In Vivo Base Editing of PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) as a Therapeutic Alternative to Genome Editing

Abstract

Objective High-efficiency genome editing to disrupt therapeutic target genes such as PCSK9 has been demonstrated in preclinical animal models, but there are safety concerns due to 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 non-proliferating cells in vivo. Base editors comprising CRISPR-Cas9 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.

Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

“…Together, these components enable efficient and permanent C•G to T•A base pair conversion in bacteria, yeast 4,9 , plants 10,11 , zebrafish 8,12 , mammalian cells 3–8,13,14 , mice 8,15,16 , and even human embryos 17,18 . Base editing capabilities have expanded through the development of base editors with different protospacer-adjacent motif (PAM) compatibilities 7 , narrowed editing windows 7 , enhanced DNA specificity 8 , and small-molecule dependence 19 .…”

What Do We Really Think About Human Germline Genome Editing, and What Does It Mean for Medicine?

“…The objective of the session was to provide attendees with sufficient background regarding CRISPR-Cas9 genome editing so that they could then meaningfully explore and discuss how genome editing might be relevant to clinical practice. We (the authors) gave short presentations that covered: the basic concepts of CRISPR-Cas9 genome editing; how CRISPR-Cas9 has transformed the way in which mouse models of human physiology and disease are made, making the process far more rapid and efficient; 2 proof-of-concept studies demonstrating that CRISPR-Cas9 targeting of genes in the mouse liver can beneficially modify lipid traits, heralding possible one-shot, lifelong treatments for dyslipidemia and coronary heart disease; 3, 4 potential dangers of genome editing (e.g., unintended mutations) and strategies to reduce those dangers; 4, 5 potential clinical scenarios in which one might contemplate performing GGE, including the preemption of devastating genetic diseases in one’s offspring, the reduction of risk of common disorders such as coronary heart disease and Alzheimer disease, and the addition of desired non-medical traits (“enhancements”); the possibility of implementing GGE not in embryos but rather in germ cells such as sperm and oocytes, which might be less morally objectionable to some people; and potential adverse consequences of modifying the human germline, such as spreading susceptibility to certain diseases or exacerbating societal inequities. In particular, it was pointed out that although many commentators have noted that in vitro fertilization paired with pre-implantation genetic diagnosis can often avoid any need for GGE, there are situations where all unmodified embryos will yield offspring with disease—two parents with a recessive disorder such as sickle cell disease or cystic fibrosis, or a parent homozygous or compound heterozygous for dominant mutations such as the Huntington disease repeat expansion.…”

A CRISPR edit for heart disease

“…The technique has been used in labs to correct genes in yeast, plants, zebrafish, mice and even human embryos. A proof-of-concept study by Alexandra Chadwick, a postdoctoral researcher in Musunuru's lab, delivered a base editor into the livers of adult mice to disable Pcsk9, halving the level of Pcsk9 and cutting LDL cholesterol by almost one-third 12 . Musunuru adds that he has preliminary results showing base editing of Angptl3 in mice using Liu's C-to-T method.…”