Reversible attachment of a membrane active polymer to a polynucleotide
View Patent ↗Described is a process for delivering a biologically active compound to a cell by reversibly linking the compound to a membrane active polymer. In particular, polymer-polynucleotide conjugates are described. Methods for reversibly modifying the polymers to decrease cellular toxicity and improve efficacy are provided.
1. A composition for delivering a polynucleotide to a mammalian cell in vivo comprising: a reversibly-modified membrane active polyamine-polynucleotide conjugate wherein:
a) the reversibly-modified membrane active polyamine is linked to the polynucleotide via a labile covalent bond; and,
b) the reversibly-modified membrane active polyamine consists of a negatively charged polymer that is not membrane active wherein the negative charge comes from a plurality of carboxyl groups linked to the polymer via pH-labile covalent linkages wherein cleavage of the pH-labile covalent bonds in response to a decrease in pH results in cleavage of the carboxyl groups from the polymer and formation of amine groups thereby forming a membrane active polyamine, and wherein
c) the membrane active polyamine has a molecular weight greater than 10,000 daltons.
2. The composition of claim 1 wherein the polynucleotides consists of an oligonucleotide.
3. The composition of claim 2 wherein the polynucleotide is selected from the group consisting of: dsRNA, siRNA, microRNA, siRNA expression cassette, antisense oligonucleotide and ribozyme.
4. The composition of claim 1 wherein the membrane active polyamine consists of a polyvinyl ether.
5. The composition of claim 1 wherein the membrane active polyamine consists of an amphipathic polymer.
6. A composition for delivering a biologically active compound to a mammalian cell in vivo comprising: a reversibly-modified membrane active polyamine-biologically active compound conjugate wherein:
a) the reversibly-modified membrane active polyamine is linked to the biologically active compound via a labile covalent bond; and,
b) the reversibly-modified membrane active polyamine consists of a negatively charged polymer that is not membrane active wherein the negative charge comes from a plurality of carboxyl groups linked to the polymer via pH-labile covalent linkages wherein cleavage of the pH-labile covalent bonds in response to a decrease in pH results in cleavage of the carboxyl groups from the polymer and formation of amine groups thereby forming a membrane active polyamine; and wherein
c) the membrane active polyamine has a molecular weight greater than 10,000 daltons.
7. The composition of claim 6 wherein the biologically active compound comprises a polynucleotide.
8. The composition of claim 7 wherein the polynucleotides consists of an oligonucleotide.
9. The composition of claim 8 wherein the polynucleotide is selected from the group consisting of: dsRNA, siRNA, microRNA, siRNA expression cassette, antisense oligonucleotide and ribozyme.
10. The composition of claim 6 wherein the membrane active polyamine consists of an amphipathic polymer.
11. The composition of claim 6 wherein the membrane active polyamine consists of a polyvinyl ether.
12. A method for delivering a polynucleotide to a mammalian cell in vivo comprising:
a) attaching the biologically polynucleotide to an amphipathic membrane active polyamine having a molecular weight greater than 10,000 daltons via a labile bond to form a conjugate,
b) reversibly modifying a plurality of amines on the amphipathic membrane active polyamine by covalent attachment of carboxyl groups to the amines via very pH-labile bonds to form a negatively charged polymer that is not membrane active wherein cleavage of the pH-labile covalent bonds in response to a decrease in pH results in cleavage of the carboxyl groups from the polymer and formation of amine groups thereby regenerating the amphipathic membrane active polyamine; and,
c) contacting the cell with the conjugate.
13. The method of claim 12 wherein the polynucleotide is selected from the group consisting of: dsRNA, siRNA, microRNA, siRNA expression cassette, antisense oligonucleotide and ribozyme.