Pharmaceutical compositions comprising gene-corrected primary cells

A pharmaceutical composition comprising a population of primary hematopoietic stem and progenitor cells (HSPCs) isolated from an individual having sickle cell disease, wherein the population comprises:

• (a) a first plurality of primary HSPCs comprising an E6V mutation in an HBB gene;
• (b) a second plurality of primary HSPCs comprising at least one insertion and/or deletion (INDEL) within a target sequence formerly comprising SEQ ID NO:1 of an HBB gene comprising a nucleic acid sequence encoding an E6V mutation, wherein the INDEL is generated by introducing into a plurality of primary HSPCs:
• (i) a modified single guide RNA (sgRNA) comprising a first nucleotide sequence complementary to the target sequence 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;
• and
• (ii) the Cas polypeptide, a m RNA encoding the Cas polypeptide, or a recombinant expression vector comprising a nucleotide sequence encoding the Cas polypeptide,
• wherein the Cas polypeptide cleaves the target sequence in the HBB gene comprising the nucleic acid sequence encoding the E6V mutation, and wherein at least one INDEL is generated at the site of cleavage by non-homologous end-joining;
• and
• (c) a third plurality of primary HPSCs comprising a heterologous polynucleotide integrated into a target sequence formerly comprising SEQ ID NO:1 in an HBB gene comprising a nucleic acid sequence encoding an E6V mutation, wherein the heterologous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:73 and the heterologous polynucleotide corrects the nucleic acid sequence encoding the E6V mutation formerly in the HBB gene of the third plurality of primary HPSCs.