecule described herein comprises RNA or DNA. In some cases, the polynucleotide molecule comprises RNA. In some cases, the 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 cases, the RNA comprises shRNA. In some cases, the RNA comprises miRNA. In some cases, the RNA comprises dsRNA. In some cases, the RNA comprises tRNA. In some cases, the RNA comprises rRNA. In some cases, the RNA comprises hnRNA. In some cases, the RNA comprises siRNA. In some cases, the polynucleotide molecule comprises siRNA.

In some embodiments, the length of the polynucleotide molecule is about 10 to about 50 nucleotides. In some cases, the length of the polynucleotide molecule is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides.

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

In some embodiments, the polynucleotide molecule comprises a first polynucleotide. In some cases, the polynucleotide molecule comprises a second polynucleotide. In some cases, the polynucleotide molecule comprises both a first polynucleotide and a second polynucleotide. In some cases, the first polynucleotide is a sense strand or a transit strand. In some cases, the second polynucleotide is an antisense strand or a guide strand.

In some embodiments, the polynucleotide molecule is a first polynucleotide. In some embodiments, the length of the first polynucleotide is about 10 to about 50 nucleotides. In some cases, the length of the first polynucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, about 19 to about 30, about 19 to about 25, about 19 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides.

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

In some embodiments, the polynucleotide molecule is a second polynucleotide. In some embodiments, the length of the second polynucleotide is about 10 to about 50 nucleotides. In some cases, the length of the second polynucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides.

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

In some embodiments, the polynucleotide molecule comprises a first polynucleotide and a second polynucleotide. In some cases, the polynucleotide molecule further comprises a blunt end, a protruding end, or a combination thereof. In some cases, the blunt end is a 5' blunt end, a 3' blunt end, or both. In some cases, the protruding end is a 5' protruding end, a 3' protruding end, or both. In some cases, the protruding end comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-base-paired nucleotides. In some cases, the protruding end comprises 1, 2, 3, 4, 5, or 6 non-base-paired nucleotides. In some cases, the protruding end comprises 1, 2, 3, or 4 non-base-paired nucleotides. In some cases, the protruding end comprises 1 non-base-paired nucleotide. In some cases, the protruding end comprises 2 non-base-paired nucleotides. In some cases, the protruding end comprises 3 non-base-paired nucleotides. In some cases, the protruding end comprises 4 non-base-paired nucleotides.

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

In some embodiments, the sequence of the polynucleotide molecule has five or fewer mismatches with the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule has four or fewer mismatches with the target sequence described herein. In some cases, the sequence of the polynucleotide molecule has three or fewer mismatches with the target sequence described herein. In some cases, the sequence of the polynucleotide molecule has two or fewer mismatches with the target sequence described herein. In some cases, the sequence of the polynucleotide molecule has one or fewer mismatches with the target sequence described herein.

In some implementations, the specificity of the polynucleotide molecule hybridizing with the target sequence described herein is that the polynucleotide molecule is 95%, 98%, 99%, 99.5%, or 100% complementary to the target sequence. In some cases, the hybridization is performed under highly stringent conditions.

In some embodiments, the polynucleotide molecule has reduced off-target effects. In some cases, "off-target" or "off-target effect" refers to any situation where a polynucleotide polymer targeting a given target causes an unintended effect through direct or indirect interaction with another mRNA sequence, DNA sequence, or cellular protein or other part. In some cases, an "off-target effect" occurs when other transcripts are simultaneously degraded due to partial homology or complementarity between other transcripts and the sense and/or antisense strands of the polynucleotide molecule.

In some embodiments, the polynucleotide molecule comprises natural or synthetic or artificial nucleotide analogs or bases. In some cases, the polynucleotide molecule comprises a combination of DNA, RNA, and/or nucleotide analogs. In some cases, the synthetic or artificial nucleotide analog or base contains modifications at one or more of the ribose moiety, phosphate moiety, nucleoside moiety, or combinations thereof.

In some embodiments, the nucleotide analog or artificial nucleotide base comprises a nucleic acid having a modified 2' hydroxyl group at the ribose moiety. In some cases, the modification includes H, OR, R, halogen, SH, SR, NH2, NHR, NR2, or CN, where R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogens, sulfur, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary amines), amides, ethers, esters, alcohols, and oxygen. In some cases, the alkyl moiety further comprises a modification. In some cases, the modification includes an azo group, a ketone group, an aldehyde group, a carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocyclic group (e.g., imidazole, hydrazine, or hydroxyamino), an isocyanate or cyanate group, or a sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, and disulfide). In some cases, the alkyl moiety further comprises a heterosubstituted group. In some cases, the carbon atom of the heterocyclic group is replaced by nitrogen, oxygen, or sulfur. In some cases, the heterocyclic substitution includes, but is not limited to, morpholino, imidazole, and pyrrolidino.

In some cases, the modification at the 2' hydroxyl group is either a 2'-O-methyl modification or a 2'-O-methoxyethyl (2'-O-MOE) modification. In some cases, the 2'-O-methyl modification adds a methyl group to the 2' hydroxyl group of the ribose moiety, while the 2'-O-methoxyethyl modification adds a methoxyethyl group to the 2' hydroxyl group of the ribose moiety. Exemplary chemical structures of 2'-O-methyl modifications in adenosine molecules and 2'-O-methoxyethyl modifications in uridine molecules are shown below.

In some cases, the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification, in which an extended amine group of the propyl linker binds the amine group to the 2' oxygen. In some cases, this modification neutralizes the total negative charge derived from the phosphate ester of the oligonucleotide molecule by introducing a positive charge from the amine group with each sugar, and improves cellular uptake properties due to its zwitterionic nature. An exemplary chemical structure of 2'-O-aminopropyl nucleoside phosphoramidide is shown below.

In some cases, the modification at the 2' hydroxyl group is a locked or bridged ribose modification (e.g., locked nucleic acid or LNA), in which the oxygen molecule bound to the 2' carbon is linked to the 4' carbon via a methylene group, thereby forming a 2'-C, 4'-C-oxy-methylene-linked bicyclic ribonucleotide monomer. An exemplary representation of the chemical structure of LNA is shown below. The representation shown on the left highlights the chemical linkage of the LNA monomer. The representation shown on the right highlights the locked 3'-internal ( ^3E ) conformation of the furanose ring of the LNA monomer.

In some cases, modifications at the 2' hydroxyl group include ethylene nucleic acids (ENAs), such as 2'-4'-ethylene-bridged nucleic acids, which lock the sugar conformation to a C _3' -puckering conformation. ENAs are part of a class of modified nucleic acids that are bridged nucleic acids and also contain LNAs. Exemplary chemical structures of ENAs and bridged nucleic acids are shown below.

In some embodiments, additional modifications at the 2' hydroxyl group include 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA).

In some embodiments, the nucleotide analog comprises modified bases such as, but not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N'-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having modifications at the 5 position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenine, 2-methyladenine, 3-methylcytidine, 6-methyluridine, 2-methyluridine, 7-methyluridine, 2,2-dimethyluridine, 5-methylaminoethyluridine, 5-methyloxyuridine, denitronucleotides such as 7-denitro- Adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine, other thiobases such as 2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine, pseudouridine, braided glycoside, archapurin, naphthyl and substituted naphthyl, any O-alkylated and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-hydroxyacetic acid, pyridin-4-one, pyridin-2-one, phenyl and modified phenyl such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosine acting as G-clamp nucleotides, 8-substituted adenine and guanine, 5-substituted uracil and thymine, azapyrimidine, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include nucleotides modified at the sugar moiety, as well as nucleotides having a non-ribosyl sugar or analogue. For example, in some cases, the sugar moiety is or is based on mannose, arabinose, pyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbon rings. The term nucleotide also includes universal bases known in the art. For example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or muscarin.

In some embodiments, the modified nucleotide analogue further comprises morpholino, peptide nucleic acid (PNA), methylphosphonate nucleotide, thiol phosphonate nucleotide, 2'-fluoroN3-P5'-phosphamide, 1',5'-anhydrohexyl alcohol nucleic acid (HNA), or combinations thereof. Morpholino or diaminophosphate morpholino oligonucleotides (PMOs) comprise synthetic molecules whose structure mimics the structure of native nucleic acids but deviates from the normal sugar and phosphate ester structures. In some cases, the five-membered ribose ring is replaced by a six-membered morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, the ribose monomer is linked by a diaminophosphate group instead of a phosphate ester group. In such cases, the skeletal alteration removes all positive and negative charges, allowing the neutral morpholino molecule to cross the cell membrane without the aid of a cell delivery agent, such as the one used by charged oligonucleotides.

In some implementations, the peptide nucleic acid (PNA) does not contain sugar rings or phosphate ester linkages, and the bases are linked and appropriately spaced by oligoglycine-like molecules, thus eliminating skeletal charge.

In some embodiments, one or more modifications optionally occur at the internucleotide linker. In some cases, the modified internucleotide linker includes, but is not limited to, thiophosphates, dithiophosphates, methylphosphonates, 5'-alkylphosphonates, 5'-methylphosphonates, 3'-alkylphosphonates, boron trifluoride, 3'-5' linked or 2'-5' linked boron phosphates and selenophosphates, triphosphates, thiocarbonylalkyl phosphates, hydrogen phosphonates, alkylphosphonates, alkylthiophosphates, arylthiophosphates, phosphoroselenoate, diselenoate, hypophosphonates, aminophosphates, 3'-alkylaminophosphates, aminoalkylaminophosphates, thiocarbonylaminophosphates, piperazine phosphates, aniline thiophosphates, and aniline. Phosphate esters, ketones, sulfones, sulfonamides, carbonates, carbamates, methylenehydrazo, methylenedimethylhydrazo, formacetal, thiomethyl acetal, oximes, methyleneimine, methylenemethylimine, thioamides, linkages with riboacetyl groups, aminoethylglycine, silyl or siloxane linkages, linkages with or without heteroatoms of alkyl or cycloalkyl groups, for example, 1 to 10 saturated or unsaturated and/or substituted and/or heteroatom-containing carbons, linkages with morpholino groups, amides, polyamides, wherein the base is directly or indirectly linked to a nitrogen atom in the backbone, and combinations thereof. Phosphothiophosphate antisense oligonucleotides (PS ASOs) are antisense oligonucleotides containing phosphothiophosphate linkages. Exemplary PS ASOs are shown below.

In some cases, the modification is a methyl or thiol modification, such as a methylphosphonate or a thiolphosphonate modification. Exemplary thiolphosphonate nucleotides (left) and methylphosphonate nucleotides (right) are shown below.

In some cases, the modified nucleotides include, but are not limited to, 2’-fluoroN3-P5’-phosphoramide as shown below:

In some cases, the modified nucleotides include, but are not limited to, hexitol nucleic acids (or 1',5'-dehydrated hexitol nucleic acids (HNA)), as shown below:

In some embodiments, one or more modifications may further optionally include modifications to the ribose moiety, phosphate backbone, and nucleoside, or modifications to a nucleotide analog at the 3' or 5' end. For example, the 3' end may optionally contain a 3' cationic group, or a reverse nucleoside linked at the 3' end in 3'-3'. In another alternative, the 3' end may optionally be conjugated to an aminoalkyl group, such as 3'C5-aminoalkyl dT. In yet another alternative, the 3' end may optionally be conjugated to a debasement site, such as a depurinyl or depyrimidine site. In some cases, the 5' end may be conjugated to an aminoalkyl group, such as a 5'-O-alkylamino substituent. In some cases, the 5' end may be conjugated to a debasement site, such as a depurinyl or depyrimidine site.

In some embodiments, the polynucleotide molecule comprises one or more of the artificial nucleotide analogs described herein. In some cases, the polynucleotide molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more of the artificial nucleotide analogs described herein. In some embodiments, the artificial nucleotide analog 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-dimethylaminoethoxyethyl (2’-O-DMAEOE) or 2’-O-N-methylacetamido (2’-O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiol phosphonate nucleotide, 2’-fluoroN3-P5’-phosphamide or combinations thereof. In some cases, the polynucleotide molecule contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more artificial nucleotide analogs 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) Modified with 2’-O-dimethylaminoethyl (2’-O-DMAOE), 2’-O-dimethylaminopropyl (2’-O-DMAP), 2’-O-dimethylaminoethoxyethyl (2’-O-DMAEOE), or 2’-O-N-methylacetamido (2’-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiol phosphonate nucleotides, 2’-fluoroN3-P5’-phosphoramide, or combinations thereof. In some cases, the polynucleotide molecule contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more nucleotides modified with 2’-O-methyl. In some cases, the polynucleotide molecule contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more nucleotides modified with 2'-O-methoxyethyl (2'-O-MOE). In other cases, the polynucleotide molecule contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more thiol phosphonate nucleotides.

In some cases, the polynucleotide molecule contains at least one of the following: about 5% to about 100% modification, about 10% to about 100% modification, about 20% to about 100% modification, about 30% to about 100% modification, about 40% to about 100% modification, about 50% to about 100% modification, about 60% to about 100% modification, about 70% to about 100% modification, about 80% to about 100% modification, and about 90% to about 100% modification.

In some cases, the polynucleotide molecule contains at least one of the following: about 10% to about 90% modification, about 20% to about 90% modification, about 30% to about 90% modification, about 40% to about 90% modification, about 50% to about 90% modification, about 60% to about 90% modification, about 70% to about 90% modification, and about 80% to about 100% modification.

In some cases, the polynucleotide molecule contains at least one of the following: about 10% to about 80% modification, about 20% to about 80% modification, about 30% to about 80% modification, about 40% to about 80% modification, about 50% to about 80% modification, about 60% to about 80% modification, and about 70% to about 80% modification.

In some cases, the polynucleotide molecule contains at least one of the following: about 10% to about 70% modification, about 20% to about 70% modification, about 30% to about 70% modification, about 40% to about 70% modification, about 50% to about 70% modification, and about 60% to about 70% modification.

In some cases, the polynucleotide molecule contains at least one of the following: about 10% to about 60% modification, about 20% to about 60% modification, about 30% to about 60% modification, about 40% to about 60% modification, and about 50% to about 60% modification.

In some cases, the polynucleotide molecule contains at least one of the following: about 10% to about 50% modification, about 20% to about 50% modification, about 30% to about 50% modification, and about 40% to about 50% modification.

In some cases, the polynucleotide molecule contains at least one of the following: about 10% to about 40% modification, about 20% to about 40% modification, and about 30% to about 40% modification.

In some cases, the polynucleic acid molecule contains at least one of the following: about 10% to about 30% of modification and about 20% to about 30% of modification.

In some cases, the polynucleotide molecule contains about 10% to about 20% of modifications.

In some cases, the polynucleotide molecule contains about 15% to about 90%, about 20% to about 80%, about 30% to about 70%, or about 40% to about 60% of modifications.

In other cases, the polynucleotide molecule contains at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of modifications.

In some embodiments, the polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modifications.

In some cases, the polynucleotide molecule contains at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modified nucleotides.

In some cases, approximately 5% to approximately 100% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 5% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 10% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 15% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 20% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 25% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 30% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 35% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 40% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 45% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 50% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 55% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 60% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 65% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 70% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 75% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 80% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 85% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 90% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 95% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 96% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 97% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 98% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 99% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 100% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some embodiments, the artificial nucleotide analogue 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’-O-DMAOE), 2’-O-dimethylaminopropyl (2’-O-DMAP), 2’-O-dimethylaminoethoxyethyl (2’-O-DMAEOE) or 2’-O-N-methylacetamido (2’-O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiol phosphonate nucleotide, 2’-fluoroN3-P5’-phosphamide or combinations thereof.

In some embodiments, the polynucleotide molecule comprises about 1 to about 25 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 1 modification, wherein the modification comprises the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 2 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 3 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 4 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 5 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 6 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 7 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 8 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 9 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 10 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 11 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 12 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 13 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 14 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 15 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 16 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 17 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 18 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 19 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 20 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 21 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 22 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 23 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 24 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 25 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein.

In some embodiments, the polynucleotide molecule is assembled from two separate polynucleotides, one of which contains a sense strand and the second polynucleotide contains an antisense strand of the polynucleotide molecule. In other embodiments, the sense strand is linked to the antisense strand via a linker molecule, which in some cases is a polynucleotide linker or a non-nucleotide linker.

In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotide in the sense strand comprises a 2'-O-methylpyrimidine nucleotide, and the purine nucleotide in the sense strand comprises a 2'-deoxypurine nucleotide. In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotide present in the sense strand comprises a 2'-deoxy-2'-fluoropyrimidine nucleotide, and the purine nucleotide present in the sense strand comprises a 2'-deoxypurine nucleotide.

In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotide, when present in the antisense strand, is a 2'-deoxy-2'-fluoropyrimidine nucleotide, and the purine nucleotide, when present in the antisense strand, is a 2'-O-methylpurine nucleotide.

In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotide, when present in the antisense strand, is a 2'-deoxy-2'-fluoropyrimidine nucleotide, and wherein the purine nucleotide, when present in the antisense strand, comprises a 2'-deoxy-purine nucleotide.

In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand includes a terminal cap portion at the 5' end, 3' end, or both the 5' and 3' ends. In other embodiments, the terminal cap portion is a reverse deoxygenated debasement portion.

In some implementations, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand contains a phosphate backbone modification at its 3' end. In some cases, this phosphate backbone modification is a thiophosphate ester.

In some implementations, the polynucleotide molecule contains a sense strand and an antisense strand, wherein the antisense strand contains a glycerol group modification at its 3' end.

In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein 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 phosphate thioester nucleotides linked together, 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) universally modified nucleotides, and optionally at the 3′ end, 5′ end, or both the 3′ and 5′ ends of the sense strand. The antisense strand comprises about 1 to about 10 or more, particularly about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more thiophosphate nucleotides linked together, 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) universally modified nucleotides, and optionally end cap molecules at the 3' end, 5' end, or both the 3' and 5' ends of the antisense strand. In other embodiments, one or more sense strands and/or antisense strands, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides, are chemically modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, and may or may not have one or more terminal cap molecules, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, linked between phosphate thioester nucleotides and/or present in the same or different strands, at the 3′ end, 5′ end, or both the 3′ and 5′ ends.

In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein 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 phosphate thioester nucleotides linked together, 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) universally modified nucleotides, and optionally at the 3′ end, 5′ end, or 3′ and 5′ ends of the sense strand. The antisense strand contains about 1 to about 25 or more, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more thiophosphate nucleotides linked together, 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) universally modified nucleotides, and optionally terminal cap molecules at the 3′ end, 5′ end, or both the 3′ and 5′ ends of the antisense strand. In other embodiments, one or more sense strands and/or antisense strands, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides, are chemically modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, having or not having about 1 to about 25 or more, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, linked between phosphate thioester nucleotides and/or present in the same or different strands, at the 3’ end, 5’ end, or at both the 3’ and 5’ ends.

In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein 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 phosphate thioester nucleotides linked together, 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) universally modified nucleotides, and optionally, in the sense strand... The antisense strand comprises about one to about ten or more, particularly about one, two, three, four, five, six, seven, eight, nine, ten or more, phosphate thioester nucleotides linked together, and/or one or more (e.g., about one, two, three, four, five, six, seven, eight, nine, ten or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about one, two, three, four, five, six, seven, eight, nine, ten or more) universally modified nucleotides, and optionally, end cap molecules at the 3′ end, the 5′ end, or both the 3′ and 5′ ends of the antisense strand. In other embodiments, one or more sense strands and/or antisense strands, such as 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, are chemically modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, and have or do not have one or more terminal cap molecules, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, linked together and/or present in the same or different strands, at the 3′ end, 5′ end or at both the 3′ and 5′ ends.

In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein 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 phosphate thioester nucleotides linked together, 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) universally modified nucleotides, and optionally at the 3′, 5′ or 3′ end of the sense strand. The antisense strand contains about 1 to about 25 or more, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more thiophosphate nucleotides linked together, 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) universally modified nucleotides, and optionally, end cap molecules at the 3′ end, 5′ end, or both the 3′ and 5′ ends of the antisense strand. In other embodiments, one or more sense strands and/or antisense strands, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides, are chemically modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, having or not having about 1 to about 5, such as about 1, 2, 3, 4, 5 or more, phosphate thioester nucleotides linked together and/or present in the same or different strands, at the 3′ end, 5′ end or at both the 3′ and 5′ ends.

In some embodiments, the polynucleotide molecule 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 phosphate thioester nucleotides linked together in each chain of the polynucleotide molecule.

In another embodiment, the polynucleotide molecule described herein includes a 2'-5' nucleotide linker. In some cases, the 2'-5' nucleotide linker is located at the 3' end, 5' end, or both 3' and 5' ends of one or both strands of the sequence. In other instances, the 2'-5' nucleotide linker is present at various other locations within one or both strands of the sequence, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides in one or both strands of the polynucleotide molecule, including each nucleotide linker, including a 2'-5' nucleotide linker; or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more purine nucleotides in one or both strands of the polynucleotide molecule, including each nucleotide linker, including a 2'-5' nucleotide linker.

In some embodiments, the polynucleotide molecule is a single-stranded polynucleotide molecule that mediates RNAi activity in cells or reconstituted in vitro systems, wherein the polynucleotide molecule contains a single-stranded polynucleotide complementary to the target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the polynucleotide are 2'-deoxy-2'-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides, or multiple pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides), and wherein any purine nucleotides present in the polynucleotide are 2'-deoxypurine nucleotides (e.g., whose purine nucleotides are 2'-deoxypurine nucleotides). All purine nucleotides in the polynucleotide are 2'-deoxypurine nucleotides, or multiple purine nucleotides are 2'-deoxypurine nucleotides), and optionally, terminal cap modifications are present at the 3' end, 5' end, or both 3' and 5' ends of the antisense sequence. The polynucleotide molecule optionally also includes about one to about four (e.g., about one, two, three, or four) terminal 2'-deoxynucleotides at the 3' end of the polynucleotide molecule, wherein the terminal nucleotide further includes one or more (e.g., one, two, three, or four) phosphate thioester nucleotides linked together, and wherein the polynucleotide molecule optionally also includes a terminal phosphate group, such as a 5' terminal phosphate group.

In some cases, one or more artificial nucleotide analogs described herein are resistant to nucleases, such as ribonucleases like RNase H, deoxyribonucleases like DNase, or exonucleases like 5'-3' and 3'-5' exonucleases, compared to natural polynucleotide molecules. In some cases, they contain 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), or... Artificial nucleotide analogs modified with 2'-O-N-methylacetamide (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides, 2'-fluoroN3-P5'-phosphinoamide, or combinations thereof, are resistant to nucleases such as ribonucleases like RNase H, deoxyribonucleases like DNase, or exonucleases such as 5'-3' and 3'-5' exonucleases. In some cases, 2'-O-methyl-modified polynucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, 2'-O-methoxyethyl (2'-O-MOE)-modified polynucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, polynucleotide molecules modified with 2'-O-aminopropyl are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, polynucleotide molecules modified with 2'-deoxy are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, polynucleotide molecules modified with 2'-deoxy-2'-fluoro are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, polynucleotide molecules modified with 2'-O-aminopropyl (2'-O-AP) are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, polynucleotide molecules modified with 2'-O-dimethylaminoethyl (2'-O-DMAOE) are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, polynucleotide molecules modified with 2'-O-dimethylaminopropyl (2'-O-DMAP) are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, polynucleotide molecules modified with 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, polynucleotide molecules modified with 2'-O-N-methylacetamide (2'-O-NMA) are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, LNA-modified polynucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, ENA-modified polynucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, HNA-modified polynucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, morpholinonucleotides are resistant to nucleases (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, PNA-modified polynucleotides are resistant to nucleases (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, methylphosphonate nucleotide-modified polynucleotides are resistant to nucleases (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, thiolphosphonate nucleotide-modified polynucleotides are resistant to nucleases (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, polynucleotide molecules containing 2'-fluoroN3-P5'-phosphoramide are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, the 5' conjugates described herein inhibit 5'-3' exonuclease cleavage. In some cases, the 3' conjugates described herein inhibit 3'-5' exonuclease cleavage.

In some implementations, one or more artificial nucleotide analogs described herein have increased binding affinity to their mRNA targets relative to equivalent natural polynucleotide molecules. Compared to equivalent natural polynucleotide molecules, one or more artificial nucleotide analogs containing 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-dimethylaminoethoxyethyl (2’-O-DMAEOE) or 2’-O-N-methylacetamido (2’-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides or 2’-fluoroN3-P5’-phosphamide may have increased binding affinity to their mRNA targets. In some cases, 2'-O-methyl-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, 2'-O-methoxyethyl (2'-O-MOE)-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, 2'-O-aminopropyl-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, 2'-deoxy-2'-fluoro-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, 2'-O-aminopropyl (2'-O-AP)-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, polynucleotide molecules modified with 2'-O-dimethylaminoethyl (2'-O-DMAOE) exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotide molecules. In some cases, polynucleotide molecules modified with 2'-O-dimethylaminopropyl (2'-O-DMAP) exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotide molecules. In some cases, polynucleotide molecules modified with 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotide molecules. In some cases, polynucleotide molecules modified with 2'-O-N-methylacetamido (2'-O-NMA) exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotide molecules. In some cases, LNA-modified polynucleotide molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotide molecules. In some cases, ENA-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, PNA-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, HNA-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, morpholino-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, methylphosphonate nucleotide-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, thiol phosphonate nucleotide-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, polynucleotides containing 2'-fluoroN3-P5'-phosphine exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, the increased affinity is described by a lower Kd, a higher melting temperature (Tm), or a combination thereof.

In some embodiments, the polynucleotide molecules described herein are chiral (or stereopure) polynucleotide molecules, or polynucleotide molecules comprising a single enantiomer. In some cases, the polynucleotide molecule comprises an L-nucleotide. In some cases, the polynucleotide molecule comprises a D-nucleotide. In some cases, the polynucleotide molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its mirror enantiomer. In some cases, the polynucleotide molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic mixture. In some cases, the polynucleotide molecule is the polynucleotide molecule described in U.S. Patent Publications 2014/194610 and 2015/211006 and PCT Publication WO2015107425.

In some embodiments, the polynucleotide molecules described herein are further modified to include an aptamer conjugate portion. In some cases, the aptamer conjugate portion is a DNA aptamer conjugate portion. In some cases, the aptamer conjugate portion is an alphamer (Centauri Therapeutics) that includes an aptamer portion that recognizes a specific cell surface target and a portion that presents a specific epitope for linking to a circulating antibody. In some cases, the polynucleotide molecules described herein are further modified to include an aptamer conjugate portion as described in U.S. Patents 8,604,184, 8,591,910, and 7,850,975.

In other embodiments, the polynucleotide molecule described herein is modified to increase its stability. In some embodiments, the polynucleotide molecule is RNA (e.g., siRNA). In some cases, the polynucleotide molecule is modified to increase its stability by one or more of the above modifications. In some cases, the polynucleotide molecule is modified at the 2' hydroxyl position, such as by modification with 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA), or by modification with locked or bridged ribose conformations (e.g., LNA or ENA). In some cases, polynucleotide molecules are modified with 2'-O-methyl and/or 2'-O-methoxyethyl ribose. In some cases, polynucleotide molecules also contain morpholino, PNA, HNA, methylphosphonate nucleotides, thiophosphonate nucleotides, and/or 2'-fluoroN3-P5'-phosphamide to increase their stability. In some cases, the polynucleotide molecules are chiral (or stereopure) polynucleotide molecules. In some cases, chiral (or stereopure) polynucleotide molecules are modified to increase their stability. Appropriate modifications to RNA to increase delivery stability will be readily apparent to those skilled in the art.

In some cases, the polynucleotide molecule is a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region contains a nucleotide sequence complementary to a nucleotide sequence or a portion thereof in the target nucleic acid molecule, and the sense region has a nucleotide sequence corresponding to a portion thereof in the target nucleic acid sequence. In some cases, the polynucleotide molecule is assembled from two separate polynucleotides, one strand being a sense strand and the other an antisense strand, wherein the antisense and sense strands are self-complementary (e.g., each strand contains a nucleotide sequence complementary to a nucleotide sequence in the other strand; e.g., wherein the antisense and sense strands form a double helix or double-stranded structure, e.g., wherein the double-stranded region is about 19, 20, 21, 22, 23 or more base pairs); the antisense strand contains a nucleotide sequence complementary to a nucleotide sequence or a portion thereof in the target nucleic acid molecule, and the sense strand contains a nucleotide sequence corresponding to a portion thereof in the target nucleic acid sequence. Alternatively, the polynucleotide molecule is assembled from a single oligonucleotide, wherein the self-complementary sense and antisense regions of the polynucleotide molecule are linked by a nucleic acid-based or non-nucleic acid-based linker.

