NUCLEIC ACID DELIVERY COMPOSITIONS AND METHODS OF USE THEREOF

This disclosure relates to nucleic acid constructs modified to have a reduced net anionic charge. The constructs comprise phosphotriester and/or phosphothioate protecting groups. The disclosure also provides methods of making and using such constructs. 4. The nucleotide compound of claim 2 , wherein the N-SATE moiety is conjugated to the phosphate group of any of the nucleic acid bases A claim 2 , G claim 2 , C claim 2 , T or U.71. An oligonucleotide or polynucleotide comprising a nucleotide having an N-SATE moiety of claim .8. The oligonucleotide or polynucleotide of claim 7 , wherein the oligonucleotide or polynucleotide comprises a neutral or a more cationic charge when compared to the same oligonucleotide or polynucleotide lacking an N-SATE moiety.9. An oligonucleotide or polynucleotide comprising an amino alkyl S-acyl thio alkyl (N-SATE) moiety that reduces the net anionic charge of the oligonucleotide or polynucleotide backbone.10. The oligonucleotide or polynucleotide of claim 9 , wherein the oligonucleotide or polynucleotide comprise an siRNA molecule.11. The oligonucleotide or polynucleotide of claim 9 , wherein the oligonucleotide comprises a plurality modified nucleotides having an N-SATE moiety.12. The oligonucleotide or polynucleotide of claim 11 , wherein the oligonucleotide or polynucleotide comprises a plurality of adjacent nucleotides having an N-SATE moiety.13. The oligonucleotide or polynucleotide of claim 11 , wherein the oligonucleotide or polynucleotide comprises a plurality of nucleotides having an N-SATE moiety separated from one another by 1 or more nucleotide bases.14. The oligonucleotide or polynucleotide of claim 9 , further comprising at least one protein transduction domain (PTD) comprising a membrane transport function conjugated or operably linked to the oligonucleotide or polynucleotide domain.15. The oligonucleotide of comprising a plurality of protein transduction domains.16. A pharmaceutical composition comprising the ...

IMMUNOMODULATING POLYNUCLEOTIDES, ANTIBODY CONJUGATES THEREOF, AND METHODS OF THEIR USE

Immunomodulating polynucleotides are disclosed. The immunomodulating polynucleotides may contain 5-modified uridine, 5-modified cytidine, a total of from 6 to 16 nucleotides, and/or one or more abasic spacers and/or internucleoside phosphotriesters. Also disclosed are conjugates containing a targeting moiety and one or more immunomodulating polynucleotides. The immunomodulating polynucleotides and conjugates may further contain one or more auxiliary moieties. Also disclosed are compositions containing the immunomodulating polynucleotides or the conjugates containing one or more stereochemically enriched internucleoside phosphorothioates. Further disclosed are pharmaceutical compositions containing the immunomodulating polynucleotides or the conjugates and methods of their use. 1446-. (canceled)449. The compound of claim 448 , wherein Z is S.450. The compound of claim 448 , wherein d is 1.451. The compound of claim 447 , wherein the PEG comprises a total of at least 20 ethylene glycol repeating units and a total of 30 or fewer ethylene glycol repeating units.452. The compound of claim 451 , wherein the PEG comprises a total of 25 ethylene glycol repeating units.453. The compound of claim 451 , wherein the PEG comprises a total of 24 ethylene glycol repeating units.454. The compound of claim 451 , wherein the PEG comprises a total of 23 ethylene glycol repeating units.455. The compound of claim 447 , wherein b is an integer ranging from 1 to 15 and the sum of b and c is an integer ranging from 5 to 15.456. The compound of claim 455 , wherein sum of b and c is 11.457. The compound of claim 455 , wherein sum of b and c is 12.458. The compound of claim 447 , wherein each Xis independently a 2′-deoxyribonucleotide selected from the group consisting of 2′-deoxyadenosine claim 447 , 2′-deoxyguanosine claim 447 , 2′-deoxycytidine claim 447 , a 5-halo-2′-deoxycytidine claim 447 , 2′-deoxythymidine claim 447 , 2′-deoxyuridine claim 447 , and a 5-halo-2′-deoxyuridine or a 2′- ...

