Gene Therapy Targeting PCSK9

Abstract

The last decades of research in cardiovascular prevention have been characterized by successful bench-to-bedside developments for the treatment of low-density lipoprotein (LDL) hypercholesterolemia. Recent examples include the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) with monoclonal antibodies, small interfering RNA and antisense RNA drugs. The cumulative effects of LDL cholesterol on atherosclerosis make early, potent, and long-term reductions in LDL cholesterol desirable-ideally without the need of regular intake or application of medication and importantly, without side effects. Current reports show durable LDL cholesterol reductions in primates following one single treatment with PCSK9 gene or base editors. Use of the CRISPR/Cas system enables precise genome editing down to single-nucleotide changes. Provided safety and documentation of a reduction in cardiovascular events, this novel technique has the potential to fundamentally change our current concepts of cardiovascular prevention. In this review, the application of the CRISPR/Cas system is explained and the current state of in vivo approaches of PCSK9 editing is presented.

Keywords: CRISPR/Cas; LDL cholesterol; PCSK9; base editing; cardiovascular disease; gene editing; hypercholesterolemia; in vivo.

Figure 1

Shown is the principle of using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system for genome editing. The programmable guide RNA (gRNA) complementary binds to a specific DNA sequence termed “protospacer”. The DNA–gRNA complex recruits a nuclease such as Cas9. The nuclease has to interact with a specific DNA sequence next to the protospacer termed “protospacer-adjacent motif” (PAM) in order to work. If these requirements are fulfilled, the nuclease introduces a double strand break in the DNA. Endogenous DNA repair mechanisms follow. These repair mechanisms are error-prone and lead to random insertions or deletions, which, in turn, render the gene and the encoded protein dysfunctional. In a further development, the catalytic domain of Cas9 has been deactivated and fused to a base editor, which does not introduce double strand breaks, but precise single-nucleotide changes.

Figure 2

Genome editing of PCSK9 using a base editor that introduces a single-nucleotide change at the splice site of exon 1 and intron 1. This results in retention of intron 1 in the final mRNA, which contains an in-frame stop codon, leading to premature termination of protein synthesis.