In some cases, the polynucleotide molecule is a polynucleotide having a double-stranded, asymmetric double-stranded, hairpin, or asymmetric hairpin secondary structure, with self-complementary sense and antisense regions, wherein the antisense region contains a nucleotide sequence complementary to or partially complementary to a nucleotide sequence in a separate target nucleic acid molecule, and the sense region has a nucleotide sequence corresponding to or partially corresponding to the target nucleic acid sequence. In other cases, the polynucleotide molecule is a cyclic single-stranded polynucleotide having two or more loop structures and a stem containing self-complementary sense and antisense regions, wherein the antisense region contains a nucleotide sequence complementary to or partially complementary to a nucleotide sequence in a target nucleic acid, and the sense region has a nucleotide sequence corresponding to or partially corresponding to the target nucleic acid sequence, and wherein the cyclic polynucleotide is processed in vivo or in vitro to generate an active polynucleotide molecule capable of mediating RNAi. In other cases, the polynucleotide molecule further comprises a single-stranded polynucleotide having a nucleotide sequence complementary to or partially complementary to a nucleotide sequence in the target nucleic acid molecule (e.g., when such a polynucleotide molecule does not require the presence of a nucleotide sequence or partially corresponding to the target nucleic acid sequence within the polynucleotide molecule), wherein the single-stranded polynucleotide further comprises a terminal phosphate group, such as 5'-phosphate (see, for example, Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568) or 5′,3′-bisphosphate.

In some cases, asymmetric hairpins are linear polynucleotide molecules containing an antisense region, a loop portion containing nucleotides or nonnucleotides, and a sense region containing fewer nucleotides than the antisense region, such that the sense region has enough complementary nucleotides to base-pair with the antisense region and form a double helix with the loop. For example, an asymmetric hairpin polynucleotide molecule contains an antisense region (e.g., about 19 to about 22 nucleotides) long enough to mediate RNAi in a cellular or in vitro system, a loop region containing about 4 to about 8 nucleotides, and a sense region having about 3 to about 18 nucleotides complementary to the antisense region. In some cases, asymmetric hairpin polynucleotide molecules also contain a chemically modified 5' terminal phosphate group. In other cases, the loop portion of the asymmetric hairpin polynucleotide molecule contains nucleotides, nonnucleotides, linker molecules, or conjugate molecules.

In some implementations, an asymmetric duplex is a polynucleotide molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region contains fewer nucleotides than the antisense region, such that the sense region has sufficient complementary nucleotides to base-pair with the antisense region and form a duplex. For example, an asymmetric duplex polynucleotide molecule comprises an antisense region (e.g., about 19 to about 22 nucleotides) of sufficient length to mediate RNAi in a cellular or in vitro system and a sense region having about 3 to about 18 nucleotides complementary to the antisense region.

In some cases, a universal base refers to a nucleotide base analog that forms a base pair with each native DNA/RNA base with little difference. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides and nitrazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole and 6-nitroindole, as known in the art (see, for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

Polynucleic acid molecule synthesis

In some embodiments, the polynucleotide molecules described herein are constructed using procedures known in the art, employing chemical synthesis and/or enzymatic ligation reactions. For example, polynucleotide molecules are chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the double strand formed between the polynucleotide molecule and the target nucleic acid. Exemplary methods include those described in U.S. Patents 5,142,047, 5,185,444, 5,889,136, 6,008,400, and 6,111,086, PCT Publication WO2009099942, or European Publication 1579015. Other exemplary methods include those described in the following literature: Griffey et al., “2’-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisenseoligonucleotides,” J. Med. Chem. 39(26):5100-5109 (1997)); Obika et al., “Synthesis of 2′-O,4′-C-methyleneuridine and cytidine. Novel bicyclic nucleosides having a fixed C3,-endo sugar Puckering. Tetrahedron Letters 38(50):8735(1997); Koizumi, M. "ENA oligonucleotides as therapy". Current opinion in molecular therapy 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, an expression vector can be used to biologically generate polynucleotide molecules, in which the polynucleotide molecule has been subcloned into the expression vector in an antisense orientation (i.e., the RNA transcribed from the inserted polynucleotide molecule will be antisense to the target polynucleotide molecule of interest).

In some implementations, polynucleotide molecules are synthesized via a tandem synthesis approach, in which two strands are synthesized as single, continuous oligonucleotide fragments or chains separated by cleavable linkers, which are then cleaved to provide individual fragments or chains that hybridize and allow for the purification of the duplex.

In some cases, polynucleotide molecules are also assembled from two different nucleic acid chains or fragments, one of which includes a sense region and the second of which includes the antisense region of the molecule.

Other modification methods used for incorporating, for example, sugars, bases, and phosphates 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 et al., International Publication PCT No. WO 93/15187; Sproat, US Patent 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International Publication PCT No. WO 97/26270; Beigelman et al., International Publication PCT No. WO 97/26270; Beigelman et al., International Publication PCT No. WO 97/26270; Beigelman et al., International Publication PCT No. WO 92/07065; ... An et al., U.S. Patent 5,716,824; Usman et al., U.S. Patent 5,627,053; Woolf et al., International PCT Publication No. WO98/13526; Thompson et al., U.S. Ser. No. 60/082,404, filed April 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, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010. These publications describe general methods and strategies for determining the location of sugar, base, and/or phosphate modifications incorporated into nucleic acid molecules without modulating catalysis.

In some cases, while chemical modifications to the internucleotide linkages of polynucleotide molecules using thiophosphates, dithiophosphates, and/or 5'-methylphosphonates improve stability, over-modification can sometimes lead to toxicity or reduced activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages is sometimes minimized. In such cases, reducing the concentration of these linkages results in reduced toxicity, increased efficacy, and higher specificity in the molecules.

Polynucleic acid conjugates

In some implementations, the polynucleotide molecule is further conjugated to peptide A for delivery to the target site. In some cases, the polynucleotide molecule is conjugated to peptide A and optionally to a polymeric moiety.

In some cases, at least one polypeptide A is conjugated to at least one B. In some cases, at least one polypeptide A is conjugated to at least one B to form an A-B conjugate. In some embodiments, at least one A is conjugated to the 5' end of B, the 3' end of B, an internal site on B, or any combination thereof. In some cases, at least one polypeptide A is conjugated to at least two Bs. In some cases, at least one polypeptide A is conjugated to at least 2, 3, 4, 5, 6, 7, 8, or more Bs.

In some embodiments, at least one polypeptide A is conjugated to one end of at least one B, while at least one C is conjugated to the opposite end of the at least one B to form an A-B-C conjugate. In some cases, at least one polypeptide A is conjugated to one end of at least one B, while at least one C is conjugated to an internal site on the at least one B. In some cases, at least one polypeptide A is directly conjugated to at least one C. In some cases, at least one B is indirectly conjugated to at least one polypeptide A via at least one C to form an A-C-B conjugate.

In some cases, at least one B and/or at least one C, and optionally at least one D, are conjugated to at least one polypeptide A. In some cases, at least one B is conjugated to at least one polypeptide A at its terminal (e.g., 5' or 3' end) or via an internal site. In some cases, at least one C is directly conjugated to at least one polypeptide A, or indirectly conjugated to at least one polypeptide A via at least one B. If conjugated indirectly via at least one B, then at least one C is conjugated to B at the same terminal as at least one polypeptide A, to the opposite terminal of at least one polypeptide A, or independently at an internal site. In some cases, at least one additional polypeptide A is further conjugated to at least one polypeptide A, B, or C. In other cases, at least one D is optionally conjugated directly or indirectly to at least one polypeptide A, at least one B, or at least one C. If directly conjugated to at least one polypeptide A, then at least one D is also optionally conjugated to at least one B to form an A-D-B conjugate, or optionally conjugated to at least one B and at least one C to form an A-D-B-C conjugate. In some cases, at least one D is directly conjugated to at least one polypeptide A and indirectly conjugated to at least one B and at least one C to form a D-A-B-C conjugate. If indirectly conjugated to at least one polypeptide A, at least one D may also optionally be conjugated to at least one B to form an A-B-D conjugate, or optionally conjugated to at least one B and at least one C to form an A-B-D-C conjugate. In some cases, at least one additional D is further conjugated to at least one polypeptide A, B, or C.

In some implementations, the polynucleic acid conjugate comprises the construct shown in Figure 19A.

In some implementations, the polynucleic acid conjugate comprises the construct shown in Figure 19B.

In some implementations, the polynucleic acid conjugate comprises the construct shown in Figure 19C.

In some implementations, the polynucleic acid conjugate comprises the construct shown in Figure 19D.

In some implementations, the polynucleic acid conjugate comprises the construct shown in Figure 19E.

In some implementations, the polynucleic acid conjugate comprises the construct shown in Figure 19F.

In some implementations, the polynucleic acid conjugate comprises the construct shown in Figure 19G.

In some implementations, the polynucleic acid conjugate comprises the construct shown in Figure 19H.

In some implementations, the polynucleic acid conjugate comprises the construct shown in Figure 19I.

In some implementations, the polynucleic acid conjugate comprises the construct shown in Figure 19J.

In some implementations, the polynucleic acid conjugate comprises the construct shown in Figure 19K.

In some implementations, the polynucleic acid conjugate comprises the construct shown in Figure 19L.

The antibody cartoon shown in Figure 19M is for illustrative purposes only and includes humanized antibodies or their binding fragments, chimeric antibodies or their binding fragments, monoclonal antibodies or their binding fragments, monovalent Fab’, bivalent Fab2, single-chain variable fragments (scFv), biantibodies, microantibodies, nanobodies, single-domain antibodies (sdAb), or camelid antibodies or their binding fragments.

Combined part

In some embodiments, binding portion A is a polypeptide. In some cases, the polypeptide is an antibody or a fragment thereof. In some cases, the fragment is a binding fragment. In some cases, the antibody or its binding fragment includes a humanized antibody or its binding fragment, a mouse antibody or its binding fragment, a chimeric antibody or its binding fragment, a monoclonal antibody or its binding fragment, a monovalent Fab', a bivalent Fab ₂ , an F(ab)' ₃ fragment, a single-chain variable fragment (scFv), a biscFv, (scFv) ₂ , a biantibody, a microantibody, a nanobody, a triantibody, a tetraantibody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or its binding fragment, a bispecific antibody or its binding fragment, or chemically modified derivatives thereof.

In some cases, A is an antibody or its binding fragment. In some cases, A is a humanized antibody or its binding fragment, a mouse antibody or its binding fragment, a chimeric antibody or its binding fragment, a monoclonal antibody or its binding fragment, a monovalent Fab', a bivalent Fab ₂ , an F(ab)' ₃ fragment, a single-chain variable fragment (scFv), a biscFv, (scFv) ₂ , a biantibody, a microantibody, a nanobody, a triantibody, a tetraantibody, a disulfide-stabilized Fv protein (“dsFv”), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or its binding fragment, a bispecific antibody or its binding fragment, or a chemically modified derivative thereof. In some cases, A is a humanized antibody or its binding fragment. In some cases, A is a mouse antibody or its binding fragment. In some cases, A is a chimeric antibody or its binding fragment. In some cases, A is a monoclonal antibody or its binding fragment. In some cases, A is a monovalent Fab'. In some cases, A is a bivalent Fab _2. In some cases, A is a single-chain variable fragment (scFv).

In some embodiments, binding portion A is a bispecific antibody or a binding fragment thereof. In some cases, the bispecific antibody is a trifunctional antibody or a bispecific microantibody. In some cases, the bispecific antibody is a trifunctional antibody. In some cases, the trifunctional antibody is a full-length monoclonal antibody containing binding sites against two different antigens.

In some cases, the bispecific antibody is a bispecific microantibody. In some cases, the bispecific microantibody includes a bivalent Fab ₂ , F(ab)' ₃ fragment, biscFv, (scFv) ₂ , a biantibody, a microantibody, a triantibody, a tetraantibody, or a bispecific T-cell conjugate (BiTE). In some embodiments, the bispecific T-cell conjugate is a fusion protein comprising two single-chain variable fragments (scFv) that target epitopes of two different antigens.

In some embodiments, the binding portion A is a bispecific microantibody. In some cases, A is a bispecific Fab _2. In some cases, A is a bispecific F(ab)' ₃ fragment. In some cases, A is a bispecific biscFv. In some cases, A is a bispecific (scFv) _2. In some embodiments, A is a bispecific biantibody. In some embodiments, A is a bispecific microantibody. In some embodiments, A is a bispecific triantibody. In other embodiments, A is a bispecific tetraantibody. In other embodiments, A is a bispecific T-cell conjugate (BiTE).

In some embodiments, the binding portion A is a trispecific antibody. In some cases, the trispecific antibody comprises an F(ab)' ₃ fragment or a triantibody. In some cases, A is a trispecific F(ab)' ₃ fragment. In some cases, A is a triantibody. In some embodiments, A is a trispecific antibody, as described in Dimas et al., “Development of atrispecific antibody designed to simultaneously and efficiently target threedifferent antigens on tumor cells,” Mol. Pharmaceutics, 12(9):3490-3501 (2015).

In some implementations, binding moiety A is an antibody or binding fragment thereof that recognizes a cell surface protein on a skeletal muscle cell.

In some implementations, exemplary antibodies include, but are not limited to, anti-myosin antibodies, anti-transferrin antibodies, and antibodies that recognize muscle-specific kinase (MuSK). In some cases, the antibody is an anti-transferrin (anti-CD71) antibody.

In some implementations, binding moiety A nonspecifically conjugates to at least one nucleic acid molecule (B). In some cases, binding moiety A conjugates to at least one nucleic acid molecule (B) in a nonsite-specific manner via lysine or cysteine residues. In some cases, binding moiety A conjugates to at least one nucleic acid molecule (B) in a nonsite-specific manner via lysine residues. In some cases, binding moiety A conjugates to at least one nucleic acid molecule (B) in a nonsite-specific manner via cysteine residues.

In some embodiments, binding moiety A is site-specifically conjugated to a polynucleotide molecule (B). In some cases, binding moiety A is site-specifically conjugated to a polynucleotide molecule (B) via lysine residues, cysteine residues, residues at the 5' end, 3' end, non-natural amino acids, or enzyme-modified or enzyme-catalyzed residues. In some cases, binding moiety A is site-specifically conjugated to a polynucleotide molecule (B) via lysine residues. In some cases, binding moiety A is site-specifically conjugated to a polynucleotide molecule (B) via cysteine residues. In some cases, binding moiety A is site-specifically conjugated to a polynucleotide molecule (B) at the 5' end. In some cases, binding moiety A is site-specifically conjugated to a polynucleotide molecule (B) at the 3' end. In some cases, binding moiety A is site-specifically conjugated to a polynucleotide molecule (B) via non-natural amino acids. In some cases, binding moiety A is site-specifically conjugated to a polynucleotide molecule (B) via enzyme-modified or enzyme-catalyzed residues.

In some implementations, one or more polynucleotide molecules (B) are conjugated to binding site A. In some cases, approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more polynucleotide molecules are conjugated to binding site A. In some cases, approximately 1 polynucleotide molecule is conjugated to binding site A. In some cases, approximately 2 polynucleotide molecules are conjugated to binding site A. In some cases, approximately 3 polynucleotide molecules are conjugated to binding site A. In some cases, approximately 4 polynucleotide molecules are conjugated to binding site A. In some cases, approximately 5 polynucleotide molecules are conjugated to binding site A. In some cases, approximately 6 polynucleotide molecules are conjugated to binding site A. In some cases, approximately 7 polynucleotide molecules are conjugated to binding site A. In some cases, approximately 8 polynucleotide molecules are conjugated to binding site A. In some cases, approximately 9 polynucleotide molecules are conjugated to binding site A. In some cases, approximately 10 polynucleotide molecules conjugate to a single binding site A. In some cases, approximately 11 polynucleotide molecules conjugate to a single binding site A. In some cases, approximately 12 polynucleotide molecules conjugate to a single binding site A. In some cases, approximately 13 polynucleotide molecules conjugate to a single binding site A. In some cases, approximately 14 polynucleotide molecules conjugate to a single binding site A. In some cases, approximately 15 polynucleotide molecules conjugate to a single binding site A. In some cases, approximately 16 polynucleotide molecules conjugate to a single binding site A. In some cases, the one or more polynucleotide molecules are identical. In other cases, the one or more polynucleotide molecules are different.

In some implementations, the number of polynucleotide molecules (B) conjugated to binding site A forms a ratio. In some cases, this ratio is referred to as the DAR (drug-antibody) ratio, where the drug, as mentioned herein, is the polynucleotide molecule (B). In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or greater. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is about 1 or greater. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is about 2 or greater. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is about 3 or greater. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is about 4 or greater. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is about 5 or greater. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is about 6 or greater. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is about 7 or greater. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is about 8 or greater. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is about 9 or greater. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is about 10 or greater. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is about 11 or greater. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is about 12 or greater.

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

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

In some cases, conjugates containing both a polynucleotide molecule (B) and a binding motif (A) exhibit improved activity compared to conjugates containing a polynucleotide molecule (B) without a binding motif A. In some cases, this improved activity leads to enhanced biologically relevant functions, such as improved stability, affinity, binding, functional activity, and efficacy in treating or preventing disease states. In some cases, this disease state is a result of one or more mutated exons in a gene. In some cases, conjugates containing both a polynucleotide molecule (B) and a binding motif (A) result in an increase in exon skipping of one or more mutated exons compared to conjugates containing a polynucleotide molecule (B) without a binding motif A. In some cases, the increase in exon skipping in conjugates containing both a polynucleotide molecule (B) and a binding motif A is at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% compared to conjugates containing a polynucleotide molecule (B) without a binding motif A.

In some embodiments, the antibody or its binding fragment is further modified using conventional techniques known in the art, for example, by using amino acid deletions, insertions, substitutions, additions, and/or by recombination and/or any other modifications known in the art (e.g., post-translational and chemical modifications such as glycosylation and phosphorylation), alone or in combination. In some cases, the modification further includes modifications for modulating the interaction with the Fc receptor. In some cases, the one or more modifications include, for example, those described in International Publication WO97/34631, which discloses amino acid residues involved in the interaction between the Fc domain and the FcRn receptor. Methods for introducing such modifications into the nucleic acid sequence of the amino acid sequence of the antibody or its binding fragment are well known to those skilled in the art.

In some cases, the antibody-binding fragment further includes its derivatives and includes a polypeptide sequence containing at least one CDR.

In some cases, as used herein, the term "single-stranded" refers to the covalent connection of the first and second domains of a bispecific single-stranded construct, preferably in the form of a collinear amino acid sequence that can be encoded by a single nucleic acid molecule.

In some cases, bispecific single-chain antibody constructs involve constructs comprising two antibody-derived binding domains. In such embodiments, the bispecific single-chain antibody construct is a tandem dual scFv or a dual antibody. In some cases, the scFv contains VH and VL domains linked by a linker peptide. In some cases, the length and sequence of the linker are sufficient to ensure that each of the first and second domains can independently retain its differential binding specificity.

In some embodiments, the binding or interaction as used herein defines the binding/interaction of at least two antigen-interacting sites with each other. In some cases, the antigen-interacting site defines a motif of a polypeptide that exhibits the ability to specifically interact with a particular antigen or a particular group of antigens. In some cases, binding/interaction is also understood to define specific recognition. In such cases, specific recognition means that an antibody or its binding fragment is able to specifically interact with and/or bind to at least two amino acids of each target molecule. For example, specific recognition involves the specificity of an antibody molecule or its ability to identify a specific region of a target molecule. In other cases, the specific interaction of an antigen-interacting site with its specific antigen results in signal initiation, for example, due to inducing a conformational change in the antigen, oligomerization of the antigen, etc. In further embodiments, an example of binding is the specificity of a "key-lock principle". Thus, in some cases, the antigen-interacting site and a specific motif in the amino acid sequence of the antigen bind to each other due to their primary, secondary, or tertiary structures and secondary modifications of said structures. In such cases, the specific interaction of the antigen-interacting site with its specific antigen also results in the simple binding of the site to the antigen.

In some cases, specific interactions further refer to reduced cross-reactivity or off-target effects of antibodies or their binding fragments. For example, antibodies or their binding fragments that bind to a polypeptide/protein of interest but not, or substantially not, any other polypeptide are considered specific to that polypeptide/protein. Examples of specific interactions between antigen-interacting sites and specific antigens include the specificity of a ligand to its receptor, such as the interaction between an antigenic determinant (epitope) and the antigen-binding site of an antibody.

Other parts of the combination

In some embodiments, the binding moiety is a plasma protein. In some cases, the plasma protein includes albumin. In some cases, binding moiety A is albumin. In some cases, albumin conjugates to polynucleotide molecules via one or more conjugation chemistry methods described herein. In some cases, albumin conjugates to polynucleotide molecules via native linker chemistry. In some cases, albumin conjugates to polynucleotide molecules via lysine conjugation.

In some cases, the binding moiety is a steroid. Exemplary steroids include cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, saturated, unsaturated, substituted hydrocarbons, or combinations thereof. In some cases, the steroid is cholesterol. In some cases, the binding moiety is cholesterol. In some cases, cholesterol is conjugated to a polynucleotide molecule via one or more conjugation chemistry methods described herein. In some cases, cholesterol is conjugated to a polynucleotide molecule via natural linking chemistry. In some cases, cholesterol is conjugated to a polynucleotide molecule via lysine conjugation.

In some cases, the binding moiety is a polymer, including but not limited to polynucleotide aptamers that bind to specific surface markers on cells. In this case, the binding moiety is a polynucleotide that does not hybridize with the target gene or mRNA, but rather resembles an antibody that binds to a specific epitope of a cell surface marker, enabling selective binding to the cell surface marker.

In some cases, the binding moiety is a peptide. In some cases, the peptide has a size of about 1 to about 3 kDa. In some cases, the peptide has a size of about 1.2 to about 2.8 kDa, about 1.5 to about 2.5 kDa, or about 1.5 to about 2 kDa. In some cases, the peptide is a bicyclic peptide. In some cases, the bicyclic peptide is a constrained bicyclic peptide. In some cases, the binding moiety is a bicyclic peptide (e.g., a bicyclic compound from Bicycle Therapeutics).

In other cases, the binding portion is a small molecule. In some cases, this small molecule is a small molecule that recruits antibodies. In some cases, the small molecule that recruits antibodies comprises a target-binding end and an antibody-binding end, wherein the target-binding end is capable of recognizing and interacting with cell surface receptors. For example, in some cases, a target-binding end containing a glutamate compound enables interaction with PSMA, thereby enhancing antibody interaction with cells expressing PSMA. In some cases, the binding site is the small molecule described in Zhang et al., “A remote arene-binding site on prostate-specific membrane antigen revealed by antibody-recruiting small molecules,” J Am Chem Soc. 132(36):12711-12716 (2010); or McEnaney et al., “Antibody-recruiting molecules: an emerging paradigm for engaging immune function in treating human disease,” ACS Chem Biol. 7(7):1139-1151 (2012).

Production of antibodies or their binding fragments

In some embodiments, the polypeptides described herein (e.g., antibodies and their binding fragments) are generated using any method known in the art for synthesizing polypeptides (e.g., antibodies), particularly by chemical synthesis or by recombinant expression, and preferably by recombinant expression techniques.

In some cases, antibodies or their binding fragments are recombinantly expressed and the nucleic acids encoding the antibody or its binding fragments are assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242). This method involves synthesizing overlapping oligonucleotides containing the sequence portion encoding the antibody, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

Alternatively, nucleic acid molecules encoding antibodies may be generated from suitable sources (e.g., antibody cDNA libraries, or cDNA libraries generated from any tissue or cell expressing immunoglobulins) by using PCR amplification with synthetic primers that hybridize to the 3' and 5' ends of the sequence, or by using the cloning of oligonucleotide probes that are specific to a particular gene sequence.

In some cases, polyclonal antibodies are optionally generated by immunizing animals such as rabbits, or more preferably by generating monoclonal antibodies, to generate antibodies or their binding fragments, for example, 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, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Alternatively, clones encoding at least the Fab portion of an antibody can be obtained by screening Fab expression libraries for clones of Fab fragments that bind to a specific antigen (e.g., as described in Huse et al., 1989, Science 246:1275-1281) or by screening antibody libraries (see, for example, Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

In some implementations, techniques developed to generate "chimeric antibodies" are used by splicing genes from mouse antibody molecules with appropriate antigen specificity to genes from human antibody molecules with appropriate biological activity (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). Chimeric antibodies are molecules in which different parts are derived from different animal species, such as antibodies having a variable region derived from mouse monoclonal antibodies and a constant region of human immunoglobulins, such as humanized antibodies.

In some embodiments, the techniques described for generating single-chain antibodies (US Patent 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 suitable for generating single-chain antibodies. Single-chain antibodies are formed by generating single-chain polypeptides by linking the heavy and light chain fragments of the Fv region via amino acid bridges. Alternatively, techniques for assembling functional Fv fragments in *E. coli* (Skerra et al., 1988, Science 242:1038-1041) are also optionally used.

In some embodiments, an expression vector containing the antibody's nucleotide sequence or the antibody's nucleotide sequence is transferred into host cells using conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured using conventional techniques to produce the antibody. In specific embodiments, antibody expression is regulated by constitutive, inducible, or tissue-specific promoters.

In some embodiments, the antibodies or their binding fragments described herein are expressed using a variety of host expression vector systems. Such host expression systems represent vectors through which the coding sequence of the antibody is generated and subsequently purified, and also represent cells that express the antibody or its binding fragment in situ when transformed or transfected with an appropriate nucleotide coding sequence. These include, but are not limited to, microorganisms such as bacteria (e.g., *Escherichia coli* and *Bacillus subtilis*) transformed with recombinant phage DNA, plasmid DNA, or copious DNA expression vectors containing the coding sequence of the antibody or its binding fragment; yeast (e.g., *Pichia pastoris*) transformed with recombinant yeast expression vectors containing the coding sequence of the antibody or its binding fragment; insect cell systems infected with recombinant viral expression vectors (e.g., baculoviruses) containing the coding sequence of the antibody or its binding fragment; and cells expressed using recombinant viral expression vectors (e.g.,...). Plant cell systems infected with or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing an antibody or a binding fragment encoding a sequence thereof; or mammalian cell systems carrying recombinant expression constructs (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) containing a promoter derived from a mammalian cell genome (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some cases, cell lines stably expressing antibodies are optionally engineered. Instead of using expression vectors containing viral origins of replication, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selective markers. After the introduction of exogenous DNA, engineered cells are grown in enrichment medium for 1–2 days, then switched to selective medium. Selective markers in the recombinant plasmid confer resistance to the selected medium and allow cells to stably integrate the plasmid into their chromosomes and grow to form foci, which are then cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines expressing antibodies or their binding fragments.

In some cases, multiple selection systems are used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine transphosphoribosylase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine transphosphoribosylase (Lowy et al., 1980, Cell 22:817) genes used in tk-, hgprt-, or aprt- cells, respectively. In addition, antimetabolite resistance was used as the basis for selecting 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); and neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505). ; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215); and hygro, conferring resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Well-known and usable methods in the field of recombinant DNA technology 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 Chapters 12 and 13, Dracopoli et al. (eds.), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1.

In some cases, antibody expression levels are increased through vector amplification (see Bebbington and Henschel, 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 the marker in the antibody-expressing vector system is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody's nucleotide sequence, antibody production will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

In some cases, any method known in the art for purifying or analyzing antibodies or antibody conjugates may be used, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly affinity chromatography for specific antigens following protein A, and column-size chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Exemplary chromatographic methods include, but are not limited to, strong anion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and rapid protein liquid chromatography.

Conjugation Chemistry

In some embodiments, a polynucleotide molecule is conjugated to a binding moiety. In some cases, the binding moiety includes amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, and analogs or derivatives of all these categories. Other examples of binding moieties include steroids such as cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, hydrocarbons (e.g., saturated, unsaturated, or substituent-containing), enzyme substrates, biotin, digitalisin, and polysaccharides. In some cases, the binding moiety is an antibody or a binding fragment thereof. In some cases, the polynucleotide molecule is further conjugated to a polymer and optionally an endosome dissolution moiety.

In some embodiments, the polynucleotide molecule is conjugated to the binding site via a chemical ligation process. In some cases, the polynucleotide molecule is conjugated to the binding site via a natural linker. In some cases, 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 straight-forward methodology., Proc. Natl. Acad. Sci. USA 1999, 96, 10068–10073; or Wu et al., “Building complex glycopeptides: Development of acysteine-free native chemical ligation protocol,” Angew. Chem. Int. Ed. 2006, 45, 4116–4125. In some cases, the conjugation is as described in U.S. Patent 8,936,910. In some embodiments, the polynucleotide molecule is conjugated to the binding site specifically or nonspecifically via natural linker chemistry.

In some cases, the polynucleotide molecule is conjugated to the binding site using a site-specific method employing a "traceless" conjugation technique (Philochem). In some cases, this "traceless" conjugation technique utilizes the N-terminal 1,2-aminothiol group on the binding site, which is subsequently conjugated to a polynucleotide molecule containing an aldehyde group (see Casi et al., "Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery," JACS134(13):5887-5892(2012)).

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

In some cases, the polynucleotide molecule is conjugated to the binding site using a site-directed method that utilizes an enzymatic catalytic process. In some cases, this site-directed method utilizes SMARTag ^™ technology (Catalent, Inc.). In some cases, SMARTag ^™ technology involves the oxidation of cysteine to formylgly (FGly) residues via a formylglyase (FGE) in the presence of an aldehyde tag, followed by conjugation of FGly to an alkylhydrazine-functionalized polynucleotide molecule via a hydrazine-pictet-Spengler (HIPS) linker (see Wu et al., “Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag,” PNAS 106(9):3000-3005 (2009); Agarwal et al., “A Pictet-Spengler ligation for protein chemical modification,” PNAS 110(1):46-51 (2013)).

