Lowering cholesterol levels via genome editingAugust 12, 2020
Some people naturally carry mutations in the genes associated with blood cholesterol and LDL cholesterol, and have a reduced risk of coronary artery disease (CAD) without any adverse effects to their health. The discovery of this phenomenon has changed the way we approach the development of therapies in this area. Several studies have found that nonsense mutation carriers of the proprotein convertase subtilisin / kexin type 9 (PCSK9) gene have significantly decreased levels (30-40%) of low-density lipoprotein cholesterol (LDL-C) in comparison with normal people. Another set of mutations in gene apolipoprotein C3 (APOC3) is also associated with a 40% lower chance of getting CAD. Together, these mutations give a protection of more than 80% and can potentially be used as a treatment for cardiovascular diseases through genome editing.
Editing out, precisely
Genome editing — for a variety purposes such as engineering bacteria to biomedical uses — has emerged as a revolutionary technology, especially in the past 10 years. Genome editing can be achieved in vitro or in vivo by delivering specific combinations of gene-editing machinery. This process can be used for adding, deleting or correcting genes in a targeted fashion. Targeted DNA alteration happens naturally due to double-strand breaks (DSB) which induces a highly efficient recombination mechanism for repair. The two major mechanisms which are involved in DSB repair are homology-directed repair (HDR) and nonhomologous end-joining (NHEJ). HDR uses a template DNA to correct or repair and this mechanism can be used in targeted gene addition, replacement or inactivation. However, this is a very inefficient method and needs double-strand breaks to increase the gene alteration frequencies. On the other hand, NHEJ mediated repair tends to result in errors as it can efficiently insert or delete regions (indels) of DSB sites, usually causing gene inactivation. This method needs no repair matrix and occurs with higher frequency and is thus similar to RNA interference. It can be used for gene inactivation by creating a loss-of-function mutation leading to permanent gene inactivation.
In the early stages of development, zinc-finger nucleases (ZNFs) or meganucleases were used to induce DSBs at a particular DNA target site and engineer HDR. Subsequently, the use of a catalytic domain of bacterial transcription activator-like effectors (TALEs) from Flavobacterium okeanokites (FoKI) gave new programmable nucleases that can cleave any DNA at a specific site with higher frequencies. However, the major challenge with the TALEN approach was the design of complex molecular cloning for each new DNA target and the low efficacy of genome screening in successfully targeted cells. This was addressed in a new method of using clustered regularly interspaced short palindromic repeat (CRISPR)-associated 9 (Cas9) nuclease from bacterial adaptive immune defense system. This system uses the RNA-guided DNA cleavage module and has emerged as a potential alternative to ZNFs and TALENs. Now CRISPR/Cas9 has rapidly evolved into a method for modulating gene expression, ranging from genomic sequence correction or alteration to epigenetic and transcriptional changes. This has now brought forth an opportunity of using programmable nucleases for gene editing in clinical practice. The present use of this technology is in pre-clinical research on genome editing, primarily for viral infections, cardiovascular diseases, metabolic disorders, immune system defects, hemophilia, muscular dystrophy and T-cell based anticancer immunotherapies.
No off-target effects
Cardiovascular diseases (CVDs) are major causes of mortality and morbidity in the world and several types of CVDs are usually associated with a single genetic mutation or a combination of rare, inherited heterozygous mutations. Presently, clinical treatments are focused on the relief of the symptoms without addressing the underlying genetic defects. The development of in vivo CVD models using gene-editing tools has given an in-depth understanding of molecular mechanisms which can further help develop therapeutic modalities to control specific gene expressions and improve gene functions, thus improving clinical outcomes. As mentioned above, PCSK9 gene associated mutations are known to have a protective effect and it is also known that pharmacological inhibition of PCSK9 in vivo reduces LDL and cardiovascular events in subjects with elevated baseline risk. It was shown earlier (Ding et al. Circ Res. 2014) that when Cas9 and gRNA is targeted towards PCSK9 and delivered to mice using adenovirus vector, almost 50% of PCSK9 alleles in the liver were successfully edited resulting in loss-of-function of the gene. Most importantly, there were no off-target effects and a substantial reduction in the levels of plasma PCSK9 and total plasma cholesterol in the edited mice. Another similar study (Ran et al. Nature 2015) showed ~95% decrease in blood PCSK9 and a ~40% decrease in blood cholesterol levels without any major off-target effects. These proof-of-concept studies suggest that it might soon be possible to immunise patients against atherosclerotic cardiovascular disease by using somatic genome editing.
This attractive therapeutic approach can help in correcting several genetically driven diseases. However, there are a few technical challenges in translating these therapies into clinical practice — primarily in terms of accuracy, efficacy and delivery hurdles. The accuracy of gene editing is defined by its ability to edit the desired locus of interest and not cause undesired or off-target effects which are rather pernicious and can potentially lead to genomic toxicity, instability and disruption of normal functions or epigenetic alteration or some cases carcinogenesis. As these therapies depend on generating DSBs at the specific target sites, assays of identifying specificities need to be integrated such as next-generation sequencing (NGS) and whole-genome sequencing (WGS).