In Vivo Base Editing of PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) as a Therapeutic Alternative to Genome Editing.
OBJECTIVE
High-efficiency genome editing to disrupt therapeutic target genes, such as PCSK9 (proprotein convertase subtilisin/kexin type 9), has been demonstrated in preclinical animal models, but there are safety concerns because of the unpredictable nature of cellular repair of double-strand breaks, as well as off-target mutagenesis. Moreover, precise knock-in of specific nucleotide changes-whether to introduce or to correct gene mutations-has proven to be inefficient in nonproliferating cells in vivo. Base editors comprising CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats [CRISPR]-CRISPR-associated 9) fused to a cytosine deaminase domain can effect the alteration of cytosine bases to thymine bases in genomic DNA in a sequence-specific fashion, without the need for double-strand DNA breaks. The efficacy of base editing has not been established in vivo. The goal of this study was to assess whether in vivo base editing could be used to modify the mouse Pcsk9 gene in a sequence-specific fashion in the liver in adult mice.


APPROACH AND RESULTS
We screened base editors for activity in cultured cells, including human-induced pluripotent stem cells. We then delivered a base editor into the livers of adult mice to assess whether it could introduce site-specific nonsense mutations into the Pcsk9 gene. In adult mice, this resulted in substantially reduced plasma PCSK9 protein levels (>50%), as well as reduced plasma cholesterol levels (≈30%). There was no evidence of off-target mutagenesis, either cytosine-to-thymine edits or indels.


CONCLUSIONS
These results demonstrate the ability to precisely introduce therapeutically relevant nucleotide variants into the genome in somatic tissues in adult mammals, as well as highlighting a potentially safer alternative to therapeutic genome editing.
['Chadwick|Alexandra C|AC|', 'Wang|Xiao|X|', 'Musunuru|Kiran|K|']
D000818:Animals D001482:Base Composition D015415:Biomarkers D064113:CRISPR-Cas Systems D045744:Cell Line, Tumor D002784:Cholesterol D018389:Codon, Nonsense D015536:Down-Regulation D000072669:Gene Editing D015971:Gene Expression Regulation, Enzymologic D015316:Genetic Therapy D005838:Genotype D057809:HEK293 Cells D006801:Humans D057026:Induced Pluripotent Stem Cells D008099:Liver D008810:Mice, Inbred C57BL D010641:Phenotype D000071449:Proprotein Convertase 9 D013997:Time Factors D014162:Transfection
PCSK9 nucleotides gene therapy lipids and lipoprotein metabolism molecular biology