In some cases, the enzymatic catalytic process involves microbial transglutaminase (mTG). In some cases, the polynucleotide molecule is conjugated to the binding site using a process catalyzed by microbial transglutaminase. In some cases, mTG catalyzes the formation of a covalent bond between the amide side chain of the recognition sequence and the primary amine of the functionalized polynucleotide molecule. In some cases, mTG is produced by *Streptomyces mobarensis* (see Strop et al., “Locationmatters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates,” *Chemistry and Biology* 20(2) 161-167 (2013)).

In some cases, the polynucleotide molecule is conjugated to the binding site by a method described in PCT Publication WO2014/140317, which utilizes a sequence-specific transpeptidase.

In some cases, the polynucleotide molecule is conjugated to the binding site by methods as described in U.S. Patent Publications 2015/0105539 and 2015/0105540.

Polymer conjugate

In some embodiments, polymer moiety C is further conjugated to the polynucleotide molecule described herein, the binding moiety described herein, or a combination thereof. In some cases, polymer moiety C is conjugated to the polynucleotide molecule. In some cases, polymer moiety C is conjugated to the binding moiety. In other cases, polymer moiety C is conjugated to the polynucleotide molecule binding moiety. In still other cases, polymer moiety C is conjugated as described above.

In some cases, polymer part C is a natural or synthetic polymer composed of long chains of branched or unbranched monomers and/or two-dimensional or three-dimensional cross-linked networks of monomers. In some cases, polymer part C includes polysaccharides, lignin, rubber, or polyepoxides (e.g., polyethylene glycol). In some cases, at least one polymer part C includes, but is not limited to, α-, ω-dihydroxy polyethylene glycol, biodegradable lactone-based polymers such as polyacrylic acid, polylactic acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefins, polyamides, polycyanoacrylates, polyimides, polyethylene terephthalate (also known as poly(ethylene terephthalate), PET, PETG, or PETE), polybutane glycol (PTG), or polyurethanes and mixtures thereof. As used herein, mixtures refer to the use of different polymers within the same compound and in block copolymers. In some cases, a block copolymer is a polymer in which at least a portion of the polymer is composed of monomers of another polymer. In some cases, polymer part C includes polyepoxides. In some cases, polymer part C includes PEG. In some cases, polymer part C includes polyvinylimide (PEI) or hydroxyethyl starch (HES).

In some cases, C represents the PEG moiety. In some cases, the PEG moiety is conjugated to the 5' end of the polynucleotide molecule, while the binding moiety is conjugated to the 3' end. In some cases, the PEG moiety is conjugated to the 3' end of the polynucleotide molecule, while the binding moiety is conjugated to the 5' end. In some cases, the PEG moiety is conjugated to an internal site of the polynucleotide molecule. In some cases, the PEG moiety, the binding moiety, or a combination thereof is conjugated to an internal site of the polynucleotide molecule. In some cases, the conjugation is direct. In some cases, the conjugation occurs via natural linker.

In some embodiments, the polyepoxide (e.g., PEG) is a polydisperse or monodisperse compound. In some cases, the polydisperse material comprises a dispersion of materials with different molecular weights, characterized by average weight (weight-average) size and dispersibility. In some cases, the monodisperse PEG comprises molecules of a single size. In some embodiments, C is a polydisperse or monodisperse polyepoxide (e.g., PEG), and the indicated molecular weight represents the average molecular weight of the polyepoxide (e.g., PEG) molecules.

In some embodiments, the polyepoxide (e.g., PEG) 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, etc. 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 a polyepoxide (e.g., PEG) and has a strength 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, 2 Molecular weights of 800, 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 implementations, C is PEG, and has a strength of approximately 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, Molecular weights of 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 cases, the molecular weight of C is approximately 200 Da. In some cases, the molecular weight of C is approximately 300 Da. In some cases, the molecular weight of C is approximately 400 Da. In some cases, the molecular weight of C is approximately 500 Da. In some cases, the molecular weight of C is approximately 600 Da. In some cases, the molecular weight of C is approximately 700 Da. In some cases, the molecular weight of C is approximately 800 Da. In some cases, the molecular weight of C is approximately 900 Da. In some cases, the molecular weight of C is approximately 1000 Da. In some cases, the molecular weight of C is approximately 1100 Da. In some cases, the molecular weight of C is approximately 1200 Da. In some cases, the molecular weight of C is approximately 1300 Da. In some cases, the molecular weight of C is approximately 1400 Da. In some cases, the molecular weight of C is approximately 1450 Da. In some cases, the molecular weight of C is approximately 1500 Da. In some cases, the molecular weight of C is approximately 1600 Da. In some cases, the molecular weight of C is approximately 1700 Da. In some cases, the molecular weight of C is approximately 1800 Da. In some cases, the molecular weight of C is approximately 1900 Da. In some cases, the molecular weight of C is approximately 2000 Da. In some cases, the molecular weight of C is approximately 2100 Da. In some cases, the molecular weight of C is approximately 2200 Da. In some cases, the molecular weight of C is approximately 2300 Da. In some cases, the molecular weight of C is approximately 2400 Da. In some cases, the molecular weight of C is approximately 2500 Da. In some cases, the molecular weight of C is approximately 2600 Da. In some cases, the molecular weight of C is approximately 2700 Da. In some cases, the molecular weight of C is approximately 2800 Da. In some cases, the molecular weight of C is approximately 2900 Da. In some cases, the molecular weight of C is approximately 3000 Da. In some cases, the molecular weight of C is approximately 3250 Da. In some cases, the molecular weight of C is approximately 3350 Da. In some cases, the molecular weight of C is approximately 3500 Da. In some cases, the molecular weight of C is approximately 3750 Da. In some cases, the molecular weight of C is approximately 4000 Da. In some cases, the molecular weight of C is approximately 4250 Da. In some cases, the molecular weight of C is approximately 4500 Da. In some cases, the molecular weight of C is approximately 4600 Da. In some cases, the molecular weight of C is approximately 4750 Da. In some cases, the molecular weight of C is approximately 5000 Da. In some cases, the molecular weight of C is approximately 5500 Da. In some cases, the molecular weight of C is approximately 6000 Da. In some cases, the molecular weight of C is approximately 6500 Da. In some cases, the molecular weight of C is approximately 7000 Da. In some cases, the molecular weight of C is approximately 7500 Da. In some cases, the molecular weight of C is approximately 8000 Da. In some cases, the molecular weight of C is approximately 10,000 Da. In some cases, the molecular weight of C is approximately 12,000 Da. In some cases, the molecular weight of C is approximately 20,000 Da. In some cases, the molecular weight of C is approximately 35,000 Da. In some cases, the molecular weight of C is approximately 40,000 Da. In some cases, the molecular weight of C is approximately 50,000 Da. In some cases, the molecular weight of C is approximately 60,000 Da. In some cases, the molecular weight of C is approximately 100,000 Da.

In some embodiments, the polyepoxide (e.g., PEG) comprises discrete ethylene oxide units (e.g., 4 to about 48 ethylene oxide units). In some cases, the polyepoxide comprising discrete ethylene oxide units is linear. In other cases, the polyepoxide comprising discrete ethylene oxide units is branched.

In some cases, polymer portion C is a polyepoxide (e.g., PEG) comprising discrete ethylene oxide units. In some cases, polymer portion C comprises about 4 to about 48 ethylene oxide units. In some cases, polymer portion 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.

In some cases, polymer portion C is a discrete PEG containing, for example, about 4 to about 48 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 4 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 5 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 6 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 7 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 8 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 9 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 10 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 11 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 12 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 13 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 14 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 15 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 16 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 17 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 18 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 19 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 20 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 21 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 22 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 23 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 24 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 25 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 26 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 27 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 28 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 29 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 30 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 31 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 32 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 33 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 34 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 35 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 36 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 37 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 38 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 39 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 40 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 41 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 42 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 43 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 44 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 45 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 46 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 47 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 48 ethylene oxide units.

In some cases, polymer part C is (Quanta Biodesign Ltd).

In some embodiments, polymer portion C comprises a cationic viscosic acid-based polymer (cMAP). In some cases, the cMAP comprises one or more subunits of at least one repeating subunit, and the subunit structure is represented by formula (V):

Where m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 each time it appears, preferably 4-6 or 5; and n is independently 1, 2, 3, 4 or 5 each time it appears. In some embodiments, m and n are, for example, about 10.

In some cases, cMAP is further conjugated to the PEG moiety to form a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some cases, the PEG moiety ranges from about 500 Da to about 50,000 Da. In some cases, the PEG moiety ranges from about 500 Da to about 1,000 Da, greater than 1,000 Da to about 5,000 Da, greater than 5,000 Da to about 10,000 Da, greater than 10,000 Da 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 cases, polymer moiety C is a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In other cases, polymer moiety C is a cMAP-PEG copolymer. In still other cases, polymer moiety C is a cMAP-PEG-cMAP triblock polymer.

In some implementations, polymer portion C is conjugated to multiple nucleic acid molecules, a binding portion, and an optional endosome dissolution portion, as described above.

Endosome Dissolution

In some embodiments, the molecule of formula (I) – _AX1 -BX2 _-C – further comprises an additional conjugation portion. In some cases, this additional conjugation portion is an endosome-dissolving portion. In some cases, the endosome-dissolving portion is a compartmental release component, such as a compound capable of being released from any compartment known in the art, such as an endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubule, peroxisome, or other intracellular vesicle. In some cases, the endosome-dissolving portion comprises an endosome-dissolving polypeptide, an endosome-dissolving polymer, an endosome-dissolving lipid, or an endosome-dissolving small molecule. In some cases, the endosome-dissolving portion comprises an endosome-dissolving polypeptide. In other cases, the endosome-dissolving portion comprises an endosome-dissolving polymer.

Endosome-dissolving peptides

In some embodiments, the molecule of formula (I) – _AX1 -BX2 _-C – is further conjugated with an endosomal dissolving peptide. In some cases, the endosomal dissolving peptide is a pH-dependent membrane-active peptide. In some cases, the endosomal dissolving peptide is an amphiphilic peptide. In other cases, the endosomal dissolving peptide is a peptide mimic. In some cases, the endosomal dissolving peptide comprises INF, meliostein, meucin, or derivatives thereof. In some cases, the endosomal dissolving peptide comprises INF or a derivative thereof. In other cases, the endosomal dissolving peptide comprises meliostein or a derivative thereof. In still other cases, the endosomal dissolving peptide comprises meucin or a derivative thereof.

In some cases, INF7 is a 24-residue polypeptide whose sequence contains CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO:1) or GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO:2). In some cases, INF7 or its derivatives contain the following sequences: GLFEAIEGFIENGWEGMIWDYGSGSCG (SEQ ID NO:3), GLFEAIEGFIENGWEGMIDGWYG-(PEG)6-NH2 (SEQ ID NO:4), or GLFEAIEGFIENGWEGMIWDYG-SGSC-K(GalNAc)2 (SEQ ID NO:5).

In some cases, melivitin is a 26-residue polypeptide whose sequence includes CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 6) or GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 7). In some cases, melivitin comprises a polypeptide sequence as described in U.S. Patent 8,501,930.

In some cases, meucin is an antimicrobial peptide (AMP) derived from the venom glands of the striped scorpion (Mesobuthus eupeus). In some cases, meucin includes meucin-13 and meucin-18, with the sequence of meucin-13 containing IFGAIAGLLKNIF-NH ₂ (SEQ ID NO:8) and the sequence of meucin-18 containing FFGHLFKLATKIIPSLFQ (SEQ ID NO:9).

In some cases, the endosome-dissolving polypeptide includes a polypeptide whose sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence identity with INF7 or a derivative thereof, melitoxin or a derivative thereof, or meucin or a derivative thereof. In some cases, the endosome-dissolving portion includes INF7 or a derivative thereof, melitoxin or a derivative thereof, or meucin or a derivative thereof.

In some cases, the endosome dissolution portion is INF7 or a derivative thereof. In some cases, the endosome dissolution portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1. In some cases, the endosome dissolution portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1. In some cases, the endosome dissolution portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:2-5. In some cases, the endosome dissolution portion comprises SEQ ID NO:1. In some cases, the endosome dissolution portion comprises SEQ ID NO:2-5. In some cases, the endosome dissolution portion consists of SEQ ID NO:1. In some cases, the endosome dissolution portion consists of SEQ ID NO:2-5.

In some cases, the endosome dissolution fraction is a melitoxin or a derivative thereof. In some cases, the endosome dissolution fraction contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:6. In some cases, the endosome dissolution fraction contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:6. In some cases, the endosome dissolution portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:7. In some cases, the endosome dissolution portion comprises SEQ ID NO:6. In some cases, the endosome dissolution portion comprises SEQ ID NO:7. In some cases, the endosome dissolution portion consists of SEQ ID NO:6. In some cases, the endosome dissolution portion consists of SEQ ID NO:7.

In some cases, the endosome dissolution portion is meucin or a derivative thereof. In some cases, the endosome dissolution portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:8. In some cases, the endosome dissolution portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:8. In some cases, the endosome dissolution portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:9. In some cases, the endosome dissolution portion comprises SEQ ID NO:8. In some cases, the endosome dissolution portion comprises SEQ ID NO:9. In some cases, the endosome dissolution portion consists of SEQ ID NO:8. In some cases, the endosome dissolution portion consists of SEQ ID NO:9.

In some cases, the endosome dissolution portion contains sequences as shown in Table 1.

In some cases, the endosome lysis fraction contains the Bak BH3 peptide, which induces apoptosis by antagonizing inhibitor targets such as Bcl-2 and/or Bcl-x _L. In other cases, the endosome lysis fraction contains the Bak BH3 peptide described by Albarran et al., “Efficient intracellular delivery of a pro-apoptotic peptide with a pH-responsive carrier,” Reactive & Functional Polymers 71:261-265 (2011).

In some cases, the endosome dissolution portion contains a polypeptide (e.g., a cell-penetrating polypeptide) as described in PCT disclosures WO2013/166155 or WO2015/069587.

Endosome-dissolved lipids

In some embodiments, the endosome-dissolving portion is a lipid (e.g., a fusion-promoting lipid). In some embodiments, the molecule of formula (I) – _AX1 -BX2 _- C – is further conjugated with an endosome-dissolving lipid (e.g., a fusion-promoting lipid). Exemplary fusion-promoting lipids include 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadec-9,12-dienyl)-1,3-dioxolane-4-yl)methylamine (DLin-k-DMA), and N-methyl-2-(2,2-di((9Z,12Z)-octadec-9,12-dienyl)-1,3-dioxolane-4-yl)ethylamine (XTC).

In some cases, the endosome-dissolving portion is a lipid (e.g., a fusion-promoting lipid) as described in PCT Publication WO09/126,933.

Endosome Dissolution of Small Molecules

In some embodiments, the endosome dissolving moiety is a small molecule. In some embodiments, the molecule of formula (I) – _AX1 -BX2 _-C – is further conjugated with the endosome dissolving small molecule. Exemplary small molecules suitable as endosome dissolving moieties include, but are not limited to, quinine, chloroquine, hydroxychloroquine, carnoquine, amopiquine, primaquine, mefloquine, nivaquine, halopanthracenes, quinone imines, or combinations thereof. In some cases, the endosome dissolved portion of quinoline includes, but is not limited to, 7-chloro-4-(4-diethylamino-1-methylbutyl-amino)quinoline (chloroquine); 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyl-amino)quinoline (hydroxychloroquine); 7-fluoro-4-(4-diethylamino-1-methylbutyl-amino)quinoline; 4-(4-diethylamino-1-methylbutyl-amino)quinoline; 7-hydroxy-4-(4-diethylamino-1-methylbutyl-amino)quinoline; 7-chloro-4-(4-diethylamino)-amino-1-methylbutyl-amino)quinoline; 7-fluoro-4-(4-diethylamino-1-butylamino)quinoline (demethylchloroquine); 4-(4-diethylamino-1-butylamino)quinoline; 7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline; 7-fluoro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline; 4-(1-carboxy-4-diethylamino-1-butylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline; 4-Diethylamino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline; 7-fluoro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline; 4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline; 7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 4-(4-ethyl-( 2-Hydroxy-ethyl)-amino-1-methylbutylamino-)quinoline; 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; hydroxychloroquine phosphate; 7-chloro-4-(4-ethyl-(2-hydroxyethyl-1)-amino-1-butylamino)quinoline (demethylhydroxychloroquine); 7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-hydroxy-4-(4-ethyl- (2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)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-methoxyquinoline dihydrochloride; 1-acetyl-1,2,3,4-tetrahydroquinoline; 8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride; 1-butyryl-1,2,3,4-tetrahydroquinoline; 3-chloro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-dimethylaminoquinoline, 4-[(4-diethyl-amino)-1-methylbutyl-amino]-6-methoxyquinoline; 3-fluoro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-dimethylaminoquinoline, 4-[(4-diethyl-amino)-1-methylbutyl-amino]- 6-Methoxyquinoline; 4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolyl)-2,5-dimethylaminoquinoline;4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline; 3,4-dihydro-1-(2H)-quinoline carboxyl aldehyde; 1,1′-pentamethylenediquinoline diiodide; 8-hydroxyquinoline sulfate and their amino, aldehyde, carboxyl, hydroxyl, halogen, ketone, thiol, and vinyl derivatives or analogs. In some cases, the endosome-dissolved moiety is a small molecule as described in Naisbitt et al. (1997, J Pharmacol Exp Therapy 280:884-893) and U.S. Patent 5,736,557.

Connector

In some embodiments, the connector described herein is either a cuttable connector or a non-cuttable connector. In some cases, the connector is a cuttable connector. In other cases, the connector is a non-cuttable connector.

In some cases, the linker is a non-polymeric linker. A non-polymeric linker is a linker that does not contain repeating monomeric units generated through a polymerization process. Exemplary non-polymeric linkers include, but are not limited to, _C1 - _C6 alkyl (e.g., _C5 , _C4 , _C3 , _C2 , or _C1 alkyl), homobifunctional crosslinkers, heterobifunctional crosslinkers, peptide linkers, traceless linkers, self-immolative linkers, maleimide-based linkers, or combinations thereof. In some cases, the non-polymeric linker comprises _C1 - _C6 alkyl (e.g., _C5 , _C4 , _C3 , _C2 , or _C1 alkyl), homobifunctional crosslinkers, heterobifunctional crosslinkers, peptide linkers, traceless linkers, self-immolative linkers, maleimide-based linkers, or combinations thereof. In other cases, the non-polymeric linker does not contain more than two linkers of the same type, for example, more than two homobifunctional crosslinkers or more than two peptide linkers. In other cases, the nonpolymeric linker optionally contains one or more reactive functional groups.

In some cases, the non-polymeric linker does not contain the polymers described above. In some cases, the non-polymeric linker does not contain the polymer contained in polymer portion C. In some cases, the non-polymeric linker does not contain polyepoxides (e.g., PEG). In some cases, the non-polymeric linker does not contain PEG.

In some cases, the linker comprises a homobifunctional linker. Exemplary homobifunctional linkers include, but are not limited to, Lomant reagents: dithiobis(succinimide propionate)DSP, 3′3′-dithiobis(sulfosuccinimide propionate) (DTSSP), disuccinimide octanoate (DSS), bis(sulfosuccinimide) octanoate (BS), disuccinimide tartrate (DST), disulfosuccinimide tartrate (sulfonated DST), bis(succinimide) ethylene glycol ester (EGS), disuccinimide glutarate (DSG), N,N′-disuccinimide carbonate (DSC), dimethyl adipamide (DMA), dimethyl heptamethamide (DMP), dimethyl octanoide (DMS), and dimethyl-3,3′-dithiobispropionide. (DTBP), 1,4-di-3′-(2′-pyridyldithio)propamido)butane (DPDPB), bismaleimide hexane (BMH), aryl halogenated compounds (DFDNB) such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4′-difluoro-3,3′-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylic acid)ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbazide, o-toluidine, 3,3′-dimethylbenzidine, benzidine, α,α′-p-diaminobiphenyl, diiodo-p-xylenesulfonic acid, N,N′-ethylene-bis(iodoacetamide) or N,N′-hexamethylene-bis(iodoacetamide).

In some embodiments, the linker comprises a heterobifunctional linker. Exemplary heterobifunctional linkers include, but are not limited to, amine-reactive and thiol-crosslinkers, such as N-succinimidyl 3-(2-pyridyl dithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyl dithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyl dithio)propionate (sulfonyl-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl dithio)toluene (sMPT), and sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyl dithio)toluene]. [Amide group] Hexanoate (sulfon-LC-sMPT), succinimide-4-(N-maleimide methyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimide-4-(N-maleimide methyl)cyclohexane-1-carboxylate (sulfon-sMCC), m-maleimide benzoyl-N-hydroxysuccinimide (MBs), m-maleimide benzoyl-N-hydroxysulfosuccinimide (sulfon-MBs), N-succinimide (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimide (4-iodoacetyl)aminobenzoate Acyl)aminobenzoate (sulfon-sIAB), succinimide-4-(p-maleimide-phenyl)butyrate (sMPB), sulfosuccinimide-4-(p-maleimide-phenyl)butyrate (sulfon-sMPB), N-(γ-maleimide-butyryloxy)succinimide (GMBs), N-(γ-maleimide-butyryloxy)sulfosuccinimide (sulfon-GMBs), succinimide-6-((iodoacetyl)amino)hexanoate (sIAX), succinimide-6-[6-(((iodosulfosuccinimide)amino]amino] [4-(((iodosulfonyl)amino]hexanoate (sIAXX), succinimide-4-(((iodosulfonyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimide-6-((((4-iodosulfonyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA); carbonyl reactive and thiol reactive crosslinkers, such as 4-(4-N-maleimide-phenyl)butyric acid hydrazide (MPBH), 4-(N-maleimide-methyl)cyclohexane-1-carboxy-hydrazide-8 ( _{M2C2 )} H), 3-(2-pyridyldithio)propionylhydrazide (PDPH); amine-reactive and photoreactive crosslinkers, such as N-hydroxysuccinimide-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimide-4-azidosalicylic acid (sulfon-NHs-AsA), sulfosuccinimide-(4-azidosalicylamido)hexanoate (sulfon-NHs-LC-AsA), sulfosuccinimide-2-(ρ-azidosalicylamido)ethyl-1,3′-dithiopropionate (sAsD), N-hydroxysuccinimide-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimide-4-azidobenzoate, etc. Imide-4-azidobenzoate (sulfon-HsAB), N-succinimide-6-(4′-azido-2′-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimide-6-(4′-azido-2′-nitrophenylamino)hexanoate (sulfon-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimide-2-(m-azido-o-nitrobenzoamide)-ethyl-1,3′-dithiopropionate (sAND), N-succinimide-4-(4-azidophenyl)1,3′-dithiopropionate (sAD) P), N-sulfosuccinimide (4-azidophenyl)-1,3′-dithiopropionate (sulfon-sADP), sulfosuccinimide 4-(ρ-azidophenyl)butyrate (sulfon-sAPB), sulfosuccinimide 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate (sAED), sulfosuccinimide 7-azido-4-methylcoumarin-3-acetate (sulfon-sAMCA), ρ-nitrophenyldiazopyruvate (ρNPDP), ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP); thiol reaction Reactive and photoreactive crosslinkers, such as 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3′-(2′-pyridinyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimide; carbonyl reactive and photoreactive crosslinkers, such as ρ-azidobenzoylhydrazide (ABH); carboxylic acid ester reactive and photoreactive crosslinkers, such as 4-(ρ-azidosalicylamido)butylamine (AsBA); and arginine reactive and photoreactive crosslinkers, such as ρ-azidophenylglyoxal (APG).

In some cases, 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 the binding moiety. Exemplary electrophilic groups include carbonyl groups, such as aldehydes, ketones, carboxylic acids, esters, amides, ketenes, acyl halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include acylhydrazides, oximes, amino groups, hydrazines, thioureas, hydrazide carboxylates, and arylhydrazides.

In some embodiments, the linker comprises a maleimide group. In some cases, the maleimide group is also referred to as a maleimide spacer group. In some cases, the maleimide group further comprises hexanoic acid, forming a maleimide hexanoyl (mc). In some cases, the linker comprises a maleimide hexanoyl (mc). In some cases, the linker is a maleimide hexanoyl (mc). In other cases, the maleimide group comprises a maleimide methyl group, such as succinimidyl-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (sulfon-sMCC) as described above.

In some embodiments, the maleimide group is a self-stabilized maleimide. In some cases, the self-stabilized maleimide utilizes diaminopropionic acid (DPR) incorporated into the basic amino group adjacent to the maleimide to provide intramolecular catalysis for the thiosuccinimide ring hydrolysis, thereby preventing the maleimide from undergoing elimination reactions via the reverse Michael reaction. In some cases, the self-stabilized maleimide is the maleimide group described by Lyon et al., “Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates,” Nat. Biotechnol. 32(10):1059-1062 (2014). In some cases, the linker comprises a self-stabilized maleimide. In some cases, the linker is a self-stabilized maleimide.

In some embodiments, the linker comprises a peptide moiety. In some cases, the peptide moiety comprises at least 2, 3, 4, 5, or 6 or more amino acid residues. In some cases, the peptide moiety comprises up to 2, 3, 4, 5, 6, 7, or 8 or more amino acid residues. In some cases, the peptide moiety comprises about 2, about 3, about 4, about 5, or about 6 amino acid residues. In some cases, the peptide moiety is a cleavable peptide moiety (e.g., enzymatically or chemically). In some cases, the peptide moiety is an incleavable peptide moiety. In some cases, the peptide moiety comprises Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO:865), Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO:866), or Gly-Phe-Leu-Gly (SEQ ID NO:867). In some cases, the linker comprises a peptide moiety such as: Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 865), Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 866), or Gly-Phe-Leu-Gly (SEQ ID NO: 867). In some cases, the linker comprises Val-Cit. In some cases, the linker is Val-Cit.

In some embodiments, the linker comprises a benzoic acid group or a derivative thereof. In some cases, the benzoic acid group or its derivative comprises p-aminobenzoic acid (PABA). In some cases, the benzoic acid group or its derivative comprises γ-aminobutyric acid (GABA).

In some embodiments, the linker comprises one or more of any combination of maleimide groups, peptide moieties, and/or benzoic acid groups. In some embodiments, the linker comprises a combination of maleimide groups, peptide moieties, and/or benzoic acid groups. In some cases, the maleimide group is maleimide hexanoyl (mc). In some cases, the peptide group is val-cit. In some cases, the benzoic acid group is PABA. In some cases, the linker comprises an mc-val-cit group. In some cases, the linker comprises a val-cit-PABA group. In other cases, the linker comprises an mc-val-cit-PABA group.

In some embodiments, the connector is a self-sacrificing connector or a self-eliminating connector. In some cases, the connector is a self-sacrificing connector. In other cases, the connector is a self-eliminating connector (e.g., a circumferential self-eliminating connector). In some cases, the connector comprises the connector described in U.S. Patent 9,089,614 or PCT Publication WO2015038426.

In some embodiments, the linker is a dendritic linker. In some cases, the dendritic linker comprises a branched multifunctional linker portion. In some cases, the dendritic linker is used to increase the molar ratio of polynucleotide B to binding moiety A. In some cases, the dendritic linker comprises a PAMAM dendritic polymer.

In some embodiments, the linker is a traceless linker or a linker that leaves no linker portion (e.g., atoms or linker groups) on the binding portion A, polynucleotide B, polymer C, or endosome dissolution portion D after cleavage. Exemplary traceless linkers include, but are not limited to, germanium linkers, silicon linkers, sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers, boron linkers, chromium linkers, or phenylhydrazine linkers. In some cases, the linker is a traceless aryl-triazene linker as described by Hejesen et al., “A traceless aryl-triazene linker for DNA-directed chemistry,” Org Biomol Chem 11(15):2493-2497 (2013). In some cases, the linker is a traceless linker as described by Blanky et al., “Traceless solid-phase organic synthesis,” Chem. Rev. 102:2607-2024 (2002). In some cases, the connector is the seamless connector described in U.S. Patent 6,821,783.

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

In some implementations, _X1 and _X2 are each independently a bonded or non-polymeric linker. In some cases, _X1 and _X2 are each independently a bonded linker. In some cases, _X1 and _X2 are each independently a non-polymeric linker.

In some cases, _X1 is a bonded or non-polymeric linker. In some cases, _X1 is a bonded linker. In some cases, _X1 is a non-polymeric linker. In some cases, the linker is a _C1 - _C6 alkyl group. In some cases, _X1 is a _C1 - _C6 alkyl group, for example, a _C5 , _C4 , _C3 , _C2 , or _C1 alkyl group. In some cases, the _C1 - _C6 alkyl group is an unsubstituted _C1 - _C6 alkyl group. As used in the context of a linker, particularly in the context of _X1 , alkyl means a saturated straight-chain or branched hydrocarbon group containing up to six carbon atoms _. In some cases, _X1 includes homo-bifunctional or hetero-bifunctional linkers as described above. In some cases, _X1 includes hetero-bifunctional linkers. In some cases, X1 includes sMCC. In other cases, _X1 includes hetero-bifunctional linkers optionally conjugated to a _C1 - _C6 alkyl group. In other cases, _X1 includes sMCCs optionally conjugated to _C1 - _C6 alkyl groups. In still other cases, _X1 does not include homo- or hetero-bifunctional linkers as described above.