POLYNUCLEOTIDE CONSTRUCTS HAVING AN AUXILIARY MOIETY NON-BIOREVERSIBLY LINKED TO AN INTERNUCLEOSIDE PHOSPHATE OR PHOSPHOROTHIOATE

The invention features a hybridized polynucleotide construct including a passenger strand, a guide strand loadable into a RISC complex, and one or more auxiliary moieties. At least one of the auxiliary moieties is non-bioreversibly linked to an internucleoside phosphate or phosphorothioate in the passenger strand. The invention further features methods of delivery a polynucleotide construct to a cell and methods of reducing the expression of a protein in a cell. The methods typically involve contacting the cell with the hybridized polynucleotide construct. 1. A hybridized polynucleotide construct comprising a passenger strand , a guide strand loadable into a RISC complex , and one or more auxiliary moieties;wherein at least one of said auxiliary moieties is non-bioreversibly linked to an internucleoside phosphate or phosphorothioate in said passenger strand; wherein said auxiliary moieties are independently selected from the group consisting of a targeting moiety, a cell penetrating peptide, an endosomal escape moiety, and a neutral organic polymer.2. The hybridized polynucleotide construct of claim 1 , comprising 5 or fewer auxiliary moieties claim 1 , at least one of said auxiliary moieties being linked non-bioreversibly to an internucleoside phosphate or phosphorothioate in said passenger strand claim 1 , and the remaining of said auxiliary moieties being independently linked bioreversibly to a phosphate or phosphorothioate in said guide strand or linked bioreversibly or non-bioreversibly to a phosphate or phosphorothioate in said passenger strand.3. The hybridized polynucleotide construct of claim 1 , wherein said passenger strand comprises a pattern —N-p-(—N-p-)-N-p-(—N-p-)-N-p-[(—N-p-)-N-p-]- claim 1 ,whereineach N is independently a nucleoside;{'sup': 'L', 'each pis a phosphate or phosphorothioate linked non-bioreversibly to an auxiliary moiety;'}each p is independently a phosphate, phosphorothioate, phosphoramidate, or phosphonate;each z is independently 0, ...

POLYNUCLEOTIDE CONSTRUCTS

Disclosed are polynucleotide constructs having a strand linked to a moiety carrying one or more auxiliary moieties. Also disclosed are polynucleotide constructs interrupted with a sugar analogue, and polynucleotide constructs with stereochemical{circumflex over ( )}enriched phosphorothioates. The polynucleotide constructs may be provided as hybridized polynucleotide constructs. Also featured are methods of delivery a polynucleotide construct to a cell and methods of reducing the expression of a protein in a cell by contacting the cell with the disclosed polynucleotide construct or hybridized polynucleotide construct. 3. The polynucleotide construct of or , or a salt thereof , or a stereoisomer thereof , wherein at least one -LinkA(-T)is of formula (II):{'br': None, 'sup': 1', '2', '3', '4', '5', '6, 'sub': s', 'p, '-Q-Q([-Q-Q-Q]-Q-T),\u2003\u2003 (II)'}whereineach s is independently an integer from 0 to 20, wherein the repeating units are same or different;{'sup': '1', 'Qis a conjugation linker;'}{'sup': '2', 'Qis a linear group, if p is 1, or a branched group, if p is an integer from 2 to 6;'}{'sup': 3', '6, 'sub': 2', '2', '2', '2', '2', '2, 'each Qand each Qis independently absent, —CO—, —NH—, —O—, —S—, —SO—, —OC(O)—, —COO—, —NHC(O)—, —C(O)NH—, —CH—, —CHNH—, —NHCH—, —CHO—, or —OCH—;'}{'sup': '4', 'sub': 1-12', '2-12', '2-12', '2-12', '6-10', '1-9', '1-9, 'each Qis independently absent, optionally substituted Calkylene, optionally substituted Calkenylene, optionally substituted Calkynylene, optionally substituted Cheteroalkylene, optionally substituted Carylene, optionally substituted Cheteroarylene, or optionally substituted Cheterocyclylene;'}{'sup': 5', 'a', 'a, 'sub': 2', '2, 'each Qis independently absent, —CO—, —NH—, —O—, —S—, —SO—, —CH—, —C(O)O—, —OC(O)—, —C(O)NH—, —NH—C(O)—, —NH—CH(R)—C(O)—, or —C(O)—CH(R)—NH—; and'}{'sup': 'a', 'each Ris independently H or an amino acid side chain;'}{'sup': 3', '4', '5, 'provided that at least one of Q, Q, and Qis present ...

