BLOCK COPOLYMERS
Described herein are block copolymers, and methods of making and utilizing such copolymers. The described block copolymers are disruptive of a cellular membrane, including an extracellular membrane, an intracellular membrane, a vesicle, an organelle, an endosome, a liposome, or a red blood cell. Preferably, in certain instances, the block copolymer disrupts the membrane and enters the intracellular environment. In specific examples, the block copolymer is endosomolytic and capable of delivering an oligonucleotide (e.g., an mRNA) to a cell. Compositions comprising a block copolymer and an oligonucleotide (e.g., an mRNA) are also disclosed.
1 . A composition comprising:
a) a block copolymer of the formula I
T1-L1-[A] x —[B] y —Z I
wherein
T1 is a first targeting moiety;
L1 is absent or a linking moiety;
A is a first block that is a polymer formed from monomers comprising formula A2 or a random copolymer formed from monomers comprising formulae A2 and A3; A2, A4 and A5; A2 and A5; or A4 and A5;
wherein n is 1-120, R 3 is H or C 1 -C 6 alkyl, R 4 is S, O, NH or N(C 1 -C 6 alkyl), R 5 is O or S and R 6 is H, C 1 -C 6 alkyl, C 1 -C 6 alkyl-NH 2 , C 1 -C 6 alkyl-NH(C 1 -C 6 alkyl), C 1 -C 6 alkyl-N(C 1 -C 6 alkyl) 2 ;
wherein R 7 and R 10 are independently H or C 1 -C 6 alkyl, R 8 is S, O, NH or N(C 1 -C 6 alkyl), and R 9 is O or S and R 11 is an amine protecting group;
wherein n is 1-230, R 17 is H or C 1 -C 6 alkyl, R 18 is O, S, NH or N(C 1 -C 6 alkyl), R 19 is O or S, and R 20 is OH, NH, H, T2, or C 1 -C 6 alkyl, where T2 is a second targeting moiety;
wherein R 21 is H or C 1 -C 6 alkyl, R 22 is O, NH or N(C 1 -C 6 alkyl), R 23 is H, aryl, arylhalide, alkyl, alkyl alcohol;
B is a second block that is a random copolymer formed from monomers comprising formulae B1, B2, B3 and B4
wherein R 12 , R 13 , R 1 , R 15 , R 16 and R 17 are independently H or C 1 -C 6 alkyl, R 18 is O, S, NH, N(C 1 -C 6 alkyl), or (OCH 2 CH 2 ) 1-120 , and Q is selected from the group consisting of (i) S—S-G, (ii) (OCH 2 CH 2 ) 1-120 —S—S-G, (iii) V-L3-G wherein V is an amide, ester, imine, oxime, thioester, product of a [3+2] cycloaddition, product of a [4+1] cycloaddition, carbonate, carbamate, urea, acetal, ketal, or hydrazone, and L3 is C 1 -C 6 alkyl, (OCH 2 CH 2 ) 1-50 , C 1 -C 6 alkyl-(OCH 2 CH 2 ) 1-50 , or thioether,
wherein R 29 is C 1 -C 6 alkyl, (OCH 2 CH 2 ) 1-50 , C 1 -C 6 alkyl-(OCH 2 CH 2 ) 1-50 , O, NH, or N(C 1 -C 6 alkyl), and
(vii) S—S-L2-G wherein L2 is
wherein n=1-35 and designates a point of attachment of L2 to G,
wherein G is a cationic peptide, polyamine, or polycation;
x is 2-20 kDa;
y is 2-20 kDa;
the ratio of x to y is from 2:1 to 1:4; and
Z is H, SH, C(CH 3 ) 2 CN,
wherein R 24 is S—(C 1 -C 12 alkyl), aryl, arylhalide, O—(C 1 -C 12 alkyl), or NR 25 R 26
wherein R 25 and R 26 are independently H, alkyl, aryl, or heteroaryl; and
designates a point of attachment;
and
b) an oligonucleotide.
2 . The composition of claim 1 , wherein G is a cationic peptide.
3 . The composition of claim 1 , wherein the oligonucleotide is selected from the group consisting of an siRNA, an antisense oligonucleotide, a dicer substrate, a miRNA, an aiRNA, and an shRNA.
4 . The composition of claim 1 , wherein the oligonucleotide is an mRNA.
5 . The composition of claim 4 , wherein the mRNA encodes a functional protein associated with a protein deficiency disease.
6 . The composition of claim 5 , wherein the mRNA encodes a functional protein selected from the group consisting of ornithine transcarbamylase (OTC), carbamoyl phosphate synthetase I (CPS1), fumarylacetoacetase (FAH) enzyme, alanine:glyoxylate-aminotransferase (AGT), methylmalonyl CoA mutase (MUT), propionyl CoA carboxylase alpha subunit (PCCA), propionyl CoA carboxylase beta subunit (PCCB), a subunit of branched-chain ketoacid dehydrogenase (BCKDH), copper-transporting ATPase Atp7B, bilirubin uridinediphosphate glucuronyltransferase (BGT) enzyme, hepcidin, glucose-6-phosphatase (G6Pase), glucose 6-phosphate translocase, lysosomal glucocerebrosidase (GB), Niemann-Pick C1 protein (NPC1), Niemann-Pick C2 protein (NPC2), acid sphingomyelinase (ASM), alpha-1 antitrypsin (A1AT), Factor IX, galactose-1-phosphate uridylyltransferase, galactokinase, UDP-galactose 4-epimerase, transthyretin, a complement regulatory protein, phenylalanine hydroxylase (PAH), homogentisate 1,2-dioxygenase, porphobilinogen deaminase, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), argininosuccinate lyase (ASL), and P-type ATPase protein FIC-1.