In some cases, _X2 is a bond or linker. In some cases, _X2 is a bond. In other cases, _X2 is a linker. In some cases, _X2 is a non-polymeric linker. In some embodiments, _X2 is a _C1 - _C6 alkyl group. In some cases, _X2 is a homo- or hetero-bifunctional linker as described above. In some cases, _X2 is a homo-bifunctional linker as described above. In some cases, _X2 is a hetero-bifunctional linker as described above. In some cases, _X2 contains a maleimide group, such as the aforementioned maleimide-hexanoyl (mc) or a self-stabilizing maleimide group. In some cases, _X2 contains a peptide moiety, such as Val-Cit. In some cases, _X2 contains a benzoic acid group, such as PABA. In other cases, _X2 contains a combination of a maleimide group, a peptide moiety, and/or a benzoic acid group. In other cases, _X2 contains an mc group. In another case, X ₂ contains an mc-val-cit group. In another case, X ₂ contains a val-cit-PABA group. In another case, X ₂ contains an mc-val-cit-PABA group.

How to use

Muscle atrophy refers to the loss of muscle mass and/or the progressive weakening and degeneration of muscle. In some cases, muscle mass loss and/or progressive weakening and degeneration occur due to high protein degradation rates, low protein synthesis rates, or both. In some cases, high rates of muscle protein degradation are due to muscle protein catabolism (i.e., the breakdown of muscle proteins to utilize amino acids as substrates for gluconeogenesis).

In one embodiment, muscle atrophy refers to a significant loss of muscle strength. A significant loss of muscle strength is a reduction in the strength of diseased, injured, or disused muscle tissue in a subject relative to the same muscle tissue in a control subject. In one embodiment, a significant loss of muscle strength is a reduction in strength of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more, relative to the same muscle tissue in a control subject. In another embodiment, a significant loss of muscle strength is a reduction in the strength of disused muscle tissue relative to the muscle strength of the same muscle tissue in the same subject before a period of non-use. In one embodiment, a significant loss of muscle strength is a reduction in muscle strength of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more, relative to the muscle strength of the same muscle tissue in the same subject before a period of non-use.

In another embodiment, muscle atrophy refers to a significant loss of muscle mass. A significant loss of muscle mass means a reduction in the muscle volume of diseased, injured, or disused muscle tissue in a subject relative to the same muscle tissue in a control subject. In one embodiment, a significant loss of muscle volume is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more, relative to the same muscle tissue in a control subject. In another embodiment, a significant loss of muscle mass means a reduction in the muscle volume of disused muscle tissue relative to the muscle volume of the same muscle tissue in the same subject before a period of non-use. In one embodiment, a significant loss of muscle tissue is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more, relative to the muscle volume of the same muscle tissue in the same subject before a period of non-use. Optionally, muscle volume is measured by assessing the cross-sectional area of the muscle, for example, by magnetic resonance imaging (e.g., by a muscle volume/cross-sectional area (CSA) MRI method).

Myotonic dystrophy is a multisystemic neuromuscular disease comprising two main types: myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM1 is caused by the expansion of a dominant “CTG” repeat in the gene DM protein kinase (DMPK), which, when transcribed into mRNA, forms hairpins that bind with high affinity to proteins of the blind muscle-like (MBNL) family. MBNL proteins are involved in posttranscriptional splicing and polyadenylation site regulation, and loss of MBNL protein function leads to downstream accumulation of nucleoids and increased missplicing events, resulting in myotonia and other clinical symptoms.

In some implementations, this document describes methods for treating a subject with muscle atrophy or myotonic dystrophy, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule or polynucleotide conjugate described herein. In some cases, the muscle atrophy is caused by and/or associated with cachexia (e.g., cancer cachexia), denervation, myopathy, motor neuron disease, diabetes, chronic obstructive pulmonary disease, liver disease, congestive heart failure, chronic renal failure, chronic infection, sepsis, fasting, sarcopenia, glucocorticoid-induced atrophy, disuse, or spaceflight. In some cases, myotonic dystrophy is DM1.

cachexia

Cachexia is an acquired, accelerated loss of muscle caused by an underlying disease. In some cases, cachexia refers to weight loss that cannot be reversed by nutrition and is often associated with underlying diseases such as cancer, COPD, AIDS, and heart failure. When cachexia is observed in patients with advanced cancer, it is termed "cancer cachexia." Cancer cachexia affects most patients with advanced cancer and is associated with reduced treatment tolerance, response to treatment, quality of life, and survival. In some cases, cancer cachexia is defined as a multifactorial syndrome characterized by persistent reduction in skeletal muscle mass, reduced or no reduction in fat mass, inability to be fully reversed by routine nutritional support, and progressive functional impairment. In some cases, skeletal muscle loss appears to be the most significant event in cancer cachexia. Furthermore, the classification of cancer cachexia suggests that diagnostic criteria should consider not only weight loss as a signaling event in the cachexia process but also the patient's initial reserves, such as low BMI or low levels of muscle mass.

In some embodiments, this document describes a method for treating cachexia-related muscle atrophy in a subject, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule described herein or a polynucleotide conjugate described herein. In other embodiments, this document describes a method for treating cancer cachexia-related muscle atrophy in a subject, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule described herein or a polynucleotide conjugate described herein.

Denervation

Denervation is the result of damage to peripheral motor neurons, a partial or complete interruption of nerve fibers between organs and the central nervous system, leading to the disruption of nerve conduction and motor neuron impulses, which in turn prevent skeletal muscle contraction. This loss of neural function can be localized or generalized due to the loss of entire motor neuron units. The inability of skeletal muscles to contract results in muscle atrophy. In some cases, denervation is associated with or is a consequence of degenerative, metabolic, or inflammatory neuropathy (e.g., Guillain-Barré syndrome, peripheral neuropathy, or exposure to environmental toxins or drugs). In other cases, denervation is associated with bodily injuries such as surgical procedures.

In some embodiments, this document describes a method for treating muscle atrophy in subjects that is associated with or caused by denervation, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule described herein. In other embodiments, this document describes a method for treating muscle atrophy in subjects that is associated with or caused by denervation, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule conjugate described herein.

Myopathy

Myopathy is a broad term that describes diseases of the muscles. In some cases, myopathy includes myotonia; congenital myopathy, such as linear myopathy, multinucleated/micronucleated myopathy, and myotubular (central nucleus) myopathy; mitochondrial myopathy; familial periodic paralysis; inflammatory myopathy; metabolic myopathy, for example, caused by glycogen or lipid storage diseases; dermatomyositis; polymyositis; inclusion body myositis; ossifying myositis; rhabdomyolysis; and myoglobinuria. In some cases, myopathy is caused by muscular dystrophy syndromes, such as Duchenne muscular dystrophy, Behringer's muscular dystrophy, myotonic muscular dystrophy, Emery-Dreifussian muscular dystrophy, oculopharyngeal muscular dystrophy, scapuhumeral muscular dystrophy, limb-girdle muscular dystrophy, Fukuyama muscular dystrophy, or hereditary distal myopathy. In some cases, myopathy is caused by myotonic dystrophy (e.g., myotonic dystrophy type 1 or DM1). In some cases, myopathy is caused by DM1.

In some embodiments, this document describes methods for treating myopathy-related or myopathy-induced muscle atrophy in a subject, comprising administering a therapeutically effective amount of the polynucleotide molecule described herein to the subject. In other embodiments, this document describes methods for treating myopathy-related or myopathy-induced muscle atrophy in a subject, comprising administering a therapeutically effective amount of the polynucleotide molecule conjugate described herein to the subject.

motor neuron disease

Motor neuron disease (MND) includes neurological disorders that affect motor neurons, the cells that control voluntary muscle movement in the body. Exemplary motor neuron diseases include, but are not limited to, adult motor neuron disease, infantile spinal muscular atrophy, amyotrophic lateral sclerosis (ALS), adolescent spinal muscular atrophy, autoimmune motor neuropathy with multifocal conduction block, paralysis due to stroke or spinal cord injury, or skeletal instability due to trauma.

In some embodiments, this document describes methods for treating muscle atrophy in subjects that is associated with or caused by motor neuron disease, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule described herein. In other embodiments, this document describes methods for treating muscle atrophy in subjects that is associated with or caused by motor neuron disease, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule conjugate described herein.

diabetes

Diabetes mellitus (DM) includes type 1 diabetes, type 2 diabetes, type 3 diabetes, type 4 diabetes, double diabetes, latent autoimmune diabetes (LAD), gestational diabetes, neonatal diabetes (NDM), juvenile-onset diabetes (MODY), Wolfram syndrome, prediabetes, or diabetes insipidus. Type 2 diabetes, also known as non-insulin-dependent diabetes, is the most common type of diabetes, accounting for 95% of all diabetes cases. In some cases, type 2 diabetes is caused by multiple factors, including insulin resistance due to abnormal pancreatic beta cell function, leading to hyperglycemia. In other cases, elevated glucagon levels stimulate the liver to produce abnormal amounts of unwanted glucose, resulting in hyperglycemia.

Type 1 diabetes, also known as insulin-dependent diabetes mellitus, accounts for approximately 5% to 10% of all diabetes cases. Type 1 diabetes is an autoimmune disease in which T cells attack and destroy the beta cells in the pancreas that produce insulin. In some implementations, type 1 diabetes is caused by a combination of genetic and environmental factors.

Type 4 diabetes is a recently discovered form of diabetes that affects approximately 20% of people over the age of 65. In some implementations, type 4 diabetes is characterized by age-related insulin resistance.

In some implementations, type 3 diabetes is used as the term for Alzheimer's disease, which causes insulin resistance in the brain.

In some embodiments, this document describes a method for treating diabetes-related muscular atrophy in a subject, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule described herein or a polynucleotide conjugate described herein. In other embodiments, this document describes a method for treating cancer-related diabetes-related muscular atrophy in a subject, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule described herein or a polynucleotide conjugate described herein.

Chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a type of obstructive lung disease characterized by chronic breathing problems and poor airflow. Chronic bronchitis and emphysema are two distinct types of COPD. In some cases, this document describes methods for treating muscle atrophy in subjects that is associated with or caused by COPD (e.g., chronic bronchitis or emphysema), comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule described herein. In other embodiments, this document describes methods for treating muscle atrophy in subjects that is associated with or caused by COPD (e.g., chronic bronchitis or emphysema), comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule conjugate described herein.

liver disease

Liver disease (or liver disorders) includes fibrosis, cirrhosis, hepatitis, alcoholic liver disease, hepatic steatosis, hereditary diseases, or primary liver cancer. In some cases, this document describes methods for treating liver disease-related or liver disease-induced muscle atrophy in subjects, which includes administering a therapeutically effective amount of the polynucleotide molecule described herein to the subject. In other embodiments, this document describes methods for treating liver disease-related or liver disease-induced muscle atrophy in subjects, which includes administering a therapeutically effective amount of the polynucleotide molecule conjugate described herein to the subject.

congestive heart failure

Congestive heart failure refers to a condition in which the heart is unable to pump sufficient blood and oxygen to the body's tissues. In some cases, this document describes methods for treating muscle atrophy in subjects associated with or caused by congestive heart failure, comprising administering a therapeutically effective amount of the polynucleotide molecule described herein to the subject. In other embodiments, this document describes methods for treating muscle atrophy in subjects associated with or caused by congestive heart failure, comprising administering a therapeutically effective amount of the polynucleotide molecule conjugate described herein to the subject.

Chronic renal failure

Chronic renal failure, or chronic kidney disease, is a condition characterized by the gradual loss of kidney function over time. In some embodiments, this document describes methods for treating muscle atrophy in subjects that is associated with or caused by chronic renal failure, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule described herein.

Chronic infection

In some implementations, chronic infections such as AIDS further lead to muscle atrophy. In some cases, this document describes methods for treating muscle atrophy in subjects associated with or caused by a chronic infection (e.g., AIDS), comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule described herein. In other implementations, this document describes methods for treating muscle atrophy in subjects associated with or caused by a chronic infection (e.g., AIDS), comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule conjugate described herein.

sepsis

Sepsis is an immune response to an infection that leads to tissue damage, organ failure, and/or death. In some embodiments, this document describes a method for treating sepsis-associated or sepsis-induced muscle atrophy in a subject, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule described herein. In other embodiments, this document describes a method for treating sepsis-associated or sepsis-induced muscle atrophy in a subject, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule conjugate described herein.

fasting

Fasting is the withdrawal or reduction of certain or all foods, beverages, or both for a period of time. In some embodiments, this document describes a method for treating fasting-related or fasting-induced muscle atrophy in a subject, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule described herein. In other embodiments, this document describes a method for treating fasting-related or fasting-induced muscle atrophy in a subject, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule conjugate described herein.

sarcopenia

Sarcopenia is a persistent process of muscle atrophy during normal aging, characterized by a gradual loss of muscle mass and strength over months and years. In this context, normal aging refers to the aging process that is not affected or accelerated by the presence of conditions or diseases that promote neurodegeneration of skeletal muscle.

In some embodiments, this document describes a method for treating muscle atrophy in subjects that is associated with or caused by sarcopenia, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule described herein. In other embodiments, this document describes a method for treating muscle atrophy in subjects that is associated with or caused by sarcopenia, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule conjugate described herein.

Glucocorticoid-related muscle atrophy

In some implementations, treatment with glucocorticoids further leads to muscle atrophy. Exemplary glucocorticoids include, but are not limited to, cortisol, dexamethasone, betamethasone, prednisone, methylprednisolone, and prednisolone.

In some embodiments, this document describes a method for treating glucocorticoid-related muscle atrophy in a subject, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule described herein. In other embodiments, this document describes a method for treating glucocorticoid-related muscle atrophy in a subject, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule conjugate described herein.

Disuse-related muscle atrophy

When a limb is immobilized (e.g., due to a limb or joint fracture or orthopedic surgery such as a hip or knee replacement), it results in disuse-related muscle atrophy. As used herein, “immobilization” or “fixed” means that a limb, muscle, bone, tendon, joint, or any other part of the body is partially or completely restricted in its movement for an extended period of time (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, or longer). In some cases, the duration of immobilization includes brief periods or situations where movement is unrestricted, such as bathing, changing or adjusting external equipment. Limb immobilization may be performed using a variety of external devices, including but not limited to braces, slings, casts, bandages, and splints (any of which may optionally be made of hard or soft materials, including but not limited to cloth, gauze, fiberglass, plastic, plaster, or metal), and any internal devices, including surgically implanted splints, plates, braces, etc. When a limb is immobilized, limited movement involves single or multiple joints (e.g., simple joints such as the shoulder or hip, complex joints such as the radiocarpal joint, and complex joints such as the knee), including but not limited to one or more of the following: joints of the hand, shoulder, elbow, wrist, accessory joints, sternoclavicular joint, vertebral joints, temporomandibular joint, sacroiliac joint, hip, knee, and foot joints), single or multiple tendons or ligaments (e.g., including but not limited to one or more of the following: anterior cruciate ligament, posterior cruciate ligament, rotator cuff tendons, collateral ligaments of the elbow and knee, flexor tendons of the hand, lateral ligaments of the ankle, and tendons and ligaments of the jaw or temporomandibular joint). (e.g., including but not limited to one or more of the following: skull, mandible, clavicle, ribs, radius, ulna, humor, pelvis, sacrum, femur, patella, phalanges, carpal bones, metacarpals, tarsal bones, metatarsals, fibula, tibia, scapula, and vertebrae), one or more muscles (e.g., including but not limited to one or more of the following: latissimus dorsi, trapezius, deltoid, pectoralis major, biceps major, triceps major, external oblique, abdominal muscles, gluteus maximus, hamstrings, quadriceps major, gastrocnemius, and diaphragm); one or more limbs (one or more arms and legs), or the entire skeletal muscle system or a portion thereof (e.g., in the case of a full-body cast or a herringbone cast).

In some embodiments, this document describes a method for treating disuse-related muscle atrophy in a subject, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule described herein. In other embodiments, this document describes a method for treating disuse-related muscle atrophy in a subject, comprising administering to the subject a therapeutically effective amount of the polynucleotide molecule conjugate described herein.

pharmaceutical preparations

In some embodiments, the pharmaceutical formulations described herein are administered to subjects via multiple routes of administration, including but not limited to parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal routes. In some cases, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intra-arterial, intraperitoneal, intrathecal, intracerebral, intraventricular, or intracranial) administration. In other cases, the pharmaceutical compositions described herein are formulated for oral administration. In still other cases, the pharmaceutical compositions described herein are formulated for intranasal administration.

In some embodiments, the pharmaceutical formulation includes, but is not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate-release formulations, controlled-release formulations, rapidly melting formulations, tablets, capsules, pills, delayed-release formulations, extended-release formulations, pulsed-release formulations, multi-particle formulations (e.g., nanoparticle formulations), and hybrid formulations of immediate and controlled release.

In some cases, the pharmaceutical formulation comprises a multi-particulate formulation. In some cases, the pharmaceutical formulation comprises a nanoparticle formulation. In some cases, the nanoparticles comprise cMAP, cyclodextrin, or lipids. In some cases, the nanoparticles comprise solid lipid nanoparticles, polymer nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micelle solutions. Other exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendritic polymers (such as metal chelates with covalent bonds), nanofibers, nanohorns, nanoonions, nanorods, nanowires, and quantum dots. In some cases, nanoparticles are metallic nanoparticles, such as nanoparticles 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 thereof, alloys or oxides thereof.

In some cases, nanoparticles contain a core or a core and a shell, such as in core-shell nanoparticles.

In some cases, the nanoparticles are further coated with molecules for attaching functional elements (e.g., one or more of the polynucleotide molecules or binding moieties described herein). In some cases, the coating comprises chondroitin sulfate, dextran sulfate, carboxymethyl dextran, alginic acid, pectin, carrageenan, fucoidan, agar, alginic acid, erythritol, gellan gum, xanthan gum, hyaluronic acid, glucosamine, galactosamine, chitosan (or chitosan), polyglutamic acid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, α-chymotrypsin, polylysine, polyarginine, histone, protamine, ovalbumin, dextrin, or cyclodextrin. In some cases, the nanoparticles include graphene-coated nanoparticles.

In some cases, nanoparticles have at least one dimension smaller than about 500 nm, 400 nm, 300 nm, 200 nm or 100 nm.

In some cases, nanoparticle formulations comprise paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendritic polymers (such as metal chelates with covalent bonds), nanofibers, nanohorns, nanoonions, nanorods, nanotethers, or quantum dots. In some cases, the polynucleotide molecules or binding moieties described herein are directly or indirectly conjugated to the nanoparticles. In some cases, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more polynucleotide molecules or binding moieties described herein are directly or indirectly conjugated to the nanoparticles.

In some embodiments, the pharmaceutical formulation comprises a delivery vector, such as a recombinant vector, for delivering polynucleotide molecules into cells. In some cases, the recombinant vector is a DNA plasmid. In other cases, the recombinant vector is a viral vector. Exemplary viral vectors include vectors derived from adeno-associated virus, retrovirus, adenovirus, or alphavirus. In some cases, recombinant vectors capable of expressing polynucleotide molecules provide stable expression in target cells. In other cases, viral vectors providing transient expression of polynucleotide molecules are used.

In some embodiments, the pharmaceutical formulation comprises a carrier or carrier material selected based on its compatibility with the compositions disclosed herein and the release profile properties of the desired dosage form. Exemplary carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, etc. Pharmaceutically compatible carrier materials include, but are not limited to, gum arabic, gelatin, colloidal silica, calcium glycerophosphate, calcium lactate, maltodextrin, glycerol, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurine, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium hydrogen phosphate, cellulose and cellulose conjugates, sodium saccharide stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, etc. See, for example, Remington: The Science and Practice of Pharmacy, 19th edition (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, 7th edition (Lippincott Williams & Wilkins 1999).

In some cases, the pharmaceutical formulation further comprises pH adjusters or buffers, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris(hydroxymethyl)aminomethane; and buffers such as citrate/dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

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

In some cases, the pharmaceutical formulation further comprises diluents to stabilize the compound, as they provide a more stable environment. Salts dissolved in buffer solutions (which also provide pH control or maintenance) are used in the art as diluents, including but not limited to phosphate-buffered saline solutions. In some cases, diluents increase the volume of the composition to facilitate compression or to produce sufficient volume for uniform blending for capsule filling. These compounds include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as calcium hydrogen phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugars such as Amstar; mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, confectionery sugars; basic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, glucose dextrorate; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, etc.

In some cases, the pharmaceutical formulation contains a disintegrant to facilitate the breakdown or disintegration of the substance. The term "disintegration" includes the dissolution and dispersion of the dosage form upon contact with gastrointestinal fluids. Examples of disintegrants include starches, such as natural starches like corn starch or potato starch; pregelatinized starches like National 1551 or sodium glycolate starch, or celluloses like wood products; methyl crystalline celluloses, such as PH101, PH102, PH105, P100, Ming, and methyl cellulose; cross-linked carboxymethyl cellulose, or cross-linked celluloses such as sodium cross-linked carboxymethyl cellulose; cross-linked starches such as sodium glycolate starch; cross-linked polymers such as povidone and cross-linked polyvinylpyrrolidone; alginates such as alginic acid or salts of alginic acid such as sodium alginate; clays such as HV (magnesium aluminum silicate); gums such as agar, guar gum, locust bean gum, ebony gum, pectin, or tragacanth gum; sodium glycolate starch; bentonite; natural sponges; surfactants; resins such as cation exchange resins; citrus pulp; sodium lauryl sulfate; combinations of sodium lauryl sulfate and starch; and so on.

In some cases, the pharmaceutical preparation contains fillers such as lactose, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, glucose binder, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, etc.

Lubricants and flow aids may also be optionally included in the pharmaceutical formulations described herein for preventing, reducing, or inhibiting adhesion or friction of materials. Exemplary lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearoyl fumarate, hydrocarbons such as mineral oil, or hydrogenated vegetable oils such as hydrogenated soybean oil, higher fatty acids and their alkali metal and alkaline earth metal salts such as aluminum, calcium, magnesium, zinc salts, stearic acid, sodium stearate, glycerol, talc, wax, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000) or methoxy polyethylene glycol such as Carbowax ^™ , sodium oleate, sodium benzoate, glyceryl betaine, polyethylene glycol, magnesium dodecyl sulfate or sodium dodecyl sulfate, colloidal silica such as Syloid ^™ , starch such as corn starch, silicone oil, surfactants, etc.

Plasticizers are compounds used to soften microencapsulated materials or film coatings to make them less brittle. Suitable plasticizers include, for example, polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350 and PEG 800, stearic acid, propylene glycol, oleic acid, triethylcellulose and triacetin. Plasticizers are also used as dispersants or wetting agents.

Solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl octanoate, sodium dodecyl sulfate, sodium docusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropanol, cholesterol, bile salts, polyethylene glycol 200-600, tetrahydrofuran polyethylene glycol ether (glycofurol), diethylene glycol monoethyl ether (transcutol), propylene glycol, and dimethyl isosorbide.

Stabilizers include compounds such as any antioxidants, buffers, acids, preservatives, etc.

Suspension agents include polyvinylpyrrolidone such as polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25 or polyvinylpyrrolidone K30, vinylpyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol (e.g., polyethylene glycol having a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methylcellulose. Compounds include hydroxymethyl cellulose acetate stearate, polysorbate-80, hydroxyethyl cellulose, sodium alginate, gums such as gum arabic and gum arabic, guar gum, xanthan gum (including xanthan gum), sugars, and celluloses such as sodium carboxymethyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, and povidone.

Surfactants include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin ETPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbate, poloxamer, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide such as BASF. Other surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, such as octoxynol 10 and octoxynol 40. Sometimes, surfactants are included to enhance physical stability or for other purposes.

Viscosity enhancers include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropyl methylcellulose acetate stearate, hydroxypropyl methylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, gum arabic, chitosan, and combinations thereof.

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

Treatment plan

In some embodiments, the pharmaceutical composition described herein is applied for therapeutic use. In some embodiments, the pharmaceutical composition is applied once daily, twice daily, three times daily, or more. The pharmaceutical composition is applied daily, once daily, every other day, five days a week, once a week, every other week, two weeks a month, three weeks a month, once a month, twice a month, three times a month, or more. The pharmaceutical composition is applied 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 longer.

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

In some embodiments, two or more different pharmaceutical compositions are administered together. In some cases, two or more different pharmaceutical compositions are administered simultaneously. In some cases, two or more different pharmaceutical compositions are administered sequentially without a time interval between administrations. In other cases, two or more different pharmaceutical compositions are administered sequentially with an interval of approximately 0.5 hours, 1 hour, 2 hours, 3 hours, 12 hours, 1 day, 2 days, or longer between administrations.

If the patient's condition does improve, the medication may continue to be administered based on the physician's judgment; or the dosage of the medication may be temporarily reduced or temporarily suspended for a certain period of time (i.e., a "withdrawal period"). In some cases, the length of the withdrawal period varies from 2 days to 1 year, including, for example, 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. During the withdrawal period, the dose is reduced by 10%-100%, for example only, including 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once the patient's condition improves, a maintenance dose may be administered if necessary. Subsequently, depending on changes in symptoms, the dosage or frequency of administration, or both, may be reduced to a level where the improvement in the disease, symptom, or condition can be maintained.

In some embodiments, the amount of a given agent corresponding to such a quantity varies depending on factors such as the specific compound, disease severity, and characteristics (e.g., weight) of the subject or host requiring treatment; however, it is still conventionally determined in a manner known in the art based on the specific circumstances relevant to the instance, including, for example, the specific agent administered, the route of administration, and the subject or host being treated. In some cases, the required dose is conveniently presented as a single dose or in fractions, which are administered simultaneously (or over short periods of time) or at appropriate intervals, such as two, three, four, or more sub-dose per day.

The aforementioned ranges are merely suggestive, as the number of variables relating to individual treatment regimens is enormous, and considerable deviations from these recommended values are not uncommon. Such dosages vary depending on multiple variables, including but not limited to the activity of the compound used, the disease or condition being treated, the route of administration, the individual's needs, the severity of the disease or condition being treated, and the physician's judgment.

In some embodiments, the toxicity and therapeutic efficacy of such treatment regimens are determined through standard pharmaceutical procedures in cell cultures or laboratory animals, including but not limited to the determination of LD50 (the 50% lethal dose for a population) and ED50 (the 50% therapeutically effective dose for a population). The dose ratio between toxic and therapeutic effects is the therapeutic index and is expressed as the ratio of LD50 to ED50. Compounds exhibiting a high therapeutic index are preferred. Dosage ranges for human use are established using data obtained from cell culture experiments and animal studies. The dosage of such compounds is preferably within the cyclic concentration range containing the ED50 and exhibiting minimal toxicity. The dosage varies within this range depending on the dosage form and route of administration used.

reagent kits/products

In some embodiments, kits and articles thereof are disclosed for use with one or more compositions and methods described herein. Such kits include carriers, packaging, or containers compartmentalized to receive one or more containers such as vials, tubes, etc., each containing a single element to be used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the container is formed of various materials such as glass or plastic.

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

For example, the container contains the target nucleic acid molecule described herein. Such a kit may optionally include an identifying description or label or instruction regarding its use in the methods described herein.

Kits typically include a label listing the contents and/or instructions for use, as well as a packaging insert with usage instructions. A separate instruction manual is also usually included.

In one embodiment, the label is on or associated with the container. In one embodiment, the label is on the container when the letters, numbers, or other characters constituting the label are affixed, molded, or etched onto the container itself; the label is associated with the container when it is present within a receiver or carrier that also houses the container (e.g., as a packaging insert). In one embodiment, the label is used to indicate that the contents will be used for a specific therapeutic application. The label also provides guidance regarding the use of the contents, such as in the methods described herein.

In some embodiments, the pharmaceutical composition is provided in a package or dispenser device containing one or more unit dosage forms of the compound provided herein. For example, the package may contain metal or plastic foil, such as blister packs. In one embodiment, the package or dispenser device is accompanied by instructions for use. In one embodiment, the package or dispenser is also accompanied by a container-associated notice in the form prescribed by a government agency regulating the manufacture, use, or sale of the drug, reflecting that the agency has approved the form of the drug for human or veterinary administration. Such a notice may be, for example, a label or approved product insert approved by the U.S. Food and Drug Administration for prescription drugs. In one embodiment, a composition containing the compound provided herein formulated in a compatible drug carrier is also prepared, placed in a suitable container, and labeled for the treatment of the indicated condition.

certain terms

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by one of ordinary skill in the art to which the claimed subject matter pertains. It should be understood that the foregoing general description and the following detailed description are exemplary and illustrative only and are not intended to limit any of the claimed subject matter. In this application, the singular is used to include the plural unless specifically stated otherwise. It must be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise. In this application, the use of “or” means “and/or” unless otherwise stated. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “having” is non-limiting.

As used herein, ranges and quantities can be expressed as “about” a specific value or range. “About” also includes the exact quantity. Therefore, “about 5 μL” means “about 5 μL” and also “5 μL”. Typically, the term “about” includes the quantity expected to be within experimental error.

The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

As used herein, the terms “individual,” “subject,” and “patient” refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of these terms require or are limited to situations characterized by supervision (e.g., continuous or intermittent) by a healthcare worker (e.g., a physician, registered nurse, nurse practitioner, physician assistant, caregiver, or hospice worker).