POLYNUCLEOTIDES HAVING BIOREVERSIBLE GROUPS

The disclosure provides methods and compositions for delivering polynucleotides into cells. The disclosure provides transiently protected polynucleotides comprising an anionic charge-neutralizing moiety/group, which may also confer additional functionality. These compounds can enter the cytosol of cells by endocytic or macropinocytic mechanisms. The transient protecting group is bioreversible, i.e., once inside a cell, it is designed to be removed by enzymatic activity or by passive intracellular methods (e.g., changes in pH or reductive environment). 1. A polynucleotide construct comprising a component (i) selected from the group consisting of a peptide , a polypeptide , a carbohydrate , a neutral organic polymer , a positively charged polymer , a therapeutic agent , a targeting moiety , an endosomal escape moiety , and any combination thereof ,wherein component (i) is linked to the polynucleotide construct through a bioreversible group attached to an internucleotide bridging group.2. The polynucleotide construct of claim 1 , further comprising at least one second component (ii) selected from the group consisting of a bioreversible group which comprises a hydrophilic functional group claim 1 , a bioreversible group which comprises a conjugating moiety claim 1 , and a bioreversible group which comprises a conjugating moiety and a hydrophilic group claim 1 ,wherein the conjugating moiety may further comprise a protecting group.3. The polynucleotide construct of claim 1 , wherein the bioreversible group comprises a thioester.4. The polynucleotide construct of claim 1 , wherein the component (i) allows the polynucleotide construct to be transported intracellularly claim 1 , whereupon the bioreversible group is cleaved.5. The polynucleotide construct of claim 1 , further comprising at least one third component (iii) selected from the group consisting of a small molecule claim 1 , a peptide claim 1 , a polypeptide claim 1 , a carbohydrate claim 1 , a neutral organic ...

Polynucleotide constructs having disulfide groups

The invention features polynucleotide constructs containing one or more components (i) containing a disulfide linkage, where each of the one or more components is attached to an internucleotide bridging group or a terminal group of the polynucleotide construct, and each of the one or more components (i) contains one or more bulky groups proximal to the disulfide group. The invention also features polynucleotide constructs containing one or more components (i) containing a disulfide linkage, where each of the one or more components (i) is attached to an internucleotide bridging group or a terminal group of the polynucleotide construct, and each of the one or more components (i) contains at least 4 atoms in a chain between the disulfide linkage and the phosphorus atom of the internucleotide bridging group or the terminal group; and where the chain does not contain a phosphate, an amide, an ester, or an alkenylene. The invention also features methods of delivering a polynucleotide to a cell using the polynucleotide constructs of the invention.