7 . The composition of claim 4 , wherein the mRNA encodes a functional protein selected from the group consisting of erythropoietin, alpha-galactosidase A, LDL receptor, Factor VII, Factor VIII, Factor IX, alpha-L-iduronidase, iduronate-2-sulfatase, heparan-N-sulfatase, alpha-N-acetylglucosaminidase, galactose 6-sulfatase, acid β-galactosidase, lysosomal acid lipase, ornithine transcarbamylase (OTC), alpha-1-antitrypsin, arylsulfatase A, arylsulfatase B, acid ceramidase, acid α-L-fucosidsase, acid β-glucosidase, galactocerebrosidase, acid α-mannosidase, acid β-mannosidase, N-acetylgalactosamine-6-sulfate sulfatase, acid sphingomyelinase, acid α-glucosidase, β-hexosaminidase B, acetyl-CoA:α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, alpha-N-acetylgalactosaminidase, sialidase, β-glucuronidase, and β-hexosaminidase A.
8 . The composition of claim 4 , wherein the mRNA encodes a functional tumor suppressor protein.
9 . The composition of claim 8 , wherein the functional tumor suppressor protein is selected from the group consisting of Retinoblastoma protein (pRb), p53 tumor-suppressor protein, Phosphatase and tensin homolog (PTEN), Von Hippel-Lindau tumor suppressor (pVHL), Adenomatous polyposis coli (APC), FAS receptor (FasR), Suppression of tumorigenicity 5 (ST5), YPEL3, Suppressor of tumorigenicity protein 7 (ST7), and Suppressor of tumorigenicity 14 protein (ST14).
10 . The composition of claim 2 , wherein the oligonucleotide is an mRNA.
11 . The composition of claim 10 , wherein the mRNA encodes a functional protein associated with a protein deficiency disease.
12 . The composition of claim 11 , wherein the mRNA encodes a functional protein selected from the group consisting of ornithine transcarbamylase (OTC), carbamoyl phosphate synthetase I (CPS1), fumarylacetoacetase (FAH) enzyme, alanine:glyoxylate-aminotransferase (AGT), methylmalonyl CoA mutase (MUT), propionyl CoA carboxylase alpha subunit (PCCA), propionyl CoA carboxylase beta subunit (PCCB), a subunit of branched-chain ketoacid dehydrogenase (BCKDH), copper-transporting ATPase Atp7B, bilirubin uridinediphosphate glucuronyltransferase (BGT) enzyme, hepcidin, glucose-6-phosphatase (G6Pase), glucose 6-phosphate translocase, lysosomal glucocerebrosidase (GB), Niemann-Pick C1 protein (NPC1), Niemann-Pick C2 protein (NPC2), acid sphingomyelinase (ASM), alpha-1 antitrypsin (A1AT), Factor IX, galactose-1-phosphate uridylyltransferase, galactokinase, UDP-galactose 4-epimerase, transthyretin, a complement regulatory protein, phenylalanine hydroxylase (PAH), homogentisate 1,2-dioxygenase, porphobilinogen deaminase, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), argininosuccinate lyase (ASL), and P-type ATPase protein FIC-1.
13 . The composition of claim 10 , wherein the mRNA encodes a functional protein selected from the group consisting of erythropoietin, alpha-galactosidase A, LDL receptor, Factor VII, Factor VIII, Factor IX, alpha-L-iduronidase, iduronate-2-sulfatase, heparan-N-sulfatase, alpha-N-acetylglucosaminidase, galactose 6-sulfatase, acid β-galactosidase, lysosomal acid lipase, ornithine transcarbamylase (OTC), alpha-1-antitrypsin, arylsulfatase A, arylsulfatase B, acid ceramidase, acid α-L-fucosidsase, acid β-glucosidase, galactocerebrosidase, acid α-mannosidase, acid β-mannosidase, N-acetylgalactosamine-6-sulfate sulfatase, acid sphingomyelinase, acid α-glucosidase, β-hexosaminidase B, acetyl-CoA:α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, alpha-N-acetylgalactosaminidase, sialidase, β-glucuronidase, and β-hexosaminidase A.
14 . The composition of claim 10 , wherein the mRNA encodes a functional tumor suppressor protein.
15 . The composition of claim 14 , wherein the functional tumor suppressor protein is selected from the group consisting of Retinoblastoma protein (pRb), p53 tumor-suppressor protein, Phosphatase and tensin homolog (PTEN), Von Hippel-Lindau tumor suppressor (pVHL), Adenomatous polyposis coli (APC), FAS receptor (FasR), Suppression of tumorigenicity 5 (ST5), YPEL3, Suppressor of tumorigenicity protein 7 (ST7), and Suppressor of tumorigenicity 14 protein (ST14).
16 . The composition of claim 1 , wherein T1 is selected from the group consisting of an antibody, a peptide, a sugar, and a vitamin.
17 . The composition of claim 1 , wherein T1 specifically binds to the asialoglycoprotein receptor.
18 . The composition of claim 17 , wherein T1 is a galactose-containing targeting moiety.
19 . The composition of claim 17 , wherein T1 comprises a N-acetyl galactoseamine (NAG) moiety.
20 . The composition of claim 1 , wherein L1 is a polymer having a molecular weight of from 0.5 kDa to 6 kDa and comprising at least 10 ethylene oxide units.