The term "therapeutic effective dose" refers to the amount of a polynucleotide conjugate sufficient to provide the desired therapeutic effect in mammalian subjects. In some cases, this dose is a single or multiple administration dose to a patient (e.g., a human) for the treatment, prevention, prevention of its onset, cure, delay, reduction of its severity, alleviation of at least one symptom of the condition or recurrence of the condition, or prolongation of the patient's survival beyond that expected without such treatment. Naturally, the dose level of a specific polynucleotide conjugate used to provide a therapeutically effective dose varies depending on the type of lesion, the subject's age, weight, sex, medical condition, severity of the condition, route of administration, and the specific inhibitor used. In some cases, as described herein, the therapeutically effective dose of the polynucleotide conjugate is initially estimated from cell cultures and animal models. For example, _IC50 values determined in cell culture methods are optionally used as a starting point for animal models, while _IC50 values determined in animal models are optionally used to identify therapeutically effective doses in humans.

Skeletal muscles, or voluntary muscles, are typically anchored to bones by tendons and are generally used to achieve skeletal movements such as motion or posture maintenance. Although some control over skeletal muscles is usually maintained in an involuntary reflex manner (e.g., postural muscles or the diaphragm), skeletal muscles respond to conscious control. Smooth or involuntary muscles are found in the walls of organs and structures such as the esophagus, stomach, intestines, uterus, urethra, and blood vessels.

Skeletal muscle is further divided into two main categories: Type I (or "slow-twitch" muscle) and Type II (or "fast-twitch" muscle). Type I muscle fibers have dense capillaries and are rich in mitochondria and myoglobin, which gives Type I muscle tissue its characteristic red color. In some cases, Type I muscle fibers carry more oxygen and use fat or carbohydrates as fuel to sustain aerobic exercise. Type I muscle fibers can contract for a long time, but with little force. Type II muscle fibers are further subdivided into three main subtypes (IIa, IIx, and IIb), which differ in contraction speed and the force generated. Type II muscle fibers contract rapidly and powerfully, but fatigue quickly, thus producing only a brief burst of anaerobic activity before the muscle contraction becomes painful.

Unlike skeletal muscle, smooth muscle is not under conscious control.

Cardiac muscle is also involuntary muscle, but structurally more similar to skeletal muscle, and is found only in the heart. Cardiac and skeletal muscles are striated muscles because they contain sarcomeres arranged in highly regular bundles. In contrast, myofibrils in smooth muscle cells are not arranged in sarcomeres and therefore lack striations.

Muscle cells include any cells that contribute to muscle tissue. Exemplary muscle cells include myoblasts, satellite cells, myotubes, and myofibrils.

As used herein, muscle strength is proportional to cross-sectional area (CSA), while muscle velocity is proportional to muscle fiber length. Therefore, comparing the cross-sectional area and muscle fiber length among various muscles can provide an indication of muscle atrophy. Various methods for measuring muscle strength and muscle weight are known in the art; for example, see Hazel M. Clarkson, “Musculoskeletal assessment: Joint range of motion and manual muscle strength,” Lippincott Williams & Wilkins, 2000. Another method for measuring muscle mass is to evaluate the generation of tomographic images from selected muscle tissue using computed axial computed tomography and ultrasound examination.

This invention provides, but is not limited to, the following embodiments:

1. A polynucleotide conjugate comprising an antibody or a binding fragment thereof conjugated to a polynucleotide molecule, said polynucleotide molecule hybridizing to a target sequence of an atrogene; wherein said polynucleotide molecule comprises at least one 2' modified nucleotide, at least one modified internucleotide linker, or at least one reverse debasement moiety; and wherein said polynucleotide conjugate mediates RNA interference against said atrogene, thereby treating a subject with myasthenia gravis or myotonic dystrophy.

2. The polynucleotide conjugate according to Embodiment 1, wherein the atrogene includes differentially regulated genes within the IGF1-Akt-FoxO pathway, glucocorticoid-GR pathway, PGC1α-FoxO pathway, TNFα-NFκB pathway, or myostatin-ActRIIb-Smad2/3 pathway.

3. The polynucleotide conjugate according to Embodiment 2, wherein the atrogene is a gene downregulated in the IGF1-Akt-FoxO pathway, glucocorticoid-GR pathway, PGC1α-FoxO pathway, TNFα-NFκB pathway, or myostatin-ActRIIb-Smad2/3 pathway.

4. The polynucleotide conjugate according to Embodiment 2, wherein the atrogene is a gene upregulated in the IGF1-Akt-FoxO pathway, glucocorticoid-GR pathway, PGC1α-FoxO pathway, TNFα-NFκB pathway, or myostatin-ActRIIb-Smad2/3 pathway.

5. The polynucleotide conjugate according to Embodiment 1, wherein the atrogene encodes an E3 ligase.

6. The polynucleotide conjugate according to Embodiment 1, wherein the atrogene encodes a forkhead box transcription factor.

7. The polynucleotide conjugate according to Embodiment 1, wherein the atrogene includes the atrogin-1 gene (FBXO32), the MuRF1 gene (TRIM63), FOXO1, FOXO3, or MSTN.

8. The polynucleotide conjugate according to Embodiment 1, wherein the atrogen comprises DMPK.

9. The polynucleotide conjugate according to Embodiment 1, wherein the antibody or its binding fragment includes a humanized antibody or its binding fragment, a chimeric antibody or its binding fragment, a monoclonal antibody or its binding fragment, a monovalent Fab’, a bivalent Fab2, a single-chain variable fragment (scFv), a biantibody, a microantibody, a nanobody, a single-domain antibody (sdAb), or a camelid antibody or its binding fragment.

10. The polynucleic acid conjugate according to Embodiment 1, wherein the antibody or its binding fragment is an anti-transferrin receptor antibody or its binding fragment.

11. The polynucleotide conjugate according to Embodiment 1, wherein the polynucleotide molecule comprises a sense strand and an antisense strand, and wherein each of the sense strand and the antisense strand independently comprises at least one 2' modified nucleotide, at least one modified internucleotide linker, or at least one reverse debasement moiety.

12. The polynucleotide conjugate according to Embodiment 1, wherein the polynucleotide hybridizes to at least 8 consecutive bases of the target sequence of atrogene.

13. The polynucleotide conjugate according to Embodiment 1, wherein the target sequence has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:28-141, 370-480, or 703-3406.

14. The polynucleotide conjugate according to Embodiment 1, wherein the polynucleotide is about 8 to about 50 nucleotides in length, or about 10 to about 30 nucleotides in length.

15. The polynucleotide conjugate according to Embodiment 1, wherein the sense strand comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 142-255, 481-591, 3407-6110, or 8815-11518.

16. The polynucleotide conjugate according to Embodiment 1, wherein the antisense strand comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 256-369, 592-702, 6111-8814, or 11519-14222.

17. The polynucleotide molecular conjugate according to embodiment 1, wherein the polynucleotide molecular conjugate comprises a linker that links the binding portion to the polynucleotide.

18. The polynucleotide molecular conjugate according to embodiment 1, wherein the polynucleotide molecular conjugate further comprises a polymer, the polymer optionally being indirectly conjugated to the polynucleotide via an additional linker.

19. The polynucleic acid molecular conjugate according to embodiment 17 or 18, wherein the linker and the other linker are each independently non-polymeric linkers.

20. The polynucleic acid molecular conjugate according to Embodiment 1, wherein the polynucleic acid molecular conjugate comprises a molecule of formula (I):

AX ₁ -BX ₂ -C

Formula I

in,

A represents the antibody or its binding fragment;

B is a polynucleotide molecule that hybridizes with the target sequence of atrogene;

C is a polymer; and

_X1 and _X2 are each independently selected from bonded or non-polymeric linkages; and

A and C are not connected to the same end of B.

21. The polynucleic acid molecular conjugate according to Embodiment 20, wherein C is polyethylene glycol (PEG).

22. The polynucleic acid conjugate according to embodiment 20, wherein _AX1 is conjugated to the 5' end of B and _X2 -C is conjugated to the 3' end of B.

23. The polynucleic acid conjugate according to embodiment 20, wherein _X2 -C is conjugated to the 5' end of B and _AX1 is conjugated to the 3' end of B.

24. The polynucleotide conjugate according to embodiment 20, wherein B comprises a sense strand and an antisense strand.

25. The polynucleic acid conjugate according to embodiment 24, wherein A and C are each attached to different ends of the sense strand.

26. The polynucleotide conjugate according to Embodiment 1, wherein the at least one 2' modified nucleotide comprises a nucleotide modified with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA).

27. The polynucleotide conjugate according to Embodiment 1, wherein the at least one 2' modified nucleotide comprises locked nucleic acid (LNA) or vinyl nucleic acid (ENA).

28. The polynucleotide conjugate according to Embodiment 1, wherein the at least one modified nucleotide linker comprises a thiophosphate linker or a dithiophosphate linker.

29. The polynucleic acid molecular conjugate according to Embodiment 1, wherein the at least one reverse debasement moiety is at at least one end.

30. The polynucleotide conjugate according to Embodiment 1, wherein the muscle atrophy is diabetes-associated muscle atrophy or cancer cachexia-associated muscle atrophy.

31. The polynucleic acid conjugate according to Embodiment 1, wherein the muscle atrophy is associated with insulin deficiency, chronic renal failure, congestive heart failure, chronic respiratory disease, chronic infection, fasting, denervation, sarcopenia, or glucocorticoid therapy.

32. The polynucleotide conjugate according to Embodiment 1, wherein the muscle atrophy is associated with myotonic dystrophy type 1 (DM1).

33. The polynucleic acid conjugate according to Embodiment 1, wherein the myotonic dystrophy is DM1.

34. A pharmaceutical composition comprising:

Polynucleic acid conjugates of embodiments 1-33; and

Pharmaceutically acceptable excipients.

35. The pharmaceutical composition according to embodiment 34, wherein the pharmaceutical composition is formulated as a nanoparticle formulation.

36. The pharmaceutical composition according to embodiment 34, wherein the pharmaceutical composition is formulated for parenteral, oral, intranasal, buccal, rectal or transdermal administration.

37. A method for treating muscle atrophy or myotonic dystrophy in a subject of need, comprising:

The subject is given a therapeutically effective amount of the polynucleic acid molecular conjugate of Embodiments 1-33 or the pharmaceutical composition of Embodiments 34-36 to treat the subject's muscle atrophy or myotonic dystrophy.

38. The method according to embodiment 37, wherein the subject is a human being.

A kit comprising the polynucleic acid molecular conjugates of embodiments 1-33 or the pharmaceutical compositions of embodiments 34-36.

Example

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

Example 1. siRNA sequence and synthesis

All siRNA single-stranded RNAs were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were then double-stranded to obtain double-stranded siRNAs. All siRNA guest strands contained different forms of constrictors at each end of the strand, namely C6- _NH2 and/or C6-SH. One or more constrictors were attached to the siRNA guest strand via reverse debasing phosphodiester or thiophosphate. The following are representative structures of the formats used in in vivo experiments.

A representative structure of siRNA with a C6- _NH2 ligature at the 5' end of the transient strand and a C6-SH ligature at the 3' end.

A representative structure of a siRNA transit strand with a C6-NH2 _ligature at the 5' end and a C6-S-PEG ligature at the 3' end.

A representative structure of a siRNA transit strand with a C6-NH2 _ligature at the 5' end and a C6-S-NEM at the 3' end.

A representative structure of an siRNA transit strand with a C6-N-SMCC ligature at the 5' end and a C6-S-NEM ligature at the 3' end.

A representative structure of a siRNA transit strand with PEG at the 5' end and C6-SH at the 3' end.

A representative structure of a siRNA transit strand with a C6-S-NEM at the 5' end and a C6-NH2 _ferrule at the 3' end.

Cholesterol-myostatin siRNA conjugate

The sequence of the guide/antisense strand is complementary to the gene sequence for the mouse mRNA transcript of MSTN starting at base position 1169 (UUAUUAUAUUGUGUUCUUUGCCUU; SEQ ID NO: 868). Base, sugar, and phosphate modifications were used to optimize the efficacy of the duplex and reduce immunogenicity. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were duplexed to obtain double-stranded siRNA. The transit strand contained 5' cholesterol conjugated as shown in Figure 1 below.

Example 2. General Experimental Scheme and Materials

animal

All animal studies were conducted at ExploraBioLabs in accordance with the Institutional Animal Management and Use Committee (IACUC) protocol, and in accordance with the provisions outlined in the USDA Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (National Research Council Publication, 8th Edition, revised 2011). All mice were obtained from Charles River Laboratories or Harlan Laboratories.

The specified ASC (or antibody-nucleic acid conjugate) and dose were administered to wild-type CD1 mice (4-6 weeks old) via intravenous (iv) injection.

Antiferritin receptor antibody

Anti-mouse transferrin receptor antibody or CD71 mAb is a rat IgG2a subclass monoclonal antibody that binds to mouse CD71 or mouse transferrin receptor 1 (mTfR1). This antibody is manufactured by BioXcell and is commercially available (catalog number BE0175).

IgG2a isotype control antibody

The rat IgG2a isotype control antibody was purchased from BioXcell (clone 2A3, catalog number BE0089). This antibody is specific for trinitrophenol and has no known antigens in mice.

Anti-EGFR antibody

The anti-EGFR antibody is a fully human IgG1κ monoclonal antibody targeting the human epidermal growth factor receptor (EGFR). It was produced in the Chinese hamster ovary cell line DJT33, which was derived from the CHO-K1SV cell line by transfection with a GS vector carrying the antibody gene from a hybridoma cell line (2F8) that produces human anti-EGFR antibodies. The anti-EGFR antibody was prepared using standard mammalian cell culture and purification techniques.

The theoretical molecular weight (MW) of the glycan-free anti-EGFR antibody is 146.6 kDa. As determined by mass spectrometry, the experimental MW of the major glycosylated isotype of this antibody is 149 kDa. SDS-PAGE under reducing conditions revealed a MW of approximately 25 kDa for the light chain and approximately 50 kDa for the heavy chain. The heavy chains are linked to each other by two interchain disulfide bonds, and a light chain is linked to each heavy chain by a single interchain disulfide bond. The light chain has two intrachain disulfide bonds, while the heavy chain has four. The antibody is N-linked glycosylated at Asn305 of the heavy chain by a glycan composed of N-acetyl-glucosamine, mannose, fucose, and galactose. The dominant glycan present is a fucoidan-like structure containing zero or one terminal galactose residue.

The charged isoform patterns of IgG1κ antibodies have been investigated using imaging capillary IEF, agarose IEF, and analytical cation exchange HPLC. Several charged isoforms were identified, with the major isoform having an isoelectric point of approximately 8.7.

The primary mechanism of action of anti-EGFR antibodies is concentration-dependent inhibition of EGF-induced EGFR phosphorylation in A431 cancer cells. Furthermore, antibody-dependent cell-mediated cytotoxicity (ADCC) has been observed at low antibody concentrations in preclinical in vitro studies.

In vitro evaluation of siRNA efficacy and efficacy

C2C12 myoblasts (ATCC) were grown in DMEM supplemented with 10% v/v FBS. For transfection, cells were seeded at a density of 10,000 cells/well in 24-well plates and transfected within 24 hours. C2C12 myoblast cultures were generated by incubating the confluent C2C12 myoblast cultures in DMEM supplemented with 2% v/v horse serum for 3–4 days. The medium was changed daily during and after differentiation. Predifferentiated primary human skeletal muscle cells were obtained from Thermo Fisher and seeded in DMEM containing 2% v/v horse serum according to the manufacturer's recommendations. Human SJCRH30 rhabdomyosarcoma myoblasts (ATCC) were grown in DMEM supplemented with 10% v/v heat-inactivated fetal bovine serum, 4.5 mg/mL glucose, 4 mM L-glutamine, 10 mM HEPES, and 1 mM sodium pyruvate. For transfection, cells were seeded at a density of 10,000–20,000 cells/well in 24-well plates and transfected within 24 hours. All cells were transfected using RNAiMax (ThermoFisher) with various concentrations of siRNA (0.0001–100 nM; 10-fold dilution) according to the manufacturer's recommendations. Transfected cells were incubated in 5% CO2 at 37°C for 2 days, then washed with PBS, harvested in 300 μL TRIzol (ThermoFisher), and stored at -80°C. RNA was prepared using the ZYMO 96-well RNA kit (ThermoFisher), and relative RNA expression levels were quantified by RT-qPCR using commercially available TaqMan probes (Fife Technology). Expression data normalized relative to Ppib expression were analyzed using the ΔΔCT method and presented as %KD relative to simulated transfected cells. The data were analyzed using a 3-parameter dose-response inhibition function (GraphPadPrism 7.02) via nonlinear regression. Under these experimental conditions, all knockdown results showed the observed maximum KD.

Myostatin ELISA

The myostatin protein in plasma was quantified using the GDF-8 (Myostatin) Quantikine EFISA Immunoassay (catalog number DGDF80) from R&D Systems, according to the manufacturer's instructions.

RISC loading determination

RISC-based specific immunoprecipitation from tissue lysates and quantification of small RNAs in immunoprecipitates were determined by stem-loop PCR, using an improved assay described in Pei et al. Quantitative evaluation of siRNA delivery in vivo. RNA (2010), 16:2553–2563.

Example 3. Synthesis of Conjugates

The following structure illustrates the exemplary AX ₁ -BX _2- Y (Equation I) architecture described herein.

Architecture-1: Antibody-Cys-SMCC-5’-passive chain. This conjugate is generated by conjugation of the antibody chain inter-cysteine residue of maleimide (SMCC) at the 5’ end of the passive chain.

Architecture-2: Antibody-Cys-SMCC-3'-passive chain. This conjugate is generated by conjugation of the antibody chain inter-cysteine residue of maleimide (SMCC) at the 3' end of the passive chain.

Structure-3: Antibody-Cys-bisMal-3'-passive chain. This conjugate is generated by conjugation of an antibody chain inter-cysteine residue with a 3'-terminal bismaleimide (bisMal) linker.

Architecture-4: Model structure of the Fab-Cys-bisMal-3' transit chain. This conjugate is generated by conjugation of an inter-chain cysteine residue of the Fab chain with a 3' end bismaleimide (bisMal) linker.

Architecture-5: Model structure of an antibody siRNA conjugate in which two distinct siRNAs are linked to an antibody molecule. This conjugate is generated by conjugating a mixture of SSB and HPRT siRNAs to a bismaleimide (bisMal) linker at the 3' end of each siRNA passchain with a reduced mAb interstrand cysteine residue.

Architecture-6: Model structure of an antibody siRNA conjugate linking two distinct siRNAs. This conjugate is generated by conjugating a mixture of SSB and HPRT siRNAs to a maleimide (SMCC) linker at the 3' end of each siRNA passer chain with a reduced mAb chain intercysteine residue.

Example 3.1 Synthesis of antibody-siRNA conjugates using SMCC linkers

Synthesis Scheme-1: Antibody-Cys-SMCC-siRNA-PEG conjugate via antibody cysteine conjugation

Step 1: Antibody interchain disulfide reduction using TCEP

The antibody was buffer-exchanged with 25 mM borate buffer (pH 8) to achieve a concentration of 10 mg/mL. Two equivalents of TCEP in water were added to this solution, and the mixture was incubated at room temperature for 2 hours. The resulting reaction mixture was then buffer-exchanged with pH 7.4 PBS containing 5 mM EDTA, and SMCC-C6-siRNA or SMCC-C6-siRNA-C6-NHCO-PEG-X kDa (2 equivalents) (X = 0.5 kDa to 10 kDa) was added to a solution of 5 mM EDTA in pH 7.4 PBS at room temperature, and the mixture was incubated overnight. Analysis of the reaction mixture by analytical SAX column chromatography revealed the antibody-siRNA conjugate as well as unreacted antibody and siRNA.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC using anion exchange chromatography method-1 as described in Example 3.4. Fractions containing DAR1 and DAR>2 antibody-siRNA-PEG conjugates were separated, concentrated, and buffer-exchanged with pH 7.4 PBS.

Step 3: Analysis of the purified conjugate

The isolated conjugates were characterized by SEC, SAX chromatography, and SDS-PAGE. The purity of the conjugates was assessed by analytical HPLC using either anion exchange chromatography method-2 or anion exchange chromatography method-3. Both methods are described in Example 3.4. The isolated DAR1 conjugates typically eluted at 9.0 ± 0.3 min in analytical SAX methods and had a purity greater than 90%. Typical DAR>2 cysteine conjugates contain more than 85% DAR2 and less than 15% DAR3.

Figure 2 shows the SAX HPLC chromatogram of TfR mAb-(Cys)-HPRT-PEG5k,DAR1.

Figure 3 shows the SEC HPLC chromatogram of TfR mAb-(Cys)-HPRT-PEG5k,DAR1.

Example 3.2. Synthesis of antibody-siRNA conjugates using bismaleimide (BisMal) linkers

Synthesis Scheme 2: Antibody-Cys-BisMal-siRNA-PEG conjugate

Step 1: Reduce antibody with TCEP

The antibody was buffer-exchanged with 25 mM borate buffer (pH 8) to achieve a concentration of 5 mg/mL. Two equivalents of TCEP in water were added to this solution, and the mixture was rotated at room temperature for 2 hours. The resulting reaction mixture was then exchanged with pH 7.4 PBS containing 5 mM EDTA, and added to a solution of BisMal-C6-siRNA-C6-S-NEM (two equivalents) in pH 7.4 PBS containing 5 mM EDTA at room temperature, and incubated overnight at 4°C. Analysis of the reaction mixture by analytical SAX column chromatography revealed the antibody-siRNA conjugate as well as unreacted antibody and siRNA.

Step 2: Purification

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

Step 3: Analysis of the purified conjugate

The separated conjugates were characterized by mass spectrometry or SDS-PAGE. The purity of the conjugates was assessed by analytical HPLC using anion exchange chromatography method-2 or 3 and size exclusion chromatography method-1.

Figure 4 shows the overlay of DAR1 and DAR2SAX HPLC chromatograms of the TfR1mAb-Cys-BisMal-siRNA conjugate.

Figure 5 shows the overlay of DAR1 and DAR2SEC HPLC chromatograms of the TfR1mAb-Cys-BisMal-siRNA conjugate.

Example 3.3. Generation of Fab’ from mAb and conjugation with siRNA

Option 3: Generation of Fab-siRNA conjugates

Step 1: Digest antibodies with pepsin

The antibody was buffer-exchanged with 20 mM sodium acetate/acetic acid buffer at pH 4.0 to achieve a concentration of 5 mg/mL. Immobilized pepsin (Thermo Scientific, product number 20343) was added and incubated at 37°C for 3 hours. The reaction mixture was filtered using a 30 kDa MWCO Amicon rotary filter and pH 7.4 PBS. The retentate was collected and purified using size exclusion chromatography to separate F(ab’)2. The collected F(ab’)2 was then reduced with 10 equivalents of TCEP and conjugated with SMCC-C6-siRNA-PEG5 in pH 7.4 PBS at room temperature. Analysis of the reaction mixture by SAX chromatography revealed the Fab-siRNA conjugate as well as unreacted Fab and siRNA-PEG.

Step 2: Purification

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

Step 3: Analysis of the purified conjugate

The characterization and purity of the separated conjugates were evaluated by analytical HPLC using anion exchange chromatography method-2 or 3 and SEC method-1.

Figure 6 shows the SEC chromatogram of CD71 Fab-Cys-HPRT-PEG5.

Figure 7 shows the SAX chromatogram of CD71 Fab-Cys-HPRT-PEG5.

Example 3.4. Purification and Analytical Methods

Anion exchange chromatography method (SAX)-1.

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

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

3. Gradient:

Anion Exchange Chromatography (SAX) Method - 2

1. Column: Thermo Scientific, ProPac ^TM SAX-10, Bio LC ^TM , 4X 250mm

2. Solvent A: 80% 10mM TRIS, pH 8, 20% ethanol; Solvent B: 80% 10mM TRIS, pH 8, 20% ethanol, 1.5M NaCl; Flow rate: 0.75 ml/min

3. Gradient:

Anion Exchange Chromatography (SAX) Method - 3

1. Column: Thermo Scientific, ProPacTM SAX-10, Bio LCTM, 4X 250mm

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

3. Flow rate: 0.75 ml/min

4. Gradient:

Size Exclusion Chromatography (SEC) Method - 1

1. Column: TOSOH Biosciences, TSKgelG3000SW XL, 7.8X 300mm, 5μM

2. Mobile phase: 150mM phosphate buffer

3. Flow rate: 1.0 ml/min, 15 min

Example 4. In vitro screening: Atrogin-1

Identifying the regulation of mouse and human/NHP atrogin-1 by siRNA

Bioinformatics screening identified 56 siRNAs (19-mers) that specifically bind to mouse atrogin-1 (Fbxo32; NM_026346.3). Additionally, 6 siRNAs targeting mouse and human atrogin-1 (FBXO32; NM_058229.3) were identified. Screening for siRNAs (19-mers) specifically targeting human/NHP atrogin-1 (FBXO32; NM_058229.3) yielded 52 candidates (Tables 2A-2B). All selected siRNA target sites were free of SNPs (positions 2-18).

Tables 2A and 2B show the identified siRNA candidates for regulating mouse and human/NHP atrogin-1.

Table 2A.

Table 2B.

atrogin-1 siRNA was evaluated in transfected mouse C2C12 myoblasts, mouse C2C12 myotubes, predifferentiated myotubes of primary human skeletal muscle cells, and human SJCRH30 rhabdomyosarcoma myoblasts.

From 62 identified siRNAs targeting mouse atrogin-1 and 52 identified siRNAs targeting human atrogin-1, 30 and 20 siRNAs, respectively, were selected for synthesis and functional analysis. The activity of these siRNAs was analyzed in transfected mouse C2C12 myoblasts, mouse C2C12 myotubes, predifferentiated myotubes of primary human skeletal muscle cells, and human SJCRH30 rhabdomyosarcoma myoblasts.

In mouse C2C12 myoblasts (10 nM), none of the tested siRNAs targeting mouse atrogin-1 showed significant activity. However, in C2C12 myoblasts, three siRNAs downregulated mouse atrogin-1 mRNA by >75% (Table 3). Conversely, the siRNA targeting Murf1 was expressed only in C2C12 myoblasts (Figure 8) and was active in C2C12 myoblasts, demonstrating that the siRNA could be transfected into C2C12 myoblasts. To determine whether atrogin-1 could be selectively spliced in C2C12 myoblasts and myoblasts, RT-qPCR was used to probe various locations in atrogin-1 mRNA, but similar results were obtained. Of the 20 tested siRNAs targeting human atrogin-1, only four produced >75% KD. For both mouse and human atrogin-1, the active siRNAs were located in or near the coding region. One of the siRNAs targeting mouse atrogin-1 (1179) exhibits strong cross-reactivity with human atrogin-1. Although this siRNA failed to show significant activity in mouse C2C12 myotubes, it effectively downregulated human atrogin-1 in human primary skeletal muscle cell myotubes. All effective siRNAs downregulated their respective targets with sub-nanomolar potency.

Table 3 shows the activity of the selected atrogin-1 siRNA in transfected mouse C2C12 myoblasts, mouse C2C12 myotubes, predifferentiated myotubes of primary human skeletal muscle cells, and human SJCRH30 rhabdomyosarcoma myoblasts. For experimental procedures, see Example 2.

Example 5. In vitro screening: MuRF-1

Identification of siRNAs targeting mouse Murf1 (Trim63) and/or human/NHP MuRF1 (TRIM63)

Bioinformatics screening was performed, and 51 siRNAs (19-mers) specifically binding to the mouse Murf1 sequence were identified. These sequences showed more than three sequences derived from mouse Murf2 (Trim55; NM_001039048.2) or Murf3 (Trim54). Additionally, nine siRNAs targeting mouse Murf1 and human MuRF1 (TRIM63; NM_032588.3) were identified. Screening for siRNAs (19-mers) specifically targeting human and NHP MuRF1 (MuRF1; NM_032588.3) yielded 52 candidates (Tables 4A-4B). All selected siRNA target sites were free of SNPs (positions 2-18).

Tables 4A and 4B show the identified siRNA candidates for regulating mouse and human/NHP MuRF1.

Table 4A

Table 4B

The activity of selected MuRF1 siRNAs in transfected mouse C2C12 myotubes and predifferentiated myotubes of primary human skeletal muscle cells

From 60 identified siRNAs targeting mouse Murf1 and 25 targeting human MuRF1, 35 and 25 siRNAs, respectively, were synthesized. The activity of these siRNAs was analyzed in transfected mouse C2C12 myotubes and predifferentiated primary human skeletal muscle cells (Table 5). Among the siRNAs targeting mouse MurF1, 14 showed >70% Murf1 knockdown in C2C12 myotubes, but <20% Murf2 and Murf3 knockdown. At least 6 of these 14 siRNAs cross-reacted with human MuRF1. Among the tested siRNAs targeting human MuRF1, 8 showed >70% MuRF1 knockdown in predifferentiated myotubes of primary human skeletal muscle cells, but <20% MuRF2 and MuRF3 knockdown. Only 1 of these 8 siRNAs showed significant cross-reactivity with mouse Muf1. All effective siRNAs downregulated their respective targets with sub-nanomolar efficacy.

Table 5 shows the activity of the selected MuRF1 siRNAs in transfected mouse C2C12 myotubes and predifferentiated myotubes of primary human skeletal muscle cells. Cells were grown and transfected as described in Example 5, and RNA was isolated and analyzed.

