Wisconsin Alumni Research Foundation

Krishanu Saha

Amr Ashraf Abdeen

Shaoqin Gong

Yuyuan Wang

Jared Matthew Carlson-Stevermer

Lucille Katherine Kohlenberg

Guojun Chen

Ruosen Xie

Described herein are guide RNAs and modified guide RNAs suitable for biallelic correction of Pompe disease. Also included are methods of modifying a target gene in a patient or in a patient-derived cell, wherein the patient has an autosomal recessive disorder with compound heterozygous mutations, the methods including delivering a first modified guide RNA, a second modified guide RNA, a Cas9 polypeptide, a biotin-binding molecule, a first biotinylated donor polynucleotide, and a second biotinylated donor polynucleotide. The first modified guide RNA and the first biotinylated donor polynucleotide correct a first diseased allele, and the second modified guide RNA and the second biotinylated donor polynucleotide correct a second diseased allele.

Gene correction of Pompe disease and other autosomal recessive disorders via RNA-guided nucleases

Provided herein are nanoplexes comprising a payload selected from a protein and/or a polynucleic acid; and a plurality of copolymers comprising a first copolymer that is poly(N,N'-bis(acryloyl)cystamine-poly(aminoalkyl)) (PBAP), a second copolymer that is poly(Cakylene glycol)-PBAP-poly(Cakylene glycol), and a third copolymer that is TG-poly(Cakylene glycol)-PBAP-poly(Cakylene glycol)-TG wherein TG at each occurrence is independently a targeting ligand, a cell penetrating peptide, an imaging agent or a capping group, provided that a plurality of TG groups is a targeting ligand; wherein the payload is non-covalently complexed to one or more of the copolymers, one or more of the first, second, and/or third copolymers comprises an endosomal escape group having a pKa of about 4.5 to about 6.5, and optionally one or more of the first, second, and/or third copolymers comprises a host and a guest non-covalent crosslinker.

Polyplex delivery system for proteins, nucleic acids and protein/nucleic acid complexes

Provided herein are nanocapsules comprising a single ribonucleoprotein (RNP) complex as a core and an biodegradable crosslinked polymer shell that encapsulates the core, wherein the RNP complex comprises a Cas9 polypeptide and a guide RNA, and the biodegradable crosslinked polymer shell comprises polymerized monomers of imidazolyl acryloyl monomers, bisacryloyl disulfide monomers (a biodegradable cross-linker), optionally PEG acryloyl monomers, and either cationic acryloyl monomers, anionic acryloyl monomers, or both cationic and anionic acryloyl monomers (optionally in combination with non-ionic acryloyl monomers) as defined herein. Also provided are methods of making the nanocapsules, kits containing the nanocapsules and methods of delivering the encapsulated RNP to cells.

Nanocapsule delivery system for ribonucleoproteins

Described herein are modified guide RNAs such as a single guide RNA including, from 5' to 3&#x2032;, a single-stranded protospacer sequence, a first complementary strand of a binding region for the Cas9 polypeptide, an aptamer that binds a biotin-binding molecule, and a second complementary strand of the binding region for the Cas9 polypeptide. Also described is an RNP complex including the modified guide RNA and a Cas9 polypeptide or active fragment thereof. Also included are methods of modifying target genes in cells using the modified guide RNAs.

Growing community of inventors

Middleton, WI, United States of America

Krishanu Saha