Multi-armed polyrotaxane platform for protected nucleic acid delivery

A polyrotaxane carrier for in vivo delivery of a nucleic acid, said carrier comprising:

• a multi-arm polyethylene glycol (PEG) backbone comprising at least three arms;
• at least one cyclic compound having a cavity, where an arm of said multi-arm PEG backbone is threaded into the cavity of said cyclic compound forming an inclusion complex;
• a bulky moiety capping the terminal of the arm(s) threaded into said cyclic compound where said moiety inhibits dethreading of the cyclodextrin from the arm(s) of said backbone;
• where at least one arm of said PEG backbone is free of cyclic compounds;
• where said carrier has a net positive charge;
• where:
• at least one arm not threaded into said cyclic compound is terminated with a protecting group, and/or a fluorophore, and/or a targeting moiety;
• and/or
• at least one arm not threaded into said cyclic compound are terminated with a protecting group selected from the group consisting of dansyl, acetyl, amide, and 3 to 20 carbon alkyl groups, Fmoc, Tboc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh),Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), 1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA);
• and/or
• at least one arm not threaded into said cyclic compound is attached to a fluorophore;
• and/or
• at least one arm not threaded into said cyclic compound is attached to a targeting moiety that specifically or preferentially binds to a cell;
• and/or
• at least one arm not threaded into said cyclic compound is attached to a at least one arm not threaded into said cyclic compound is attached to a targeting moiety selected from the group consisting of an antibody, a receptor ligand, a nucleic acid aptamer, a peptide aptamer, neural cell adhesion molecule (NCAM), a cell penetrating peptide (CPP), a peptide aptamer, and a lectin;
• and/or
• at least one arm not threaded into said cyclic compound is attached to a at least one arm not threaded into said cyclic compound is attached to a targeting moiety comprising a ligand that binds a receptor where said ligand is selected from the group consisting of transferrin, mannose, glucose, and folic acid;
• and/or
• at least one arm not threaded into said cyclic compound is attached to a targeting moiety comprising transferrin.

A construct for the treatment of Duchenne Muscular Dystrophy, said construct comprising:

• a polyrotaxane carrier comprising:
• a multi-arm polyethylene glycol (PEG) backbone comprising at least three arms;
• at least one cyclic compound having a cavity, where an arm of said multi-arm PEG backbone is threaded into the cavity of said cyclic compound forming an inclusion complex;
• a bulky moiety capping the terminal of the arm(s) threaded into said cyclic compound where said moiety inhibits dethreading of the cyclodextrin from the arm(s) of said backbone;
• where at least one arm of said PEG backbone is free of cyclic compounds;
• and
• where said carrier has a net positive charge;
• and
• where said carrier is complexed with a plasmid encoding a class 2 CRISPR/Cas endonuclease, and a guide RNA that hybridizes to a target sequence within intron 44 of a mutant dystrophin gene, and/or a second CRISPR/Cas guide RNA guide sequence that hybridizes to a target sequence within intron 55 of the mutant dystrophin gene.