Example 6. Design and synthesis of 2017-PK-279-WT-CD71 vs IgG2A isotype, HPRT vs MSTN siRNA

MSTN: A 21-base complementary double-stranded siRNA with a 19-base complement and a 3' dinucleotide overhang was designed for mouse MSTN. The sequence (5' to 3') of the guide/antisense strand is UUAUUAUUUGUUCUUUGCCUU (SEQ ID NO: 686). Base, sugar, and phosphate modifications were used to optimize the efficacy of the double-stranded siRNA and reduce immunogenicity. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were double-stranded to obtain double-stranded siRNA. The transit strand contains two concentric stalks, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both concentric stalks are attached to the siRNA transit strand via a phosphate thioester-reverse debase-phosphate thioester linker.

HPRT: A 21-base complementary double-stranded RNA with a 19-base complement and a 3' dinucleotide overhang was designed for mouse MSTN. The sequence (5' to 3') of the guide/antisense strand is UUAAAAUCUACAGUCAUAGUU (SEQ ID NO: 869). Base, sugar, and phosphate modifications were used to optimize the efficacy of the double-stranded RNA and reduce immunogenicity. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were double-stranded to obtain double-stranded siRNA. The transit strand contains two concentric stalks, namely C6- _NH2 at the 3' end and C6-SH at the 5' end. Both concentric stalks are attached to the siRNA transit strand via a phosphate thioester-reverse debase-phosphate thioester linker.

Negative control siRNA sequence (random): A 21-mer duplex with 19 complementary bases and a 3' dinucleotide overhang was used, as disclosed (Burke et al. (2014) Pharm. Res., 31(12):3445-60). The sequence of the guide/antisense strand (5' to 3') was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 870). The negative control siRNA was modified with the same bases, sugars, and phosphates used for the active MSTN siRNA duplex. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were duplexed to obtain double-stranded siRNA. The guest strand contained two stalks, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both stalks were attached to the siRNA guest strand via a phosphodiester-reverse debase-phosphodiester linker.

ASC Synthesis and Characterization

CD71 mAb-siRNA DAR1 conjugates were prepared, purified, and characterized as described in Example 3. All conjugates were linked to a thiol group via cysteine conjugation, an SMCC linker, and PEG. Structure 1 was used for MSTN and mixed siRNAs, and structure 2 was used for HPRT siRNAs (see Example 3). The conjugates were characterized chromatographically as described in Table 6.

Table 6. HPLC retention time (RT) in minutes

In vivo study design

In wild-type CD1 mice, the ability of the conjugate to mediate the downregulation of myostatin (MSTN) mRNA in skeletal muscle was evaluated in vivo. Mice were administered the PBS-mediated control and the ASC and dosage shown in Figure 9A via intravenous (iv) injection. Gastroc muscle tissue was harvested 96 hours later and thawed in liquid nitrogen. Comparative qPCR assays were used to determine mRNA knockdown in the target tissue. Total RNA was extracted from the tissue, reverse transcribed, and mRNA levels were quantified using TaqMan qPCR with appropriately designed primers and probes. The results were calculated using the comparative Ct method, where the difference (ΔCt) between the target gene Ct value and the PPIB Ct value was calculated and then further normalized relative to the PBS control by taking a second difference (ΔΔCt).

result

For gastrocnemius muscle harvested 96 hours after administration, a greater than 90% downregulation of maximum MSTN mRNA was observed after a single intravenous dose of 3 mg/kg siRNA (Figure 9B). Additionally, a dose response was observed (dose range: 0.3 to 3.0 mg/kg siRNA), and no significant mRNA downregulation was observed in the control group.

in conclusion

In the gastrocnemius muscle, ASC has been shown to downregulate muscle-specific genes. ASC is prepared from an anti-transferrin antibody conjugated to an siRNA designed to downregulate MSTN mRNA. Mouse gastrocnemius muscle expresses the transferrin receptor, and this conjugate, with a mouse-specific anti-transferrin antibody targeting the siRNA, leads to its accumulation in the gastrocnemius muscle. Receptor-mediated uptake results in siRNA-mediated MSTN mRNA knockdown.

Example 7. MSTN timeline of 2017-PK-289-WT-CD71 mAb used for phenotypic siRNA design and synthesis

MSTN: A 21-base complementary double-stranded siRNA with a 19-base complement and a 3' dinucleotide overhang was designed for mouse MSTN. The sequence (5' to 3') of the guide/antisense strand is UUAUUAUUUGUUCUUUGCCUU (SEQ ID NO: 868). Base, sugar, and phosphate modifications were used to optimize the efficacy of the double-stranded siRNA and reduce immunogenicity. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were double-stranded to obtain double-stranded siRNA. The guest strand contained two conjugates, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both conjugates were attached to the siRNA guest strand via a phosphate thioester-reverse debase-phosphate thioester linker. Since free thiol groups were not used for conjugation, fffu capping with N-ethylmaleimide was performed.

Negative control siRNA sequence (random): A 21-mer duplex with 19 complementary bases and a 3' dinucleotide overhang was used, as disclosed (Burke et al. (2014) Pharm. Res., 31(12):3445-60). The sequence of the guide/antisense strand (5' to 3') was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO:870). The negative control siRNA was modified with the same bases, sugars, and phosphates used for the active MSTN siRNA duplex. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were duplexed to obtain double-stranded siRNA. The guest strand contained two stalks, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both stalks were attached to the siRNA guest strand via a phosphate thioester-reverse debase-phosphate thioester linker. Since the free thiol group was not used for conjugation, fffu capping was performed using N-ethylmaleimide.

ASC Synthesis and Characterization

CD71 mAb-siRNA DAR1 conjugates were prepared and characterized as described in Example 3. All conjugates were prepared using architecture 1 via cysteine conjugation, SMCC linker, and thiol group, and were capped with NEM. The conjugates were characterized chromatographically as described in Table 7.

Table 7. HPLC retention time (RT) in minutes

In vivo study design

In wild-type CD1 mice, the ability of the conjugate to mediate the downregulation of myostatin (MSTN) mRNA in skeletal muscle was evaluated in vivo. Mice were administered the PBS-mediated control and the ASC and doses shown in Figure 10A via intravenous (iv) injection. Plasma and tissue samples were also obtained as shown in Figure 10A. Muscle tissue was harvested and thawed in liquid nitrogen. The mRNA knockdown in the target tissue was determined using comparative qPCR assays as described in the Methods section. Total RNA was extracted from the tissue, reverse transcribed, and the mRNA levels were quantified using TaqMan qPCR with appropriately designed primers and probes. The results were calculated using the comparative Ct method, where the difference (ΔCt) between the target gene Ct value and the PPIB Ct value was calculated, and then further normalized relative to the PBS control by taking a second difference (ΔΔCt). The quantification of tissue siRNA was determined using stem-loop qPCR assays as described in the Methods section. The antisense strand of siRNA was reverse transcribed using sequence-specific stem-loop RT primers with the TaqMan MicroRNA Reverse Transcription Kit. The cDNA from the RT step was then used for real-time PCR, and Ct values were converted to plasma or tissue concentrations using a linear equation derived from a standard curve.

Plasma myostatin levels were determined using ELISA; see Example 2 for complete experimental details. Changes in leg muscle area were determined: the leg to be tested was shaved, and the measurement area was marked with indelible ink. The mouse was restrained in a cone-shaped restraint device, with the right leg held by hand. Measurements were taken once in the sagittal plane and again in the coronal plane using digital calipers. This procedure was repeated twice weekly.

result

Quantifiable levels of siRNA accumulated in muscle tissue following a single intravenous administration of the antibody-siRNA conjugate are shown in Figure 10B. Stable MSTN mRNA downregulation was observed in the gastrocnemius muscle following a single intravenous administration of 3 mg/kg siRNA, resulting in decreased plasma MSTN protein levels, as shown in Figures 10C and 10D. Approximately 90% of the maximum mRNA downregulation was observed 7–14 days post-administration. At 6 weeks post-administration, approximately 75% of the mRNA was downregulated in the gastrocnemius muscle, corresponding to approximately 50% reduction in plasma protein levels compared to the haphazard control conjugated with PBS or anti-transferrin antibodies. MSTN downregulation resulted in a statistically significant increase in muscle size, as shown in Figures 10E and 10F.

in conclusion

In this embodiment, accumulation of siRNA in various muscle tissues was demonstrated following a single dose of an anti-transferrin antibody-targeted siRNA conjugate. Significant and durable siRNA-mediated downregulation of MSTN mRNA was observed in the gastrocnemius muscle. Mouse gastrocnemius muscle expresses a transferrin receptor, and this conjugate, containing a mouse-specific anti-transferrin antibody targeting the siRNA, resulted in its accumulation in the gastrocnemius muscle. Receptor-mediated uptake led to siRNA-mediated MSTN gene knockdown.

Example 8: Design and synthesis of 2017-PK-299-WT-MSTN Zalu vs TfR, mAb vs Fab, and DAR1 vs DAR2 siRNAs

MSTN: A 21-base complementary double-stranded siRNA with a 19-base complement and a 3' dinucleotide overhang was designed for mouse MSTN. The sequence (5' to 3') of the guide/antisense strand is UUAUUAUUUGUUCUUUGCCUU (SEQ ID NO: 868). Base, sugar, and phosphate modifications were used to optimize the efficacy of the double-stranded siRNA and reduce immunogenicity. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were double-stranded to obtain double-stranded siRNA. The guest strand contained two conjugates, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both conjugates were attached to the siRNA guest strand via a phosphate thioester-reverse debase-phosphate thioester linker. Since free thiol groups were not used for conjugation, fffu capping with N-ethylmaleimide was performed.

MSTN*: MSTN: A 21-base complementary double-stranded siRNA with a 19-base complement and a 3' dinucleotide overhang was designed for mouse MSTN. The sequence (5' to 3') of the guide/antisense strand is UUAUUAUUUGUUCUUUGCCUU (SEQ ID NO: 868). Base, sugar, and phosphate modifications were used to optimize the efficacy of the double-stranded siRNA and reduce immunogenicity. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were double-stranded to obtain double-stranded siRNA. The transit strand contains a conjugate stalk, namely the C6- _NH2 at the 5' end, which is linked to the siRNA transit strand via a phosphate thioester-reverse debase-phosphate thioester linker.

ASC Synthesis and Characterization

CD71 mAb-siRNA DAR1 and DAR2 conjugates were prepared and characterized as described in Example 3. Groups 1-8 and 17-20 were prepared using architecture 3 via cysteine conjugation and a BisMal linker. Groups 9-16 were prepared using architecture 1 via cysteine conjugation, an SMCC linker, and a free thiol group, and were capped with NEM PEG. The conjugates were characterized chromatographically as described in Table 8.

Table 8. HPLC retention time (RT) in minutes

In vivo study design

In wild-type CD-1 mice, the ability of the conjugate to mediate the downregulation of myostatin (MSTN) mRNA in skeletal muscle was evaluated in vivo. Mice were administered the PBS-mediated control and the ASC and doses shown in Figure 11A via intravenous (iv) injection. Plasma and tissue samples were also obtained as shown in Figure 11A. Gastroc muscle tissue was harvested and thawed in liquid nitrogen. The mRNA knockdown in the target tissue was determined using comparative qPCR assays as described in the Methods section. Total RNA was extracted from the tissue, reverse transcribed, and the mRNA levels were quantified using TaqMan qPCR with appropriately designed primers and probes. The results were calculated using the comparative Ct method, where the difference (ΔCt) between the target gene Ct value and the PPIB Ct value was calculated, and then further normalized relative to the PBS control by taking a second difference (ΔΔCt). The quantification of tissue siRNA was determined using stem-loop qPCR assays as described in the Methods section. The antisense strand of siRNA was reverse transcribed using sequence-specific stem-loop RT primers with the TaqMan MicroRNA Reverse Transcription Kit. The cDNA from the RT step was then used for real-time PCR, and Ct values were converted to plasma or tissue concentrations using a linear equation derived from a standard curve.

result

Quantifiable levels of siRNA accumulated in muscle tissue following a single intravenous administration of the antibody and Fab siRNA conjugate are shown in Figure 11B. Stable downregulation of MSTN mRNA was observed in the gastrocnemius muscle when the anti-transferrin antibody conjugate was administered as DAR1 or DAR2 or as a Fab DAR1 conjugate, as shown in Figure 11C.

in conclusion

In this embodiment, accumulation of siRNA in gastrocnemius muscle tissue was demonstrated following a single dose of an antitransferrin antibody and a Fab-targeted siRNA conjugate. In the gastrocnemius muscle, siRNA-mediated downregulation of MSTN mRNA was also observed using both DAR1 and DAR2 antibody conjugates, in addition to the DAR1 Fab conjugate. Mouse gastrocnemius muscle expresses the transferrin receptor, and the conjugate, containing a mouse-specific antitransferrin antibody or Fab to target the siRNA, resulted in its accumulation in the gastrocnemius muscle. Receptor-mediated uptake led to siRNA-mediated MSTN gene knockdown.

Example 9: Design and synthesis of 2017-PK-303-WT-dose-responsive MSTN mAb vs Fab vs Chol siRNA

MSTN: A 21-base complementary double-stranded siRNA with a 19-base complement and a 3' dinucleotide overhang was designed for mouse MSTN. The sequence (5' to 3') of the guide/antisense strand is UUAUUAUUUGUUCUUUGCCUU (SEQ ID NO: 868). Base, sugar, and phosphate modifications were used to optimize the efficacy of the double-stranded siRNA and reduce immunogenicity. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were double-stranded to obtain double-stranded siRNA. The guest strand contained two conjugates, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both conjugates were attached to the siRNA guest strand via a phosphate thioester-reverse debase-phosphate thioester linker. Since free thiol groups were not used for conjugation, fffu capping with N-ethylmaleimide was performed.

MSTN*: MSTN: A 21-base complementary double-stranded siRNA with a 19-base complement and a 3' dinucleotide overhang was designed for mouse MSTN. The sequence (5' to 3') of the guide/antisense strand is UUAUUAUUUGUUCUUUGCCUU (SEQ ID NO: 868). Base, sugar, and phosphate modifications were used to optimize the efficacy of the double-stranded siRNA and reduce immunogenicity. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were double-stranded to obtain double-stranded siRNA. The transit strand contains a conjugate stalk, namely the C6- _NH2 at the 5' end, which is linked to the siRNA transit strand via a phosphate thioester-reverse debase-phosphate thioester linker.

Negative control siRNA sequence (random): A 21-mer duplex with 19 complementary bases and a 3' dinucleotide overhang was used, as disclosed (Burke et al. (2014) Pharm. Res., 31(12):3445-60). The sequence of the guide/antisense strand (5' to 3') was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO:870). The negative control siRNA was modified with the same bases, sugars, and phosphates used for the active MSTN siRNA duplex. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were duplexed to obtain double-stranded siRNA. The guest strand contained two stalks, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both stalks were attached to the siRNA guest strand via a phosphate thioester-reverse debase-phosphate thioester linker. Since the free thiol group was not used for conjugation, fffu capping was performed using N-ethylmaleimide.

ASC Synthesis and Characterization

CD71 mAb-siRNA DAR1 and DAR2 conjugates were prepared and characterized as described in Example 3. Groups 5-12 were prepared using architecture 3 via cysteine conjugation and a BisMal linker. Groups 13-16 were prepared using architecture 1 via cysteine conjugation, an SMCC linker, and a free thiol group, and were capped with NEM. Groups 17-20 were prepared using architecture 3 via cysteine conjugation, a BisMal linker, and a free thiol group, and were capped with NEM. The conjugates were characterized chromatographically as described in Table 9.

Table 9. HPLC retention times (RT) in minutes

In vivo study design

In wild-type CD-1 mice, the ability of the conjugate to mediate the downregulation of myostatin (MSTN) mRNA in skeletal muscle was evaluated in vivo. Mice were administered the PBS-mediated control and the ASC doses shown in Figure 12A via intravenous (iv) injection. Tissue samples were also obtained as shown in Figure 12A. Gastrocnemius muscle tissue was harvested and thawed in liquid nitrogen. The mRNA knockdown in the target tissue was determined using comparative qPCR assays as described in the Methods section. Total RNA was extracted from the tissue, reverse transcribed, and mRNA levels were quantified using TaqMan qPCR with appropriately designed primers and probes. The results were calculated using the comparative Ct method, where the difference (ΔCt) between the target gene Ct value and the PPIB Ct value was calculated and then further normalized relative to the PBS control by taking a second difference (ΔΔCt). The quantification of tissue siRNA was determined using stem-loop qPCR assays as described in the Methods section. The antisense strand of siRNA was reverse transcribed using sequence-specific stem-loop RT primers with the TaqMan MicroRNA Reverse Transcription Kit. The cDNA from the RT step was then used for real-time PCR, and Ct values were converted to plasma or tissue concentrations using a linear equation derived from a standard curve.

Intracellular RISC loading was determined as described in Example 2.

result

Quantifiable levels of siRNA accumulated in the gastrocnemius and cardiac tissues following a single intravenous administration of the antibody and Fab siRNA conjugate are shown in Figure 12B. Stable downregulation of MSTN mRNA was observed in the gastrocnemius muscle when ASC was targeted with either the anti-transferrin receptor antibody or Fab, as shown in Figures 12B and 12C. Delivery of higher concentrations of siRNA to cardiac tissue did not result in stable downregulation of myostatin mRNA, as shown in Figure 12B. A much lower dose of ASC is required to achieve equivalent mRNA downregulation compared to the cholesterol siRNA conjugate. The RISC loading of the MSTN siRNA guide chain correlates with mRNA downregulation, as shown in Figure 12D.

in conclusion

In this embodiment, accumulation of siRNA in gastrocnemius muscle tissue was demonstrated following a single dose of an anti-transferrin antibody and a Fab-targeted siRNA conjugate. In the gastrocnemius muscle, siRNA-mediated downregulation of MSTN mRNA was observed using either the DAR1 anti-transferrin antibody or the Fab conjugate. Mouse gastrocnemius muscle expresses a transferrin receptor, and the conjugate, containing a mouse-specific anti-transferrin antibody or Fab to target the payload, resulted in the accumulation of the conjugate in the gastrocnemius muscle and loading into the RISC complex. Receptor-mediated uptake led to siRNA-mediated downregulation of MSTN mRNA.

Example 10: Design and synthesis of 2017-PK-304-WT-PK mAb with MSTN phenotype vs. Chol siRNA

MSTN: A 21-mer doublet with 19 complementary bases and a 3' dinucleotide overhang was designed for mouse MSTN. The sequence (5' to 3') of the guide/antisense strand is UUAUUAUUUGUUCUUUGCCUU (SEQ ID NO: 868). Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize the efficacy of the doublet and reduce immunogenicity. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were doubletted to obtain double-stranded siRNA. The guest strand contained two conjugates, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both conjugates were attached to the siRNA guest strand via a phosphate thioester-reverse debase-phosphate thioester linker. Since free thiol groups were not used for conjugation, fffu capping with N-ethylmaleimide was performed.

Negative control siRNA sequence (random): A 21-mer duplex with 19 complementary bases and a 3' dinucleotide overhang was used, as disclosed (Burke et al. (2014) Pharm. Res., 31(12):3445-60). The sequence of the guide/antisense strand (5' to 3') was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO:870). The negative control siRNA was modified with the same bases, sugars, and phosphates used for the active MSTN siRNA duplex. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were duplexed to obtain double-stranded siRNA. The guest strand contained two stalks, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both stalks were attached to the siRNA guest strand via a phosphate thioester-reverse debase-phosphate thioester linker. Since the free thiol group was not used for conjugation, fffu capping was performed using N-ethylmaleimide.

ASC Synthesis and Characterization

CD71 mAb-siRNA DAR1 and DAR2 conjugates were prepared and characterized as described in Example 3. Groups 5-12 were prepared using architecture 1 via cysteine conjugation, SMCC linker, and free thiol group, and were capped with NEM. Groups 13-16 were prepared using architecture 3 via cysteine conjugation, BisMal linker, and free thiol group, and were capped with NEM. The conjugates were characterized chromatographically as described in Table 10.

Table 10. HPLC retention times (RT) in minutes

In vivo study design

In wild-type CD-1 mice, the ability of the conjugate to mediate the downregulation of myostatin (MSTN) mRNA in skeletal muscle was evaluated in vivo. Mice were administered the PBS-mediated control and the ASC doses shown in Figure 13A via intravenous (iv) injection. Tissue samples were also obtained as shown in Figure 13A. Gastrocnemius muscle tissue was harvested and thawed in liquid nitrogen. The mRNA knockdown in the target tissue was determined using comparative qPCR assays as described in the Methods section. Total RNA was extracted from the tissue, reverse transcribed, and the mRNA levels were quantified using TaqMan qPCR with appropriately designed primers and probes. The results were calculated using the comparative Ct method, where the difference (ΔCt) between the target gene Ct value and the PPIB Ct value was calculated and then further normalized relative to the PBS control by taking a second difference (ΔΔCt). The quantification of tissue siRNA was determined using stem-loop qPCR assays as described in the Methods section. The antisense strand of siRNA was reverse transcribed using sequence-specific stem-loop RT primers with the TaqMan MicroRNA Reverse Transcription Kit. The cDNA from the RT step was then used for real-time PCR, and Ct values were converted to plasma or tissue concentrations using a linear equation derived from a standard curve.

Intracellular RISC loading was determined as described in Example 2. Plasma MSTN protein levels were measured by ELISA as described in Example 2.

To determine changes in leg muscle area: Shave the leg to be tested and mark the measurement area with indelible ink. Restrain the mouse in a small decapicone pouch. Take one measurement in the sagittal plane and another in the coronal plane using digital calipers. Repeat this procedure twice a week.

result

Quantifiable levels of siRNA accumulated in the gastrocnemius, triceps, quadriceps, and cardiac tissues following a single intravenous administration of the antibody siRNA conjugate at 3 mg/kg are shown in Figure 13D. Downregulation of MSTN mRNA was observed in the gastrocnemius, quadriceps, and triceps using the DAR1 and DAR2 conjugates, but not in cardiac tissue (Figure 13B). Downregulation of MSTN mRNA resulted in decreased plasma concentrations of MSTN protein, as measured by EFISA (Figure 13C). The RISC loading of the MSTN siRNA guide strand was correlated with mRNA downregulation (Figure 13E). Downregulation of MSTN resulted in a statistically significant increase in muscle size (Figures 13F and 13G).

in conclusion

In this embodiment, accumulation of siRNA in the gastrocnemius, quadriceps, and triceps muscle tissues was demonstrated following single doses of the anti-transferrin antibody siRNA conjugates DAR1 and DAR2. Measurable siRNA-mediated downregulation of MSTN mRNA was observed in all three tissues using the DAR1 and DAR2 anti-transferrin antibody conjugates. This mRNA downregulation was associated with decreased plasma MSTN protein levels and RISC loading of the siRNA guide chain. All three muscle tissues express the transferrin receptor, and the conjugates contain mouse-specific anti-transferrin antibodies that target the siRNA, leading to its accumulation in the muscle. Receptor-mediated uptake resulted in siRNA-mediated MSTN gene knockdown.

Example 11: Design and synthesis of 2017-PK-355-WT multi-siRNA drug delivery siRNA

HPRT: A 21-mer doublet with 19 complementary bases and a 3' dinucleotide overhang was designed for mouse MSTN. The sequence (5' to 3') of the guide/antisense strand is UUAAAAUCUACAGUCAUAGUU (SEQ ID NO: 869). Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize the efficacy of the doublet and reduce immunogenicity. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were doubletted to obtain double-stranded siRNA. The transit strand contains a conjugate stalk, namely the C6- _NH2 at the 5' end, which is linked to the siRNA transit strand via a phosphate thioester-reverse debasement-phosphate thioester linker.

SSB: A 21-base complementary double-stranded RNA with a 19-base complement and a 3' dinucleotide overhang was designed for mouse MSTN. The sequence (5' to 3') of the guide/antisense strand is UUACAUUAAAGUCUGUUGUUU (SEQ ID NO: 871). Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize the efficacy of the double-stranded RNA and reduce immunogenicity. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were double-stranded to obtain double-stranded siRNA. The transit strand contains a conjugate stalk, namely the C6- _NH2 at the 5' end, which is linked to the siRNA transit strand via a phosphate thioester-reverse debase-phosphate thioester linker.

ASC Synthesis and Characterization

The CD71 mAb-siRNA conjugate was prepared and characterized as described in Example 3.

Groups 1-4 and 5-8 were prepared using architecture 3, via cysteine conjugation, a BisMal linker, and without a 3' conjugation stalk on the transit chain. Groups 13-16 were prepared via cysteine conjugation, a BisMal linker, and without a 3' conjugation stalk on the transit chain, but the DAR2 conjugate was prepared using architecture 4 from a mixture of HPRT and SSB siRNA. The conjugates were characterized chromatographically as described in Table 11.

Table 11. HPLC retention times (RT) in minutes

In vivo study design

In wild-type CD1 mice, the ability of conjugates to mediate the downregulation of mRNA in two housekeeping genes (HPRT and SSB) in skeletal muscle was evaluated in vivo. Mice were administered the PBS-mediated control and the ASC doses shown in Figure 14A via intravenous (iv) injection. Tissue samples were also obtained as shown in Figure 14A. Gastrocnemius muscle tissue was harvested and thawed in liquid nitrogen. The mRNA knockdown in the target tissue was determined using comparative qPCR assays as described in the Methods section. Total RNA was extracted from the tissue, reverse transcribed, and mRNA levels were quantified using TaqMan qPCR with appropriately designed primers and probes. The results were calculated using the comparative Ct method, where the difference (ΔCt) between the target gene Ct value and the PPIB Ct value was calculated and then further normalized relative to the PBS control by taking a second difference (ΔΔCt). The quantification of tissue siRNA was determined using stem-loop qPCR assays as described in the Methods section. The antisense strand of siRNA was reverse transcribed using sequence-specific stem-loop RT primers with the TaqMan MicroRNA Reverse Transcription Kit. The cDNA from the RT step was then used for real-time PCR, and Ct values were converted to plasma or tissue concentrations using a linear equation derived from a standard curve.

RISC loading determination was performed as described in Example 2.

result

Following a single intravenous administration of the antibody-siRNA conjugate at the indicated dose, mRNA downregulation was observed in the gastrocnemius muscle and cardiac tissue (Figures 14B-14D). Co-administration of a mixture of two ASCs targeting two different genes (HPRT and SSB) resulted in effective mRNA downregulation of both targets in the gastrocnemius muscle and cardiac tissue. Furthermore, administration of a DAR2 conjugate synthesized using a 1:1 mixture of the two different siRNAs (HPRT and SSB) also resulted in effective mRNA downregulation of both targets in the gastrocnemius muscle and cardiac tissue. All delivery routes resulted in the accumulation of measurable amounts of siRNA in the gastrocnemius muscle tissue (Figure 14F).

in conclusion

In this embodiment, the accumulation of siRNA in the gastrocnemius and myocardial tissue following a single dose of the anti-transferrin antibody siRNA conjugate was demonstrated. Two genes were downregulated by co-administration of two ASCs produced from the same anti-transferrin antibody but conjugated to two different siRNAs (HPRT and SSB). Additionally, two genes were downregulated using an anti-transferrin mAb DAR2 conjugate synthesized using a 1:1 mixture of the two different siRNAs (HPRT and SSB). In some cases, simultaneous downregulation of more than one gene can be used to treat muscle atrophy.

Example 12: Atrogin-1 siRNA activity in vivo (dose-response) 2017-PK-380-WT siRNA design and synthesis

Atrogin-1 siRNA: Four different 21-mer duplexes with 19-base complementarity and 3' dinucleotide overhangs were designed for Atrogin-1. See Example 4 for sequence details. Base, sugar, and phosphate modifications well-described in the RNAi field were used to optimize duplex efficacy and reduce immunogenicity. All four siRNAs used the same design. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were duplexed to obtain double-stranded siRNA. The guest strand contained two conjugates: C6- _NH2 at the 5' end and C6-SH at the 3' end. Both conjugates were attached to the siRNA guest strand via a phosphate thioester-reverse debase-phosphate thioester linker. Since free thiol groups were not used for conjugation, fffu capping with N-ethylmaleimide was performed.

ASC Synthesis and Characterization

The CD71 mAb-siRNA conjugate was prepared and characterized as described in Example 3.

Groups 1-16 were prepared using architecture 3 via cysteine conjugation, BisMal linker, and free thiol groups, and were capped with NEM. The conjugates were characterized chromatographically as described in Table 12.

Table 12. HPLC retention times (RT) in minutes

In vivo study design

In wild-type CD1 mice, the ability of the conjugate to mediate the downregulation of Atrogin-1 mRNA in skeletal muscle was evaluated in vivo. Mice were administered the PBS-mediated control and the ASC and doses shown in Figure 15A via intravenous (iv) injection. Tissue samples were obtained as shown in Figure 15A. Gastroc muscle tissue was harvested and thawed in liquid nitrogen. Comparative qPCR assays as described in the Methods section were used to determine mRNA knockdown in the target tissue. Total RNA was extracted from the tissue, reverse transcribed, and mRNA levels were quantified using TaqMan qPCR with appropriately designed primers and probes. The results were calculated using the comparative Ct method, where the difference (ΔCt) between the target gene Ct value and the PPIB Ct value was calculated, and then further normalized relative to the PBS control by taking a second difference (ΔΔCt).

result

Following a single intravenous administration of the antibody siRNA conjugate at the indicated dose, up to 80% atrogin-1 mRNA downregulation was observed in the gastrocnemius muscle and up to 50% atrogin-1 mRNA downregulation was observed in cardiac tissue (see Figures 15B and 15C).

in conclusion

As shown in this example, the antibody siRNA conjugate differentially downregulated Atrogin-1 in muscle and heart.

