CRISPR Meets ApoA-I Milano to Make Liver Great Again

Presentation Number: MON 511
Date of Presentation: April 3rd, 2017

Carmelo Gabriele Pizzino*1, Federica Mannino2, Domenica Altavilla2, Francesco Squadrito1 and Alessandra Bitto1
1University of Messina, Messina, Italy, 2University of Messina


Atherosclerosis is determined by bloodstream lipids excess, and their deposition in to the so-called atherosclerotic plaque, which in turn is responsible for partial or total artery occlusion, affecting mainly carotid and coronary vessels. High-density lipoproteins (HDL) particles remove lipids from cells. Apolipoprotein A1 (ApoA-I), the major protein component of HDL particles in plasma, enables efflux of lipids from cells to transport back to LDL particles or to the liver. ApoA-I Milano is a naturally occurring mutated variant of the ApoA-I protein found in human HDL.(1) ApoA-I Milano has been shown to reduce atherosclerosis in animal models and in a small phase 2 human trial.(2) Recombinant adeno-associated virus 8 (AAV8) mediated ApoA-I Milano gene therapy in combination with low-cholesterol diet induces rapid and significant regression of atherosclerosis in mice.(2) Synthetic ApoA-I Milano production is challenging, time consuming, and expensive. AAV8-based gene transfer is affected by the need of repeated cycles of treatment.

CRISPR system uses non-coding RNAs (sgRNA) to guide the Cas9 nuclease to induce site-specific DNA cleavage (double strand break, DSB). These will be repaired by cells via non-homologous end joining or homology directed repair (HDR) if a donor DNA is provided to the cells.

In light of what stated before, we decided to overcome the limitations of both synthetic ApoA-I Milano, and gene transfer, by setting up a CRISPR-based strategy, in order to assess if we could let wild type hepatocytes to acquire the ApoA-I Milano genotype and produce a mutated ApoA-I protein.

We performed a first batch of in vitro experiments, on HepG2 human hepatocytes and on primary hepatocytes from C57BL/6-Tg(APOA1)1Rub/J (HepRub/J) transgenic mice, carrying the human ApoA-I gene. First, we designed an sgRNA targeting the ApoA-I gene (to let Cas9 produce a DSB) and a donor DNA carrying the Milano mutation (DNA-Milano) flanked by two regions of homology (which will be used by the cells to repair the Cas9-induced DSB). Second, we cloned the ApoA-I targeting sgRNA into a Cas9-expressing plasmid (px330 from Addgene including the ApoA-I sgRNA, hereinafter px330ApoA-I); third, we transfected both HepG2 and HepRub/J cells with the px330ApoA-I vector alone or in combination with DNA-Milano. After negative selection, we screened our cells via direct sequencing, to check whether or not we have been able to edit the DNA and introduce the Milano mutation. We estimated the ApoA-I Milano production by both RT-qPCR and western blot.

Our results indicate that by using CRISPR we can efficiently target ApoA-I human gene and edit its sequence in order to obtain a Milano genotype, at least in vitro.

We are currently engaged in in vivo experiments on C57BL/6-Tg(APOA1)1Rub/J mice, to verify if we could achieve the same results by delivering the genome editing system via AAV vectors targeting liver tissue.


Nothing to Disclose: CGP, FM, DA, FS, AB