Nuclease-mediated genome editing of primary cells and related methods of treatment

A method for preventing or treating sickle cell disease in a subject in need thereof, the method comprising the steps of:

• (a) stably integrating via homologous recombination a polynucleotide into a genome of one or more primary hematopoietic stem and progenitor cells (HSPCs) of a population of primary HSPCs comprising an E6V mutation in the HBB gene, wherein the integrating comprises introducing into one or more primary HSPCs of the population of primary HSPCs:
• (i) a modified single guide RNA (sgRNA) comprising a first nucleotide sequence that is complementary to a target nucleic acid sequence in the HBB gene of the one or more primary cells and a second nucleotide sequence that interacts with a CRISPR-associated Cas polypeptide, wherein the first nucleotide sequence, the second nucleotide sequence, or both comprise a modified nucleotide;
• (ii) the Cas polypeptide, an mRNA encoding the Cas polypeptide, or a recombinant expression vector comprising a nucleotide sequence encoding the Cas polypeptide, wherein the modified sgRNA guides the Cas polypeptide to the target nucleic acid sequence in the HBB gene;
• and
• (iii) an adeno-associated viral (AAV) vector comprising the polynucleotide, wherein the polynucleotide comprises a corrective SNP for reverting the E6V mutation;
• wherein the Cas polypeptide cleaves the target nucleic acid sequence, thereby generating a double-strand break in the target nucleic acid sequence;
• wherein the polynucleotide is integrated into the double-strand break in the target nucleic acid sequence, thereby producing the stable integration of the polynucleotide into the genome of the one or more primary cells and correcting the E6V mutation in the HBB gene;
• wherein the polynucleotide is integrated into the genome of greater than 10% of primary cells in the population of primary cells;
• and
• (b) administering to the subject primary cells comprising a stable integration of the polynucleotide.