Example 13: 2017-PK-383-WT activity of MuRF1 siRNA in vivo (dose response) siRNA design and synthesis

MuRF1 siRNA: Four different 21-mer duplexes with 19-base complementarity and 3' dinucleotide overhangs were designed for Atrogin-1. See Example 5 for sequence details. Base, sugar, and phosphate modifications well-described in the RNAi field were used to optimize duplex efficacy and reduce immunogenicity. All four siRNAs used the same design. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were duplexed to obtain double-stranded siRNAs. The guest strand contained two conjugates: C6- _NH2 at the 5' end and C6-SH at the 3' end. Both conjugates were attached to the siRNA guest strand via a phosphate thioester-reverse debase-phosphate thioester linker. Since free thiol groups were not used for conjugation, fffu capping with N-ethylmaleimide was performed.

ASC Synthesis and Characterization

The CD71 mAb-siRNA conjugate was prepared and characterized as described in Example 3.

Groups 1-16 were prepared using architecture 3 via cysteine conjugation, BisMal linker, and free thiol groups, and were capped with NEM. The conjugates were characterized chromatographically as described in Table 13.

Table 13. HPLC retention times (RT) in minutes

In vivo study design

In wild-type CD1 mice, the ability of the conjugate to mediate the downregulation of MuRF-1 mRNA in skeletal and cardiac muscle was evaluated in vivo. Mice were administered the PBS-mediated control and the ASC and doses shown in Figure 16A via intravenous (iv) injection. Tissue samples were obtained as shown in Figure 16A. Gastroc muscle tissue was harvested and thawed in liquid nitrogen. Comparative qPCR assays as described in the Methods section were used to determine mRNA knockdown in the target tissues. Total RNA was extracted from the tissues, reverse transcribed, and mRNA levels were quantified using TaqMan qPCR with appropriately designed primers and probes. The results were calculated using the comparative Ct method, where the difference (ΔCt) between the target gene Ct value and the PPIB Ct value was calculated, and then further normalized relative to the PBS control by taking a second difference (ΔΔCt).

result

Following a single intravenous administration of the antibody siRNA conjugate at the indicated dose, MuRF1 mRNA was downregulated to up to 70% in the gastrocnemius muscle and to up to 50% in the cardiac tissue (see Figures 16B and 16C).

in conclusion

As shown in this example, the antibody siRNA conjugate differentially downregulated MuRF1 in muscle and heart.

Example 14.

Table 14 shows exemplary siRNA (or atrogene) targets for regulating muscle atrophy. In some cases, polynucleotide molecules hybridize with the target regions of the atrogenes described in Table 14.

Example 15: Sequence

A 23-mer target sequence is generated within a DMPK transcript variant (NM_001288766). A set of 23-mer target sequences for this transcript variant is generated by shifting the transcript length down one base at a time, and a similar set of target sequences can be generated for other DMPK transcript variants using the same procedure. A common siRNA structure that can be used to target these sites in DMPK transcripts is a 19-mer fully complementary double helix, which has two overhanging (unpaired) nucleotides at the 3' end of each strand. Therefore, adding two 2-nucleotide overhangs to the 19-mer results in a total of 23 bases targeting the target site. Since the overhangs can consist of sequences reflecting the sequence of the target transcript or other nucleotides (e.g., unrelated dinucleotide sequences such as "UU"), the 19-mer fully complementary sequence can be used to describe the siRNA targeting each 23-mer target site.

For each site within the DMPK transcript, the siRNA duplexes targeting each site generate the first set of 19-mer sense and antisense sequences. The DMPK transcript variant NM_00l288766 has been used for illustration, but a similar set of siRNA duplexes can be generated by traversing other DMPK transcript variants. When the antisense strand of the siRNA is loaded into Ago2, the first base associates within the Ago2 binding pocket, while the other bases (starting from position 2 on the antisense strand) exhibit complementary mRNA binding. Since “U” is the thermodynamically preferred first base for binding Ago2 and does not bind the target mRNA, all antisense sequences can substitute “U” for the first base without affecting target complementarity and specificity. Accordingly, the last base of the sense strand 19-mer (position 19) is switched to “A” to ensure base pairing with the “U” at the first position on the antisense strand.

The second set of 19-mer sense sequences is similar to the first set of 19-mer sense sequences targeting DMPK transcripts, except that the last position of the 19-mer sense chain is replaced by the base "A".

The second set of 19-mer antisense sequences is similar to the first set of 19-mer antisense sequences targeting DMPK transcripts, except that the first position of the 19-mer antisense strand is replaced by the base "U".

Example 16: Preliminary screening of a group of selected DMPK siRNAs for in vitro activity

Using bioinformatics analysis aimed at selecting sequences with the highest probability of on-target activity and the lowest probability of off-target activity, a second set of DMPK siRNAs was narrowed down to a list of 81 siRNA sequences (SEQ ID NO: 28-189). Bioinformatics methods for selecting active and specific siRNAs are well-described in the RNAi field, and those skilled in the art can generate similar DMPK siRNA sequence lists for any other DMPK transcript variant. DMPK siRNAs from the set of 81 sequences were synthesized on a small scale using standard solid-phase synthesis methods described in the oligonucleotide synthesis literature. Both unmodified and chemically modified siRNAs are known to produce effective knockdown after in vitro transfection. DMPK siRNA sequences were synthesized using base, sugar, and phosphate modifications well-described in the RNAi field to optimize duplex potency and reduce immunogenicity. The in vitro activity of DMPK siRNA was evaluated using two human cell lines: the first was the SJCRH30 human rhabdomyosarcoma cell line (CRL-2061 ^™ ); the second was uncontrolled proliferating human skeletal muscle myoblasts derived from patients with ankylosing dystrophy type 1 (DM1). For initial screening of the DMPK siRNA library, each DMPK siRNA was transfected into SJCRH30 cells at final concentrations of 1 nM and 0.01 nM, and into DM1 myoblasts at final concentrations of 10 nM and 1 nM. The siRNA was prepared using the Lipofectamine RNAiMAX transfection reagent (Life Technologies) according to the manufacturer's "forward transfection" instructions. Twenty-four hours prior to transfection, cells were seeded in triplicate onto 96-well tissue culture plates: 8500 cells per well for SJCRH30 and 4000 cells per well for DM1 myoblasts. Cells were washed with PBS and harvested using reagent (Life Technologies) at 48 h (SJCRH30) or 72 h (DM1 myoblasts) post-transfection. RNA was isolated using the Direct-zol-96 RNA Kit (Zymo Research) according to the manufacturer's instructions. 10 μl of RNA was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. cDNA samples were evaluated by qPCR using DMPK-specific and PPIB-specific TaqMan human gene expression probes (Thermo Fisher) using Fast Advanced Master Mix (Applied Biosystems). DMPK values were normalized relative to PPIB gene expression within each sample. Quantification of HPRT downregulation was performed using the standard 2 ^-ΔΔCt method. All experiments were performed in triplicate; Tables 15A and 15B present the average of three replicates.

Table 15A

The position of the 19-mer in ^{NM_001288766.1}

Table 15B

² DM1 myoblasts; 10 nM; % DMPK mRNA

³ DM1 myoblasts; 1 nM; % DMPK mRNA

⁴ SJCRH30; 1nM; %DMPK mRNA

⁵ SJCRH30; 0.01nM; %DMPK mRNA

Example 17: In vitro dose-response curves of a selected set of DMPK siRNAs

To further validate the activity of DMPK siRNA, several sequences that showed optimal activity in the initial screening were selected for subsequent evaluation in dose-response form. Again, the in vitro activity of DMPK siRNA was assessed using two human cell lines: the first was the SJCRH30 human rhabdomyosarcoma cell line; the second was uncontrolled proliferation of human skeletal muscle myoblasts derived from patients with ankylosing dysplasia type 1 (DM1). The selected siRNAs were transfected at 10-fold dose-response concentrations of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 nM, or at 9-fold dose-response concentrations of 50, 5.55556, 0.617284, 0.068587, 0.007621, 0.000847, and 0.000094 nM. The siRNAs were prepared using the Lipofectamine RNAiMAX transfection reagent (Life Technologies) according to the manufacturer's "forward transfection" instructions. Twenty-four hours prior to transfection, cells were seeded in triplicate onto 96-well tissue culture plates: 8500 cells per well for SJCRH30 and 4000 cells per well for DM1 myoblasts. At 48 h (SJCRH30) or 72 h (DM1 myoblasts) post-transfection, cells were washed with PBS and harvested using Life Technologies. RNA was isolated using the Direct-zol-96 RNA Kit (Zymo Research) according to the manufacturer's instructions. 10 μl of RNA was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. cDNA samples were evaluated by qPCR using the FastAdvanced Master Mix (Applied Biosystems) with DMPK-specific and PPIB-specific TaqMan human gene expression probes (Thermo Fisher). DMPK values were normalized relative to PPIB gene expression within each sample. Quantification of HPRT downregulation was performed using the standard 2 ^{^-ΔΔCt} method. All experiments were performed in triplicate. Tables 16A-B, 17A-B, and 18A-B present the average of the three replicates and the calculated _IC50 values determined by fitting the curves to the dose-response data using nonlinear regression.

Table 16A

The position of the 19-mer in ^{NM_001288766.1}

Table 16B

² SJCRH30;0.0001nM;%DMPK mRNA ³ SJCRH30;0.001nM;%DMPK mRNA ⁴ SJCRH30;0.01nM;%DMPK mRNA ⁵ SJCRH30;0.1nM;%DMPK mRNA ⁶ SJCRH30;1nM;%DMPK mRNA ⁷ SJCRH30;10nM;%DMPK mRNA ⁸ SJCRH30; 100 nM; % DMPK mRNA Table 17A

The position of the 19-mer in ^{NM_001288766.1}

Table 17B

² SJCRH30; 0.000094nM; %DMPK mRNA ³ SJCRH30; 0.000847nM; %DMPK mRNA ⁴ SJCRH30; 0.007621nM; %DMPK mRNA ⁵ SJCRH30; 0.068587nM; %DMPK mRNA ⁶ SJCRH30; 0.617284nM; %DMPK mRNA ⁷ SJCRH30; 5.55556nM; %DMPK mRNA Table 18A

The position of the 19-mer in ^{NM_001288766.1}

Table 18B

² DM1 myoblasts; 0.000094 nM; % DMPK mRNA

³ DM1 myoblasts; 0.000847 nM; % DMPK mRNA

⁴ DM1 myoblasts; 0.007621 nM; % DMPK mRNA

⁵ DM1 myoblasts; 0.068587 nM; % DMPK mRNA

⁶ DM1 myoblasts; 0.617284 nM; % DMPK mRNA

⁷ DM1 myoblasts; 5.55556 nM; %DMPK mRNA

Example 18: In vitro experiments to determine species cross-reactivity in mice

The selected siRNAs were transfected into C2C12 mouse myoblasts (CRL-1772 ^™ ) at final concentrations of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 nM. The siRNAs were prepared using the Lipofectamine RNAiMAX transfection reagent (Life Technologies) according to the manufacturer's "Forward Transfection" instructions. Twenty-four hours before transfection, cells were seeded in triplicate onto 96-well tissue culture plates, with 4000 cells per well for C2C12 cells. Forty-eight hours after transfection, the cells were washed with PBS and harvested using Life Technologies reagent. RNA was isolated using the Direct-zol-96 RNA Kit (ZymoResearch) according to the manufacturer's instructions. 10 μl of RNA was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. cDNA samples were evaluated by qPCR using Fast Advanced Master Mix (Applied Biosystems) with DMPK-specific and PPIB-specific TaqMan mouse gene expression probes (Thermo Fisher). DMPK values were normalized relative to PPIB gene expression within each sample. Quantification of HPRT downregulation was performed using the standard 2 ^-ΔΔCt method. All experiments were performed in triplicate, and results are shown in Figure 17. Four DMPK siRNAs (the numbers indicated in the legend of Figure 17 correspond to ID# listed in Table 15A) were shown to effectively cross-react with mouse DMPK mRNA, resulting in stable mRNA knockdown in the mouse C2C12 myoblast cell line. Two siRNAs (ID#535 and 1028) were slightly less potent, producing only about 70% of the maximum mRNA knockdown. Two siRNAs (ID#2628 and 2636) were more potent, producing about 90% of the maximum mRNA knockdown.

Example 19: In vivo experiments to determine species cross-reactivity in mice

animal

All animal studies were conducted at ExploraBioLabs in accordance with the Institutional Animal Management and Use Committee (IACUC) protocol, and in accordance with the provisions outlined in the USDA Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (National Research Council Publication, 8th Edition, revised 2011). All mice were obtained from Charles River Laboratories or Harlan Laboratories.

Preparation of conjugates

Five siRNAs were used in the in vivo studies: four DMPK siRNAs that showed cross-reactivity with mice in vitro (Figure 17), and one siRNA with a disordered sequence that did not produce DMPK knockdown and could be used as a negative control. All siRNAs were synthesized using standard solid-phase synthesis methods described in the oligonucleotide synthesis literature. Single strands were purified by HPLC using standard methods, and then the pure single strands were mixed in equimolar ratios to produce pure duplexes. All siRNAs were synthesized with a hexylamine linker at the 5' end of the guest (sense) strand, which could be used as a conjugation stalk for antibody attachment. The siRNAs were synthesized using optimal base, sugar, and phosphate modifications well described in the field of RNAi to maximize duplex potency, metabolic stability, and immunogenicity.

The anti-mouse transferrin receptor (TfR1, also known as CD71) monoclonal antibody (mAh) is a rat IgG2a subclass monoclonal antibody that binds to mouse CD71 protein with high affinity. This CD71 antibody is manufactured by BioXcell and is commercially available (catalog number BE0175). Antibody-siRNA conjugates were synthesized using the BioXcell-derived CD71 mAh and the corresponding DMPK or scrambled siRNA. All conjugates were synthesized using a bismaleimide-TFP ester linker via cysteine conjugation to the antibody and amine conjugation to the siRNA (via hexylamine). All conjugates were purified by strong cation exchange (SAX) to isolate only those conjugates with a drug-antibody ratio (DAR) of 1 (i.e., a molar ratio of 1 siRNA/mAh). All antibody-siRNA conjugates were formulated for in vivo administration by dilution in PBS.

In vivo administration and analysis

Purified DAR1 antibody-siRNA conjugates were administered to female wild-type CD1 mice (4–6 weeks old) at doses of 0.1, 0.3, 1, and 3 mg/kg (based on siRNA weight) via a single intravenous bolus injection into the tail vein at a dose volume of 5 mL/kg. A single dose of a mimicry dose of PBS was injected into a control group of female wild-type CD1 mice (also 4–6 weeks old) at a matched dose volume (n=5). Seven days post-administration, mice were euthanized by _CO2 asphyxiation, and 20–30 mg of various tissue slices (gastrocnemius, tibialis anterior, quadriceps, diaphragm, heart, and liver) were harvested from each mouse and thawed in liquid nitrogen. Reagents (Life Technologies) were added, and each tissue slice was homogenized using TissueLyser II (Qiagen). RNA was isolated using the Direct-zol-96 RNA Kit (Zymo Research) according to the manufacturer's instructions. Using the HighCapacity cDNA Reverse Transcription Kit (Applied Biosystems), 10 μl of RNA was reverse transcribed into cDNA according to the manufacturer's instructions. cDNA samples were evaluated by qPCR using DMPK-specific and PPIB-specific TaqMan mouse gene expression probes (Thermo Fisher) using the Fast Advanced Master Mix (Applied Biosystems). DMPK values were normalized relative to PPIB gene expression within each sample. DMPK downregulation was quantified using the standard 2 ^-ΔΔCt method by comparing treated animals to a PBS control group. In vivo DMPK mRNA knockdown results are shown in Figures 18A–18F. All four DMPK siRNAs (the numbers shown in the legends of Figures 18A–18F correspond to the ID# listed in Table 15A) showed a dose-dependent reduction in DMPK mRNA levels in all analyzed skeletal muscles (gastrocnemius, tibialis anterior, quadriceps, and diaphragm). At the highest dose (3 mg/kg), the most active siRNA achieved greater than 75% DMPK mRNA knockdown in all skeletal muscles. The in vivo DMPK knockdown observed in mouse skeletal muscle (Fig. 18A–18F) was closely correlated with the in vitro DMPK knockdown observed in the mouse C2C12 myoblast cell line (Fig. 17), with siRNA IDs #2628 and 2636 showing higher mRNA knockdown levels than siRNA IDs #535 and 1028. In addition to DMPK mRNA knockdown in skeletal muscle, strong activity (greater than 50% mRNA knockdown) was also observed in mouse cardiomyocytes (heart). Finally, poorer activity (less than 50% mRNA knockdown) was observed in mouse liver. These results demonstrate the potential to achieve stable DMPK mRNA knockdown in multiple mouse muscle groups (including skeletal and cardiac muscle) while minimizing knockdown in off-target tissues such as the liver.

Example 20: siRNA Synthesis

All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. Base, sugar, and phosphate modifications well-described in the RNAi field were used to optimize the efficacy of the duplexes and reduce immunogenicity. All siRNA guest strands contained a C6- _NH2 stalk at the 5' end (see Figures 20A-21B). For 21-unit duplexes with 19 complementary bases and a 3' dinucleotide overhang, the stalk was attached to the siRNA guest strand via a reverse debased phosphodiester (Figures 20A-20B). For blunt-ended 21-unit duplexes with 19 complementary bases and a 3' dinucleotide overhang, the stalk was attached to the siRNA guest strand via a phosphodiester at the terminal base (Figures 21A-21B).

The purified single strands were double-stranded to obtain double-stranded siRNA.

Example 21: 2017-PK-401-C57BL6: In vivo transferrin mAb conjugate delivery of various Atrogin-1 siRNAs

For groups 1-4, referring to the study design in Figure 22, a 21-mer Atrogin-1 guide strand was designed. The sequence (5' to 3') of the guide/antisense strand is UCUACUGUAGUUGAAUCUUCUU (SEQ ID NO: 872). The guide strand and the fully complementary RNA transit strand were assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize the efficacy of the duplex and reduce immunogenicity. The purified single strand was duplexed to obtain the double-stranded siRNA described in Figure 20B. The transit strand contains two constrictors, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both constrictors are attached to the siRNA transit strand via a phosphodiester-reverse debasement-phosphodiester linker. Since free thiol groups were not used for constriction, fffu capping with N-ethylmaleimide was performed.

Antibody siRNA conjugates synthesized using bismaleimide (BisMal) linkers

Step 1: Reduce antibody with TCEP

The antibody was buffer-exchanged with 25 mM borate buffer (pH 8) containing 1 mM DTPA to achieve a concentration of 10 mg/mL. Four equivalents of TCEP in the same borate buffer were added to this solution, and the mixture was incubated at 37°C for 2 hours. At room temperature, the resulting reaction mixture was combined with a solution of BisMal-siRNA (1.25 equivalents) in 10 mM acetate buffer (pH 6.0) and incubated overnight at 4°C. Analysis of the reaction mixture by analytical SAX column chromatography revealed the antibody-siRNA conjugate as well as unreacted antibody and siRNA. The reaction mixture was treated with 10 EQ of N-ethylmaleimide (10 mg/mL in DMSO) to cap any remaining free cysteine residues.

Step 2: Purification

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

Anion exchange chromatography method (SAX)-1.

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

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

gradient:

Anion Exchange Chromatography (SAX) Method - 2

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

Solvent A: 80% 10mM TRIS, pH 8, 20% ethanol; Solvent B: 80% 10mM TRIS, pH 8, 20% ethanol, 1.5M NaCl; Flow rate: 0.75 ml/min

gradient:

Step 3: Analysis of the purified conjugate

The purity of the conjugates was assessed by analytical HPLC using anion exchange chromatography method-2. For the conjugate mTfR1-mAb-Atrogin-1 (DAR1), the SAX retention time was 9.1 min and the purity (% purity, based on peak area) was 99%.

In vivo study design

In in vivo experiments (C57BL6 mice), the ability of the conjugate to mediate the downregulation of Atrogin-1 mRNA in skeletal muscle was evaluated. Mice were administered the PBS-mediated control and the ASC and dosage shown in Figure 22 via intravenous (iv) injection. Gastrocnemius and cardiac tissues were harvested after the time points shown and thawed in liquid nitrogen. The mRNA knockdown in the target tissues was determined using comparative qPCR assays as described in the Methods section. Total RNA was extracted from the tissues, reverse transcribed, and mRNA levels were quantified using TaqMan qPCR with appropriately designed primers and probes. The results were calculated using the comparative Ct method, where the difference (ΔCt) between the target gene Ct value and the PPIB Ct value was calculated and then further normalized relative to the PBS control by taking a second difference (ΔΔCt).

result

When conjugated to an anti-TfR mAb targeting the transferrin receptor, the Atrogin-1 siRNA guide strand mediates the downregulation of target genes in the gastrocnemius and cardiac muscles, see Figures 23 and 24.

in conclusion

In this embodiment, it was demonstrated that the TfR1-siAtrogin-1 conjugate mediates specific downregulation of target genes in the gastrocnemius and cardiac muscle upon in vivo delivery. The ASC was prepared from an anti-transferrin antibody; mouse gastrocnemius and cardiac muscle express the transferrin receptor, and the conjugate possesses a mouse-specific anti-transferrin antibody targeting the siRNA, leading to its accumulation in the gastrocnemius and cardiac muscle. Receptor-mediated uptake results in siRNA-mediated knockdown of the target mRNA.

Example 22: 2017-PK-413-C57BL6: In vivo delivery of transferrin mAb conjugates for various MuRF1 sequences

For groups 1-2, referring to the study design in Figure 25, a 21-mer MuRF1(2089) guide strand was designed. The sequence (5' to 3') of the guide/antisense strand is UUUCGCACCAACGUAGAAAUU (SEQ ID NO: 873). The guide strand and the fully complementary RNA transit strand were assembled on a solid phase using standard phosphoramide chemistry and purified by HPLC. Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize the efficacy of the duplex and reduce immunogenicity. The purified single strand was duplexed to obtain the double-stranded siRNA described in Figure 20B. The transit strand contains two conjugates, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both conjugates are attached to the siRNA transit strand via a phosphodiester-reverse debase-phosphodiester linker. Since free thiol groups were not used for conjugation, fffu capping with N-ethylmaleimide was performed.

For groups 3–6, referring to the study design in Figure G, a 21-mer MuRF1(2265) guide strand was designed. The sequence (5'–3') of the guide/antisense strand is UCGUGAGACAGUAGAUGUUUU (SEQ ID NO: 874). The guide strand and fully complementary RNA transit strands were assembled on a solid phase using standard phosphoramide chemistry and purified by HPLC. Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize the efficacy of the duplex and reduce immunogenicity. The purified single strands were duplexed to obtain the double-stranded siRNA described in Figure 20B. The transit strand contains a conjugate stalk, namely the C6- _NH2 at the 5' end, which is linked to the siRNA transit strand via a phosphodiester-reverse debasement-phosphodiester linker.

For groups 7–10, referring to the study design in Figure G, a 21-mer MuRF1(2266) guide strand was designed. The sequence (5'–3') of the guide/antisense strand is UCACACGUGAGACAGUAGAUU (SEQ ID NO: 875). The guide strand and fully complementary RNA transit strands were assembled on a solid phase using standard phosphoramide chemistry and purified by HPLC. Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize the efficacy of the duplex and reduce immunogenicity. The purified single strands were duplexed to obtain the double-stranded siRNA described in Figure 20B. The transit strand contains a conjugate stalk, namely the C6- _NH2 at the 5' end, which is linked to the siRNA transit strand via a phosphodiester-reverse debasement-phosphodiester linker.

For groups 11–14, referring to the study design in Figure G, a 21-mer MuRF1(2267) guide strand was designed. The sequence (5'–3') of the guide/antisense strand is UUCACACGUGAGACAGUAGUU (SEQ ID NO: 876). The guide strand and fully complementary RNA transit strands were assembled on a solid phase using standard phosphoramide chemistry and purified by HPLC. Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize the efficacy of the duplex and reduce immunogenicity. The purified single strands were duplexed to obtain the double-stranded siRNA described in Figure 20B. The transit strand contains a conjugate stalk, namely the C6- _NH2 at the 5' end, which is linked to the siRNA transit strand via a phosphodiester-reverse debasement-phosphodiester linker.

For groups 15–18, referring to the study design in Figure G, a 21-mer MuRF1(2268) guide strand was designed. The sequence (5'–3') of the guide/antisense strand is UAAUAUUUCAUUUCGCACCUU (SEQ ID NO: 877). The guide strand and fully complementary RNA transit strands were assembled on a solid phase using standard phosphoramide chemistry and purified by HPLC. Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize the efficacy of the duplex and reduce immunogenicity. The purified single strands were duplexed to obtain the double-stranded siRNA described in Figure 20B. The transit strand contains a conjugate stalk, namely the C6- _NH2 at the 5' end, which is linked to the siRNA transit strand via a phosphodiester-reverse debasement-phosphodiester linker.

For groups 19–22, referring to the study design in Figure G, a 21-mer MuRF1(2269) guide strand was designed. The sequence (5'–3') of the guide/antisense strand is UAGCACCAAAUUGGCAUAUU (SEQ ID NO: 878). The guide strand and the fully complementary RNA transit strand were assembled on a solid phase using standard phosphoramide chemistry and purified by HPLC. Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize the efficacy of the duplex and reduce immunogenicity. The purified single strands were duplexed to obtain the double-stranded siRNA described in Figure 20B. The transit strand contains a conjugate stalk, namely the C6- _NH2 at the 5' end, which is linked to the siRNA transit strand via a phosphodiester-reverse debasement-phosphodiester linker.

Antibody siRNA conjugates synthesized using bismaleimide (BisMal) linkers

Step 1: Reduce antibody with TCEP

The antibody was buffer-exchanged with 25 mM borate buffer (pH 8) containing 1 mM DTPA to achieve a concentration of 10 mg/mL. Four equivalents of TCEP in the same borate buffer were added to this solution, and the mixture was incubated at 37°C for 2 hours. At room temperature, the resulting reaction mixture was combined with a solution of BisMal-siRNA (1.25 equivalents) in 10 mM acetate buffer (pH 6.0) and incubated overnight at 4°C. Analysis of the reaction mixture by analytical SAX column chromatography revealed the antibody-siRNA conjugate as well as unreacted antibody and siRNA. The reaction mixture was treated with 10 EQ of N-ethylmaleimide (10 mg/mL in DMSO) to cap any remaining free cysteine residues.

Step 2: Purification

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

Anion exchange chromatography method (SAX)-1.

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

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

gradient:

Anion Exchange Chromatography (SAX) Method - 2

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

Solvent A: 80% 10mM TRIS, pH 8, 20% ethanol; Solvent B: 80% 10mM TRIS, pH 8, 20% ethanol, 1.5M NaCl; Flow rate: 0.75 ml/min

gradient:

Step 3: Analysis of the purified conjugate

The purity of the conjugates was assessed by analytical HPLC using anion exchange chromatography method-2 (Table 19).

Table 19.

In vivo study design

In in vivo experiments (C57BL6 mice), the ability of the conjugate to mediate the downregulation of MuRF1 mRNA in muscle (gastrocnemius and cardiac muscle) was evaluated. Mice were administered the PBS-mediated control and the ASC and dosage shown in Figure 25 via intravenous (iv) injection. After 96 hours, gastrocnemius and cardiac muscle tissues were harvested and thawed in liquid nitrogen. The mRNA knockdown in the target tissues was determined using comparative qPCR assays as described in the Methods section. Total RNA was extracted from the tissues, reverse transcribed, and mRNA levels were quantified using TaqMan qPCR with appropriately designed primers and probes. The results were calculated using the comparative Ct method, where the difference (ΔCt) between the target gene Ct value and the PPIB Ct value was calculated and then further normalized relative to the PBS control by taking a second difference (ΔΔCt).

result

When conjugated to an anti-TfR1 mAb targeting the transferrin receptor, the MuRF1 siRNA guide strand mediates the downregulation of target genes in the gastrocnemius and cardiac muscles, see Figures 26 and 27.

in conclusion

In this embodiment, it was demonstrated that the TfR1-MuRF1 conjugate mediates specific downregulation of target genes in the gastrocnemius and cardiac muscle after in vivo delivery. The ASC was prepared from an anti-transferrin 1 antibody; mouse gastrocnemius and cardiac muscle express transferrin receptor 1, and the conjugate possesses a mouse-specific anti-transferrin antibody targeting the siRNA, leading to the accumulation of the conjugate in the gastrocnemius muscle. Receptor-mediated uptake results in siRNA-mediated knockdown of the target mRNA.

Example 23: 2017-PK-412-C57BL6: Prevention of dexamethasone-induced muscle atrophy with an Atrogin-1 and MuRF1 TfR1-mAb conjugate

For this experiment, three different siRNAs were used:

(1): A 21-mer Atrogin-1 guide strand was designed. The sequence (5' to 3') of the guide/antisense strand is UCUAGUAGUUGAAUCUUCUU (SEQ ID NO: 872). The guide strand and the fully complementary RNA transit strand were assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. Base, sugar, and phosphate modifications well described in the field of RNAi were used to optimize the efficacy of the duplex and reduce immunogenicity. The purified single strand was duplexed to obtain the double-stranded siRNA shown in Figure 20B. The transit strand contains two conjugates, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both conjugates are attached to the siRNA transit strand via a phosphodiester-reverse debase-phosphodiester linker. Since free thiol groups were not used for conjugation, fffu capping with N-ethylmaleimide was performed.