POLYNUCLEOTIDES HAVING BIOREVERSIBLE GROUPS

The disclosure provides methods and compositions for delivering polynucleotides into cells. The disclosure provides transiently protected polynucleotides comprising an anionic charge-neutralizing moiety/group, which may also confer additional functionality. These compounds can enter the cytosol of cells by endocytic or macropinocytic mechanisms. The transient protecting group is bioreversible, i.e., once inside a cell, it is designed to be removed by enzymatic activity or by passive intracellular methods (e.g., changes in pH or reductive environment). 193-. (canceled)96. (canceled)98. The nucleotide construct of claim 97 , wherein Lis optionally substituted Calkylene.99. (canceled)100. The nucleotide construct of claim 97 , wherein Lis a covalent bond.101. The nucleotide construct of claim 97 , wherein Lis optionally substituted Calkylene or optionally substituted Carylene.102. The nucleotide construct of claim 97 , wherein Lis not a bond claim 97 , and Gis bound to Lvia a bond formed by a reaction selected from the group consisting of a pericyclic reaction; an alkylation or arylation of a hydroxyl claim 97 , thiol claim 97 , or amino moiety; and a reaction of a hydroxyl claim 97 , thiol claim 97 , or amino nucleophile with an electrophile.103. The nucleotide construct of claim 97 , wherein Lis not a bond claim 97 , and Gis bound to Lvia an amide bond claim 97 , a sulfonamide bond claim 97 , a carboxylic ester claim 97 , a thioester claim 97 , an optionally substituted Caryl or Cheteroaryl claim 97 , an imine claim 97 , a hydrazone claim 97 , an oxime or a succinimide.104. The nucleotide construct of claim 97 , wherein Gis the peptide claim 97 , the polypeptide claim 97 , the neutral organic polymer claim 97 , or any combination thereof;{'sup': '1', 'sub': 2-6', '2-6', '2-6, 'Lis an optionally substituted Calkylene, optionally substituted Calkenylene, or optionally substituted Calkynylene, wherein each of alkylene, alkenylene, or alkynylene is optionally interrupted ...

IMMUNOMODULATING POLYNUCLEOTIDES, ANTIBODY CONJUGATES THEREOF, AND METHODS OF THEIR USE

Immunomodulating polynucleotides are disclosed. The immunomodulating polynucleotides may contain 5-modified uridine, 5-modified cytidine, a total of from 6 to 16 nucleotides, and/or one or more abasic spacers and/or internucleoside phosphotriesters. Also disclosed are conjugates containing a targeting moiety and one or more immunomodulating polynucleotides. The immunomodulating polynucleotides and conjugates may further contain one or more auxiliary moieties. Also disclosed are compositions containing the immunomodulating polynucleotides or the conjugates containing one or more stereochemically enriched internucleoside phosphorothioates. Further disclosed are pharmaceutical compositions containing the immunomodulating polynucleotides or the conjugates and methods of their use. 1446.-. (canceled)447. An oligonucleotide of Formula (A):{'br': None, 'sup': 5′', 'N', 'P', 'N', '3′, 'sub': b', 'c, 'X—(X)—Y—(X)—X\u2003\u2003(A)'}or a stereoisomer, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof;wherein:{'sup': 'N', 'each Xis independently a nucleotide;'}{'sup': '3′', 'X is a 3′ terminal nucleotide;'}{'sup': '5′', 'X is a 5′ terminal nucleotide;'}{'sup': 'P', 'Yis an internucleoside phosphotriester; and'}b and c are each an integer ranging from about 0 to about 25; with the proviso that their sum is no less than 5;wherein the oligonucleotide comprises a nucleotide with a modified nucleobase.448. A compound of Formula (B):{'br': None, 'sup': x', 'N, 'sub': 'e', 'R-L-(Q)\u2003\u2003(B)'}or a stereoisomer, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers; or a pharmaceutically acceptable salt, solvate, or hydrate thereof;wherein:{'sup': 'x', 'Ris a conjugating group;'}{'sup': 'N', 'Lis a linker;'}each Q is independently an oligonucleotide comprising a phosphotriester; ande is an integer of 1, 2, 3, or 4.449. A compound ...

Immunomodulating polynucleotides, antibody conjugates thereof, and methods of their use

Immunomodulating polynucleotides are disclosed. The immunomodulating polynucleotides may contain 5-modified uridine, 5-modified cytidine, a total of from 6 to 16 nucleotides, and/or one or more abasic spacers and/or internucleoside phosphotriesters. Also disclosed are conjugates containing a targeting moiety and one or more immunomodulating polynucleotides. The immunomodulating polynucleotides and conjugates may further contain one or more auxiliary moieties. Also disclosed are compositions containing the immunomodulating polynucleotides or the conjugates containing one or more stereochemically enriched internucleoside phosphorothioates. Further disclosed are pharmaceutical compositions containing the immunomodulating polynucleotides or the conjugates and methods of their use.