(2): A 21-mer MuRF1 guide strand was designed. The sequence (5' to 3') of the guide/antisense strand is UUUCGCACCAACGUAGAAAUU (SEQ ID NO: 873). The guide strand and the fully complementary RNA transit strand were assembled on a solid phase using standard phosphoramide chemistry and purified by HPLC. Base, sugar, and phosphate modifications well described in the field of RNAi were used to optimize the efficacy of the duplex and reduce immunogenicity. The purified single strand was duplexed to obtain the double-stranded siRNA shown in Figure 20B. The transit strand contains two conjugates, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both conjugates are attached to the siRNA transit strand via a phosphodiester-reverse debase-phosphodiester linker. Since free thiol groups were not used for conjugation, fffu capping with N-ethylmaleimide was performed.

(3): Negative control siRNA sequence (random): The published (Burke et al. (2014) Pharm. Res., 31(12):3445-60) 21-mer duplex with 19 complementary bases and a 3' dinucleotide overhang was used. The sequence of the guide/antisense strand (5' to 3') was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO:870). The negative control siRNA was modified with the same bases, sugars, and phosphates used for the active MSTN siRNA duplex. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were duplexed to obtain double-stranded siRNA. The guest strand contained two concentric stalks, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both concentric stalks were attached to the siRNA guest strand via a phosphodiester-reverse debase-phosphodiester linker. Since the free thiol group was not used for conjugation, fffu capping was performed using N-ethylmaleimide.

Antibody siRNA conjugates synthesized using bismaleimide (BisMal) linkers

Step 1: Reduce antibody with TCEP

The antibody was buffer-exchanged with 25 mM borate buffer (pH 8) containing 1 mM DTPA to achieve a concentration of 10 mg/mL. Four equivalents of TCEP in the same borate buffer were added to this solution, and the mixture was incubated at 37°C for 2 hours. At room temperature, the resulting reaction mixture was combined with a solution of BisMal-siRNA (1.25 equivalents) in 10 mM acetate buffer (pH 6.0) and incubated overnight at 4°C. Analysis of the reaction mixture by analytical SAX column chromatography revealed the antibody-siRNA conjugate as well as unreacted antibody and siRNA. The reaction mixture was treated with 10 EQ of N-ethylmaleimide (10 mg/mL in DMSO) to cap any remaining free cysteine residues.

Step 2: Purification

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

Anion exchange chromatography method (SAX)-1.

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

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

gradient:

Anion Exchange Chromatography (SAX) Method - 2

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

Solvent A: 80% 10mM TRIS, pH 8, 20% ethanol; Solvent B: 80% 10mM TRIS, pH 8, 20% ethanol, 1.5M NaCl; Flow rate: 0.75 ml/min

gradient:

Step 3: Analysis of the purified conjugate

The purity of the conjugates was assessed by analytical HPLC using anion exchange chromatography method-2 (Table 20).

Table 20.

Conjugate SAX retention time (min) Purity % (based on peak area) mTfR1-Atrogin-1(DAR1) 9.3 97 mTfR1-MuRF1(DAR1) 9.5 98 mTfR1-SC(DAR1) 9.0 99

In vivo study design

In in vivo experiments (C57BL6 mice), the ability of the conjugate to mediate the downregulation of MuRF1 and Atrogin-1 mRNA in muscle (gastrocnemius) was evaluated with and without muscle atrophy. Mice were administered the PBS mediator control and the ASCs and doses shown in Table 21 via intravenous (iv) injection. Muscle atrophy was induced daily by intraperitoneal injection (10 mg/kg) of dexamethasone for 21 days after conjugate delivery for groups 2–4, 9–11, and 16–18. For the control group (5–7, 12–14, and 19–21, without induced muscle atrophy), PBS was administered daily via intraperitoneal injection. Groups 1, 8, 15, and 22 were harvested on day 7 prior to inducing muscle atrophy to establish baseline measurements of mRNA expression and muscle weight. Gastrocnemius and cardiac muscle tissue were harvested, weighed, and thawed in liquid nitrogen at the time points shown. Comparative qPCR assays as described in the Methods section were used to determine mRNA knockdown in the target tissues. Total RNA was extracted from tissues, reverse transcribed, and quantified using TaqMan qPCR with appropriately designed primers and probes. PPIB (householding gene) was used as an internal RNA loading control. Results were compared using the Ct method, where the difference (ΔCt) between the target gene Ct value and the PPIB Ct value was calculated, and then the difference was further normalized relative to the PBS control group by taking a second difference (ΔΔCt).

The quantification of tissue siRNA was determined using stem-loop qPCR assays as described in the Methods section. The antisense strand of the siRNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit with sequence-specific stem-loop RT primers. The cDNA from the RT step was then used for real-time PCR, and the Ct values were converted to plasma or tissue concentrations using a linear equation derived from a standard curve.

Table 21.

result

Data are summarized in Figures 28–31. Co-delivery of Atrogin-1 and MuRF1 siRNA effectively downregulated Atrogin-1 and MuRF1 mRNA expression in both normal and atrophied muscle when delivered using the TfR1 mAh conjugate. Atrophy induction transiently induced Atrogin-1 and MuRF1 expression by approximately 4-fold. In both normal and atrophied gastrocnemius muscle, a single dose of mTfR1-Atrogin-1+TfR1.mAb-siMuRF1 (3 mg/kg, individually and in combination) reduced Atrogin-1 and MuRF1 mRNA levels by >70%. Downregulation of MuRF1 and Atrogin-1 mRNA increased gastrocnemius muscle mass by 5–10% and reduced DEX-induced gastrocnemius muscle mass loss by 50%. Downregulation of Atrogin-1 alone had no significant effect on gastrocnemius muscle mass changes. Treatment with Atrogin-1/MuRF1 siRNA induced muscle hypertrophy in the absence of muscle atrophy.

in conclusion

In this embodiment, it was demonstrated that co-delivery of Atrogin-1 and MuRF1 siRNAs, when delivered using the TfR1 mAh conjugate, effectively downregulated Atrogin-1 and MuRF1 mRNA expression in both normal and atrophied gastrocnemius muscle. The conjugate had little effect on the cardiac muscle, where downregulation of Atrogin-1 could be detrimental. Downregulation of MuRF1 and Atrogin-1 mRNA increased gastrocnemius muscle weight by 5-10% and reduced DEX-induced gastrocnemius muscle weight loss by 50%. Downregulation of Atrogin-1 alone had no significant effect on gastrocnemius muscle weight changes. ASC was prepared from an anti-transferrin antibody, and mouse gastrocnemius muscle expresses the transferrin receptor. The conjugate contained a mouse-specific anti-transferrin antibody targeting the siRNA, resulting in the accumulation of the conjugate in the gastrocnemius muscle. Receptor-mediated uptake led to siRNA-mediated knockdown of the target mRNA.

Example 24: 2017-PK-435-C57BL6: In vivo dose-response experiment for the delivery of transferrin mAb conjugates for Atrogin-1

For groups 1-12, referring to the study design in Figure 32, a 21-mer Atrogin-1 guide strand was designed. The sequence (5' to 3') of the guide/antisense strand is UCGUAGUUAAAUCUUCUGGUU (SEQ ID NO: 879). The guide strand and the fully complementary RNA transit strand were assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize the efficacy of the duplex and reduce immunogenicity. The purified single strand was duplexed to obtain the double-stranded siRNA shown in Figure A. The transit strand contains two conjugates, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both conjugates are attached to the siRNA transit strand via a phosphodiester-reverse debase-phosphodiester linker. Since free thiol groups were not used for conjugation, fffu capping with N-ethylmaleimide was performed.

For groups 13–18, referring to the study design in Figure 32, a 2l-mer negative control siRNA sequence (randomized) with 19 complementary bases and a 3' dinucleotide overhang was used (published by Burke et al. (2014) Pharm. Res., 31(12):3445–60). The sequence of the guide/antisense strand (5'–3') was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 870). The negative control siRNA was modified with the same bases, sugars, and phosphates used for the active MSTN siRNA duplex. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were duplexed to obtain double-stranded siRNA. The guest strand contained two concentric stalks, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both concentric stalks were attached to the siRNA guest strand via a phosphodiester-reverse debase-phosphodiester linker. Since the free thiol group was not used for conjugation, fffu capping was performed using N-ethylmaleimide.

Antibody siRNA conjugates synthesized using bismaleimide (BisMal) linkers

Step 1: Reduce antibody with TCEP

The antibody was buffer-exchanged with 25 mM borate buffer (pH 8) containing 1 mM DTPA to achieve a concentration of 10 mg/mL. Four equivalents of TCEP in the same borate buffer were added to this solution, and the mixture was incubated at 37°C for 2 hours. At room temperature, the resulting reaction mixture was combined with a solution of BisMal-siRNA (1.25 equivalents) in 10 mM acetate buffer (pH 6.0) and incubated overnight at 4°C. Analysis of the reaction mixture by analytical SAX column chromatography revealed the antibody-siRNA conjugate as well as unreacted antibody and siRNA. The reaction mixture was treated with 10 EQ of N-ethylmaleimide (10 mg/mL in DMSO) to cap any remaining free cysteine residues.

Step 2: Purification

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

Anion exchange chromatography method (SAX)-1.

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

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

gradient:

Anion Exchange Chromatography (SAX) Method - 2

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

Solvent A: 80% 10mM TRIS, pH 8, 20% ethanol; Solvent B: 80% 10mM TRIS, pH 8, 20% ethanol, 1.5M NaCl; Flow rate: 0.75 ml/min

gradient:

Step 3: Analysis of the purified conjugate

The purity of the conjugates was assessed by analytical HPLC using anion exchange chromatography method-2 (Table 22).

Table 22.

Conjugate SAX retention time (min) Purity % (based on peak area) TfR1-Atrogin-1DAR1 9.2 99 TfR1-Scramble DAR1 8.9 93

In vivo study design

In in vivo experiments (C57BL6 mice), the ability of the conjugate to mediate the downregulation of Atrogin-1 mRNA in muscle (gastrocnemius) was evaluated with and without muscle atrophy. Mice were administered the PBS-mediated control and the ASCs and doses shown in Figure 32 via intravenous (iv) injection. Muscle atrophy was induced daily by intraperitoneal injection (10 mg/kg) of dexamethasone for 3 days for groups 3, 6, 9, 12, and 15 7 days after conjugate delivery. For control groups 2, 5, 8, 11, and 14 (without induced muscle atrophy), PBS was administered daily via intraperitoneal injection. Groups 1, 4, 7, 10, and 13 were harvested on day 7 prior to induction of muscle atrophy to establish baseline measurements of mRNA expression and muscle weight. Gastrocnemius muscles were harvested, weighed, and thawed in liquid nitrogen 3 days after atrophy induction (or 10 days after conjugate delivery). Comparative qPCR assays as described in the Methods section were used to determine mRNA knockdown in the target tissue. Total RNA was extracted from tissues, reverse transcribed, and quantified using TaqMan qPCR with appropriately designed primers and probes. PPIB (householding gene) was used as an internal RNA loading control. Results were compared using the Ct method, where the difference (ΔCt) between the target gene Ct value and the PPIB Ct value was calculated, and then the difference was further normalized relative to the PBS control group by taking a second difference (ΔΔCt).

The quantification of tissue siRNA was determined using stem-loop qPCR assays as described in the Methods section. The antisense strand of the siRNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit with sequence-specific stem-loop RT primers. The cDNA from the RT step was then used for real-time PCR, and the Ct values were converted to plasma or tissue concentrations using a linear equation derived from a standard curve.

result

Data are summarized in Figures 33 through 35. When conjugated to an anti-TfR mAb targeting the transferrin receptor, the Atrogin-1 siRNA guide chain mediates the downregulation of target genes in the gastrocnemius muscle (see Figure 33). Increasing the dose from 3 mg/kg to 9 mg/kg reduced atrophy-induced Atrogin-1 mRNA levels by 2–3 fold. The maximum KD achievable with siRNA was 80%, requiring a tissue concentration of 40 nM to reach the maximum KD in atrophied muscle. This highlights that conjugate delivery methods can alter disease-induced Atrogin-1 mRNA expression levels by increasing the dose (see Figure 34). Figure 35 highlights that the mRNA downregulation is mediated by the RISC loading of the Atrogin-1 guide chain and is concentration-dependent.

in conclusion

In this embodiment, it was demonstrated that the TfR1-Atrogin-1 conjugate, upon in vivo delivery, mediates specific downregulation of target genes in the gastrocnemius muscle in a dose-dependent manner. Following atrophy induction, the conjugate was able to mediate disease-induced Atrogin-1 mRNA expression levels at higher doses. Higher RISC loading of the Atrogin-1 guide chain was associated with increased mRNA downregulation.

Example 25: 2017-PK-381-C57BL6: Downregulation of myostatin (MSTN) reduces muscle loss in dexamethasone-treated mice.

For groups 1-12, referring to the study design in Table 24, a 21-mer Atrogin-1 guide strand was designed. The sequence (5' to 3') of the guide/antisense strand was UCGUAGUUAAAUCUUCUGGUU (SEQ ID NO: 879). The guide strand and the fully complementary RNA transit strand were assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. Base, sugar, and phosphate modifications well described in the field of RNAi were used to optimize the efficacy of the duplex and reduce immunogenicity. The purified single strand was duplexed to obtain the double-stranded siRNA as shown in Figure 20B. The transit strand contained two constrictors, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both constrictors were attached to the siRNA transit strand via a phosphodiester-reverse debasement-phosphodiester linker. Since free thiol groups were not used for constriction, fffu capping with N-ethylmaleimide was performed.

For groups 13–18, referring to the study design in Table 24, a 2l-mer negative control siRNA sequence (randomized) with 19 complementary bases and a 3' dinucleotide overhang was used (published by Burke et al. (2014) Pharm. Res., 31(12):3445–60). The sequence of the guide/antisense strand (5' to 3') was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO: 870). The negative control siRNA was modified with the same bases, sugars, and phosphates used for the active MSTN siRNA duplex. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were duplexed to obtain double-stranded siRNA. The guest strand contained two concentric stalks, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both concentric stalks were attached to the siRNA guest strand via a phosphodiester-reverse debase-phosphodiester linker. Since the free thiol group was not used for conjugation, fffu capping was performed using N-ethylmaleimide.

Antibody siRNA conjugates synthesized using bismaleimide (BisMal) linkers

Step 1: Reduce antibody with TCEP

The antibody was buffer-exchanged with 25 mM borate buffer (pH 8) containing 1 mM DTPA to achieve a concentration of 10 mg/mL. Four equivalents of TCEP in the same borate buffer were added to this solution, and the mixture was incubated at 37°C for 2 hours. At room temperature, the resulting reaction mixture was combined with a solution of BisMal-siRNA (1.25 equivalents) in 10 mM acetate buffer (pH 6.0) and incubated overnight at 4°C. Analysis of the reaction mixture by analytical SAX column chromatography revealed the antibody-siRNA conjugate as well as unreacted antibody and siRNA. The reaction mixture was treated with 10 EQ of N-ethylmaleimide (10 mg/mL in DMSO) to cap any remaining free cysteine residues.

Step 2: Purification

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

Anion exchange chromatography method (SAX)-1.

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

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

gradient:

Anion Exchange Chromatography (SAX) Method - 2

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

Solvent A: 80% 10mM TRIS, pH 8, 20% ethanol; Solvent B: 80% 10mM TRIS, pH 8, 20% ethanol, 1.5M NaCl; Flow rate: 0.75 ml/min

gradient:

Step 3: Analysis of the purified conjugate

The purity of the conjugates was assessed by analytical HPLC using anion exchange chromatography method-2 (Table 23).

Table 23.

In vivo study design

In in vivo experiments (C57BL6 mice), the ability of the conjugate to mediate the downregulation of MSTN mRNA in muscle (gastrocnemius) was evaluated in the presence and absence of muscle atrophy. Mice were administered the PBS mediator control and the ASCs and dosages shown in Table 24 via intravenous (iv) injection. Muscle atrophy was induced by daily administration of dexamethasone (10 mg/kg) for 13 days after conjugate delivery for groups 2, 3, 4, 9, 10, and 11. For the control group (5, 6, 7, 12, 13, and 14, without induced muscle atrophy), PBS was administered via daily intraperitoneal injection. Groups 1 and 8 were harvested on day 7 prior to induction of muscle atrophy to establish baseline measurements of mRNA expression and muscle weight. Gastrocnemius muscles were harvested, weighed, and thawed in liquid nitrogen on days 3, 7, and 14 after atrophy induction (or days 10, 14, and 21 after conjugate delivery). mRNA knockdown in target tissues was determined using comparative qPCR assays as described in the Methods section. Total RNA was extracted from the tissues, reverse transcribed, and mRNA levels were quantified using TaqMan qPCR with appropriately designed primers and probes. PPIB (householding gene) was used as an internal RNA loading control. The results were calculated using the Ct method, where the difference (ΔCt) between the target gene Ct value and the PPIB Ct value was calculated, and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt).

The quantification of tissue siRNA was determined using stem-loop qPCR assays as described in the Methods section. The antisense strand of the siRNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit with sequence-specific stem-loop RT primers. The cDNA from the RT step was then used for real-time PCR, and the Ct values were converted to plasma or tissue concentrations using a linear equation derived from a standard curve.

Table 24

result

Data are summarized in Figures 36 and 37. In the presence and absence of dexamethasone-induced atrophy, the MSTN siRNA guideline, when conjugated to an anti-TfR mAb targeting the transferrin receptor, mediated the downregulation of target genes in the gastrocnemius muscle (see Figure 36). A single dose of 3 mg/kg siRNA downregulated MSTN mRNA levels by >75%. In the presence of dexamethasone-induced atrophy, MSTN downregulation increased muscle mass and mitigated Dex-induced muscle loss (see Figure 37).

in conclusion

In this embodiment, it was demonstrated that the TfR1-MSTN conjugate mediates specific downregulation of target genes in the gastrocnemius muscle after in vivo delivery. Following atrophy induction, the conjugate was able to increase muscle mass and alleviate Dex-induced muscle loss.

Example 26: 2017-PK-496-C57BL6: Downregulation of Atrogin-1 and MuRF1 reduces leg muscle loss during sciatic nerve denervation in mice.

For groups 1-4, referring to the study design in Figure 38, a 21-mer Atrogin-1 guide strand was designed. The sequence (5' to 3') of the guide/antisense strand is UUGGGUAACAUCGUACAAGUU (SEQ ID NO: 880). The guide strand and the fully complementary RNA transit strand were assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize the efficacy of the duplex and reduce immunogenicity. The purified single strand was duplexed to obtain the double-stranded siRNA described in Figure 20B. The transit strand contains two constrictors, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both constrictors are attached to the siRNA transit strand via a phosphodiester-reverse debasement-phosphodiester linker. Since free thiol groups were not used for constriction, fffu capping with N-ethylmaleimide was performed.

For groups 5-6, referring to the study design in Figure V, a 21-mer MuRF1 guide strand was designed. The sequence (5' to 3') of the guide/antisense strand is UUUCGCACCAACGUAGAAAUU (SEQ ID NO: 873). The guide strand and the fully complementary RNA transit strand were assembled on a solid phase using standard phosphoramide chemistry and purified by HPLC. Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize the efficacy of the duplex and reduce immunogenicity. The purified single strand was duplexed to obtain the double-stranded siRNA described in Figure 20B. The transit strand contains two conjugates, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both conjugates are attached to the siRNA transit strand via a phosphodiester-reverse debase-phosphodiester linker. Since free thiol groups were not used for conjugation, fffu capping with N-ethylmaleimide was performed.

For groups 7-12, Atrogin-1 and MuRF1 were designed as described above. After conjugation with TfR1 mAh, and after purification and isolation of individual DAR1 substances, they were mixed and co-administered.

For group 13, referring to the study design in Figure 38, a 2l-mer negative control siRNA sequence (randomized) with 19 complementary bases and a 3' dinucleotide overhang was used (published by Burke et al. (2014) Pharm. Res., 31(12):3445-60). The sequence of the guide/antisense strand (5' to 3') was UAUCGACGUGUCCAGCUAGUU (SEQ ID NO:870). The negative control siRNA was modified with the same bases, sugars, and phosphates used for the active MSTN siRNA duplex. All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were duplexed to obtain double-stranded siRNA. The guest strand contained two concentric stalks, namely C6- _NH2 at the 5' end and C6-SH at the 3' end. Both concentric stalks were attached to the siRNA guest strand via a phosphodiester-reverse debase-phosphodiester linker. Since the free thiol group was not used for conjugation, fffu capping was performed using N-ethylmaleimide.

Antibody siRNA conjugates synthesized using bismaleimide (BisMal) linkers

Step 1: Reduce antibody with TCEP

The antibody was buffer-exchanged with 25 mM borate buffer (pH 8) containing 1 mM DTPA to achieve a concentration of 10 mg/mL. Four equivalents of TCEP in the same borate buffer were added to this solution, and the mixture was incubated at 37°C for 2 hours. At room temperature, the resulting reaction mixture was combined with a solution of BisMal-siRNA (1.25 equivalents) in 10 mM acetate buffer (pH 6.0) and incubated overnight at 4°C. Analysis of the reaction mixture by analytical SAX column chromatography revealed the antibody-siRNA conjugate as well as unreacted antibody and siRNA. The reaction mixture was treated with 10 EQ of N-ethylmaleimide (10 mg/mL in DMSO) to cap any remaining free cysteine residues.

Step 2: Purification

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

Anion exchange chromatography method (SAX)-1.

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

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

gradient:

Anion Exchange Chromatography (SAX) Method - 2

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

Solvent A: 80% 10mM TRIS, pH 8, 20% ethanol; Solvent B: 80% 10mM TRIS, pH 8, 20% ethanol, 1.5M NaCl; Flow rate: 0.75 ml/min

gradient:

Step 3: Analysis of the purified conjugate

The purity of the conjugates was assessed by analytical HPLC using anion exchange chromatography method-2 (Table 25).

Table 25

Conjugate SAX retention time (min) Purity % (based on peak area) TfR1-Atrogin-1DAR1 9.2 95 TfR1-MuRF1 DAR1 9.3 92 mTfR1-SC(DAR1) 8.9 76

In vivo study design

In in vivo experiments (C57BL6 mice), the ability of the conjugate to mediate the downregulation of MuRF1 and Atrogin-1 mRNA in the gastrocnemius muscle was evaluated with and without sciatic nerve denervation. Mice were administered the PBS mediator control and the ASCs and dosages shown in Figure 38 via intravenous (iv) injection. Seven days after conjugate delivery, sciatic nerve denervation induced leg muscle atrophy in groups 2–4, 6–8, 10–12, and 14–16. No denervation was induced in control groups 1, 5, 9, 13, and 17.

For the denervation procedure on day 7, mice were anesthetized (with 5% isoflurane) and administered a subcutaneous dose of 0.1 mg/kg buprenorphine. The right dorsopelvic region was shaved from the sciatic notch to the knee. This area was disinfected with alternating alcohol and povidone-iodine. The sciatic notch was identified by palpation and an incision of approximately 1 cm from the sciatic notch to the knee. The biceps femoris muscle was split to expose the sciatic nerve, and approximately 1 cm of the nerve was removed from both ends by cauterization. The muscle and skin were then sutured to close the incision. The operated limb was then examined daily to observe the condition of the surgical wound and the overall health of the animal.

For groups 4, 8, 12, and 16, changes in leg muscle area were determined: the legs to be tested were shaved, and the measurement areas were marked with indelible ink. Mice were restrained in a cone-shaped restraint device, with the right leg held by hand. Measurements were taken once in the sagittal plane and again in the coronal plane using digital calipers. This procedure was repeated twice weekly. For all groups, at the indicated time points, gastrocnemius and cardiac muscle tissues were harvested, weighed, and thawed in liquid nitrogen. Comparative qPCR assays as described in the Methods section were used to determine mRNA knockdown in the target tissues. Total RNA was extracted from the tissues, reverse transcribed, and mRNA levels were quantified using TaqMan qPCR with appropriately designed primers and probes. The results were calculated using the comparative Ct method, where the difference (ΔCt) between the target gene Ct value and the PPIB Ct value was calculated, and then further normalized relative to the PBS control group by taking a second difference (ΔΔCt).

The quantification of tissue siRNA was determined using stem-loop qPCR assays as described in the Methods section. The antisense strand of the siRNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit with sequence-specific stem-loop RT primers. The cDNA from the RT step was then used for real-time PCR, and the Ct values were converted to plasma or tissue concentrations using a linear equation derived from a standard curve.

Figure 39A shows that a single treatment with 4.5 mg/kg (siRNA) of Atrogin-1 siRNA or MuRF1 siRNA, or a single dose of a combination of the two siRNAs, resulted in downregulation of up to 75% of each target in the gastrocnemius muscle.

Figure 39B shows that, up to 37 days after ASC administration, the mRNA knockdown of the two targets in the gastrocnemius muscle remained at 75% throughout the leg.

In the denervated leg, Atroginl mRNA knockdown was maintained for 3 days after denervation, but decreased to 20% by 10 days and to 0% by 30 days. MuRF1 mRNA knockdown was enhanced to 80-85% in the denervated leg 3 days after denervation, but decreased to 50% by 10 days and to 40% by 30 days (Figure 39C).

When a combination of two siRNAs is used for treatment, the knockdown of mRNA for each target is not affected by the knockdown of the other target (Figure 39D).

Based on measurements of leg muscle area, siRNA-mediated downregulation of MuRF1 and the combination of MuRF1 and Atrogin-1 reduced denervation-induced muscle atrophy by up to 30%. Treatment with MuRF1 siRNA alone showed a similar response compared to treatment with the combination of MuRF1 and Atrogin-1. Downregulation of Atrogin-1 alone had no significant effect on leg muscle area. Statistical analysis compared the treatment group with the disordered siRNA control group using Welch’s t-test. See Figure 39E.

Based on gastrocnemius muscle weight, only MuRF1 showed a statistically significant difference compared to the disordered siRNA control group. Similar to results obtained by measuring leg muscle area, downregulation of MuRF1 showed a reduction of up to 35% in denervation-induced muscle atrophy. These results are consistent with the effects of MuRF1 knockout in mice (Bodine et al., Science 291, 2001). See Figure 39F.

Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from the scope of this disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practice. The scope of this disclosure is intended to be defined by the appended claims, thereby covering the methods and structures within the scope of those claims and their equivalents.

Claims (10)

1. A polynucleotide conjugate comprising an antibody or a binding fragment thereof conjugated to a polynucleotide molecule, said polynucleotide molecule hybridizing to a target sequence of an atrogene; wherein said polynucleotide molecule comprises at least one 2' modified nucleotide, at least one modified internucleotide linker, or at least one reverse debasement moiety; and wherein said polynucleotide conjugate mediates RNA interference against said atrogene, thereby treating a subject with myasthenia gravis or myotonic dystrophy. 2. The polynucleotide conjugate according to claim 1, wherein the atrogene includes differentially regulated genes within the IGF1-Akt-FoxO pathway, glucocorticoid-GR pathway, PGC1α-FoxO pathway, TNFα- pathway, or myostatin-ActRIIb-Smad2/3 pathway. 3. The polynucleotide conjugate according to claim 2, wherein the atrogene is a gene downregulated in the IGF1-Akt-FoxO pathway, glucocorticoid-GR pathway, PGC1α-FoxO pathway, TNFα- pathway, or myostatin-ActRIIb-Smad2/3 pathway. 4. The polynucleotide conjugate according to claim 2, wherein the atrogene is a gene upregulated in the IGF1-Akt-FoxO pathway, glucocorticoid-GR pathway, PGC1α-FoxO pathway, TNFα- pathway, or myostatin-ActRIIb-Smad2/3 pathway. 5. The polynucleotide conjugate according to claim 1, wherein the atrogene encodes an E3 ligase. 6. The polynucleotide conjugate according to claim 1, wherein the antibody or its binding fragment comprises a humanized antibody or its binding fragment, a chimeric antibody or its binding fragment, a monoclonal antibody or its binding fragment, a monovalent Fab’, a bivalent Fab2, a single-chain variable fragment (scFv), a biantibody, a microantibody, a nanobody, a single-domain antibody (sdAb), or a camelid antibody or its binding fragment. 7. The polynucleotide conjugate according to claim 1, wherein the polynucleotide conjugate comprises a molecule of formula (I): AX ₁ -BX ₂ -C Formula I in, A represents the antibody or its binding fragment; B is a polynucleotide molecule that hybridizes with the target sequence of atrogene; C is a polymer; and _X1 and _X2 are each independently selected from bonded or non-polymeric linkages; and A and C are not connected to the same end of B. 8. A pharmaceutical composition comprising: The polynucleic acid conjugates of claims 1-7; and Pharmaceutically acceptable excipients. 9. A method for treating muscle atrophy or myotonic dystrophy in a subject of need, comprising: The subject is given a therapeutically effective amount of the polynucleic acid conjugate of claims 1-7 or the pharmaceutical composition of claim 8 to treat the subject's muscle atrophy or myotonic dystrophy. 10. A kit comprising the polynucleic acid conjugate of claims 1-7 or the pharmaceutical composition of claim 8.