Immunomodulating polynucleotides, antibody conjugates thereof, and methods of their use

Immunomodulating polynucleotides are disclosed. The immunomodulating polynucleotides may contain 5-modified uridine, 5-modified cytidine, a total of from 6 to 16 nucleotides, and/or one or more abasic spacers and/or internucleoside phosphotriesters. Also disclosed are conjugates containing a targeting moiety and one or more immunomodulating polynucleotides. The immunomodulating polynucleotides and conjugates may further contain one or more auxiliary moieties. Also disclosed are compositions containing the immunomodulating polynucleotides or the conjugates containing one or more stereochemically enriched internucleoside phosphorothioates. Further disclosed are pharmaceutical compositions containing the immunomodulating polynucleotides or the conjugates and methods of their use.

Transducible delivery of nucleic acids by reversible phosphotriester charge neutralization protecting groups

This disclosure relates to nucleic acid constructs modified to have a red uced net anionic charge. In some aspects the constructs comprise phosphodies ter and/or phosphothioate protecting groups. The disclosure also provide met hods of making and using such constructs.

Transducible delivery of nucleic acids by reversible phosphotriester charge neutralization protecting groups

This disclosure relates to nucleic acid constructs modified to have a reduced net anionic charge. In some aspects the constructs comprise phosphodiester and/or phosphothioate protecting groups. The disclosure also provide methods of making and using such constructs.

Transducible delivery of nucleic acids by reversible phosphotriester charge neutralization protecting groups

This disclosure relates to nucleic acid constructs modified to have a reduced net anionic charge. In some aspects the constructs comprise phosphodiester and/or phosphothioate protecting groups. The disclosure also provide methods of making and using such constructs.

Transducible delivery of nucleic acids by reversible phosphotriester charge neutralization protecting groups

This disclosure relates to nucleic acid constructs modified to have a reduced net anionic charge. In some aspects the constructs comprise phosphodiester and/or phosphothioate protecting groups. The disclosure also provide methods of making and using such constructs.

Immunomodulating polynucleotides, antibody conjugates thereof, and methods of their use

Immunomodulating polynucleotides are disclosed. The immunomodulating polynucleotides may contain 5-modified uridine, 5-modified cytidine, a total of from 6 to 16 nucleotides, and/or one or more abasic spacers and/or internucleoside phosphotriesters. Also disclosed are conjugates containing a targeting moiety and one or more immunomodulating polynucleotides. The immunomodulating polynucleotides and conjugates may further contain one or more auxiliary moieties. Also disclosed are compositions containing the immunomodulating polynucleotides or the conjugates containing one or more stereochemically enriched internucleoside phosphorothioates. Further disclosed are pharmaceutical compositions containing the immunomodulating polynucleotides or the conjugates and methods of their use.

Polynucleotide constructs having an auxiliary moiety non-bioreversibly linked to an internucleoside phosphate or phosphorothioate

The invention features a hybridized polynucleotide construct including a passenger strand, a guide strand loadable into a RISC complex, and one or more auxiliary moieties. At least one of the auxiliary moieties is non-bioreversibly linked to an internucleoside phosphate or phosphorothioate in the passenger strand. The invention further features methods of delivery a polynucleotide construct to a cell and methods of reducing the expression of a protein in a cell. The methods typically involve contacting the cell with the hybridized polynucleotide construct.

Transducible delivery of nucleic acids by reversible phosphotriester charge neutralization protecting groups

This disclosure relates to nucleic acid constructs modified to have a reduced net anionic charge. In some aspects the constructs comprise phosphodiester and/or phosphothioate protecting groups. The disclosure also provide methods of making and using such constructs.

Polynucleotides having bioreversible groups

The disclosure provides methods and compositions for delivering polynucleotides into cells. The disclosure provides transiently protected polynucleotides comprising an anionic charge- neutralizing moiety/group, which may also confer additional functionality. These compounds can enter the cytosol of cells by endocytic or macropinocytic mechanisms. The transient protecting group is bioreversible, i.e., once inside a cell, it is designed to be removed by enzymatic activity or by passive intracellular methods (e.g., changes in pH or reductive environment).

Polynucleotide constructs

Disclosed are polynucleotide constructs having a strand linked to a moiety carrying one or more auxiliary moieties. Also disclosed are polynucleotide constructs interrupted with a sugar analogue, and polynucleotide constructs with stereochemical^ enriched phosphorothioates. The polynucleotide constructs may be provided as hybridized polynucleotide constructs. Also featured are methods of delivery a polynucleotide construct to a cell and methods of reducing the expression of a protein in a cell by contacting the cell with the disclosed polynucleotide construct or hybridized polynucleotide construct.

Polynucleotide constructs having disulfide groups

The invention features polynucleotide constructs containing one or more components (i) containing a disulfide linkage, where each of the one or more components is attached to an internudeotide bridging group or a terminal group of the polynucleotide construct, and each of the one or more components (i) contains one or more bulky groups proximal to the disulfide group. The invention also features polynucleotide constructs containing one or more components (i) containing a disulfide linkage, where each of the one or more components (i) is attached to an internudeotide bridging group or a terminal group of the polynucleotide construct, and each of the one or more components (i) contains at least 4 atoms in a chain between the disulfide linkage and the phosphorus atom of the internudeotide bridging group or the terminal group; and where the chain does not contain a phosphate, an amide, an ester, or an alkenylene. The invention also features methods of delivering a polynucleotide to a cell using the polynucleotide constructs of the invention.

Polynucleotides having bioreversible groups

The disclosure provides methods and compositions for delivering polynucleotides into cells. The disclosure provides transiently protected polynucleotides comprising an anionic charge- neutralizing moiety/group, which may also confer additional functionality. These compounds can enter the cytosol of cells by endocytic or macropinocytic mechanisms. The transient protecting group is bioreversible, i.e., once inside a cell, it is designed to be removed by enzymatic activity or by passive intracellular methods (e.g., changes in pH or reductive environment).

Polynucleotide constructs having an auxiliary moiety non-bioreversibly linked to an internucleoside phosphate or phosphorothioate

Immunomodulating polynucleotides, antibody conjugates thereof, and methods of their use

Immunomodulating polynucleotides are disclosed. The immunomodulating polynucleotides may contain 5-modified uridine, 5-modified cytidine, a total of from 6 to 16 nucleotides, and/or one or more abasic spacers and/or internucleoside phosphotriesters. Also disclosed are conjugates containing a targeting moiety and one or more immunomodulating polynucleotides. The immunomodulating polynucleotides and conjugates may further contain one or more auxiliary moieties. Also disclosed are compositions containing the immunomodulating polynucleotides or the conjugates containing one or more stereochemically enriched internucleoside phosphorothioates. Further disclosed are pharmaceutical compositions containing the immunomodulating polynucleotides or the conjugates and methods of their use.

Polynucleotide constructs

Disclosed are polynucleotide constructs having a strand linked to a moiety carrying one or more auxiliary moieties. Also disclosed are polynucleotide constructs interrupted with a sugar analogue, and polynucleotide constructs with stereochemical{circumflex over ( )} enriched phosphorothioates. The polynucleotide constructs may be provided as hybridized polynucleotide constructs. Also featured are methods of delivery a polynucleotide construct to a cell and methods of reducing the expression of a protein in a cell by contacting the cell with the disclosed polynucleotide construct or hybridized polynucleotide construct.

Polynucleotide constructs

Disclosed are polynucleotide constructs having a strand linked to a moiety carrying one or more auxiliary moieties. Also disclosed are polynucleotide constructs interrupted with a sugar analogue, and polynucleotide constructs with stereochemically enriched phosphorothioates. The polynucleotide constructs may be provided as hybridized polynucleotide constructs. Also featured are methods of delivery a polynucleotide construct to a cell and methods of reducing the expression of a protein in a cell by contacting the cell with the disclosed polynucleotide construct or hybridized polynucleotide construct.

Polynucleotides having bioreversible groups

The disclosure provides methods and compositions for delivering polynucleotides into cells. The disclosure provides transiently protected polynucleotides comprising an anionic charge-neutralizing moiety/group, which may also confer additional functionality. These compounds can enter the cytosol of cells by endocytic or macropinocytic mechanisms. The transient protecting group is bioreversible, i.e., once inside a cell, it is designed to be removed by enzymatic activity or by passive intracellular methods (e.g., changes in pH or reductive environment).