The importance of gene editing

In order to fully understand how the human genome impacts on human biology, researchers need to carry out experiments in living human cells where the effects of altering the genetic code can be readily observed. Thanks to a number of tools that enable the genome of cultured human cells to be edited at acceptable frequencies, these experiments are now within the grasp of any laboratory

Much information about the role of specific genes in fundamental biological processes and the onset and progression of genetic disease has been gleaned by gene targeting. In 2007, Capecchi, Evans and Smithies were awarded a Nobel Prize in Physiology or Medicine “for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells”. However, to truly understand a gene’s role in human biology and disease, human cells and tissue must be studied. This approach will enable researchers to observe the effects of a mutation, SNP or deletion in combination with the added layers of regulation present within the cell, including post-translational modification, epigenetic changes associated with chromatin structure and transcriptional mechanisms.

[caption id="attachment_30942" align="alignright" width="200" caption="Figure 1: A zinc finger nuclease consists of two sequence specific monomers which dimerise at the target sequence to induce a double stranded break in the DNA"][/caption]

Unfortunately, editing the genome using double-stranded DNA vectors in differentiated human cells has proved to be orders of magnitude less efficient than doing so in mouse embryonic stem cells. Disrupting genes by using nucleases has been fairly successful, and the ability of nuclease-induced double strand breaks to stimulate homologous recombination (HR) as part of a locus specific DNA repair event has also been investigated as a method of introducing modified sequence into genes. An alternative approach to gene editing has been to harness the ability of recombinant Adeno-Associated Virus (rAAV) to hijack a cell’s natural repair mechanisms. As rAAV increases the rates of high-fidelity HR within cells, this approach enables any endogenous gene sequence of a human or mammalian cell line to be altered quickly and reliably.

Using rAAV gene-editing tools to create human cell lines that harbour specific mutations present in patients will be critical to enable high-throughput and systematic target identification and confirmation. Such cell lines will become increasingly important as the trend towards ‘personalised’ medicine gains momentum. Nucleases that introduce double strand breaks into genomic DNA have been highly successful at disrupting the coding region of both alleles of a target gene, in order to create gene-knockouts. Upon induction of a double strand break a cell can invoke either HR, a high fidelity method of DNA repair, or non-homologous end joining (NHEJ), which is an error prone repair pathway. The repair enzymes involved in NHEJ frequently cause the introduction of minor deletions or additions at the break site, and this can be exploited to introduce disruptions in a gene’s coding sequence. Zinc-finger nucleases (ZFNs), which combine an adaptable, sequence specific zinc-finger DNA-recognition domain fused to a dimerisation-dependent nuclease have been particularly efficient in this regard, disrupting on average 3-5% of target genes in a bulk cell population. However, as any alterations in the gene sequence cannot be predicted in advance, the researcher must screen the resultant clones to identify which of these carry a desired mutation. A second problem, which is harder to deal with, is that off-target deletions or insertions elsewhere in the genome cannot be controlled for or readily defined. Furthermore, the design of ZFNs with high specificity has been challenging.

Introducing specific activating mutations into genes using nucleases is also theoretically possible, if a nuclease is co-delivered with a transgene construct highly homologous to the target gene. The success of this method relies on the cell invoking HR following the dsDNA break and using the vector as a template. Unfortunately, there are no reliable methods to influence whether HR or NHEJ is chosen as the repair mechanism, so again the researcher must screen resultant clones. In addition, whole genome sequencing would be required to ensure that no off-target gene insertions or deletions were present to confound the resulting genotype.

Other nucleases, in particular transcription activator-like nucleases (TALENs), are also being investigated as a possible tool for use in human gene editing. One potential advantage of these nucleases over ZFNs is that TALENs are almost completely modular and deterministic in their assembly, allowing a simple design approach. Early reports suggest that these nucleases are likely to have similar specificity to ZFNs, however, and as such problems with regard to off-target cutting will remain.

[caption id="attachment_30943" align="alignleft" width="200" caption="Figure 2: Gene modification using an rAAV vector which exploits homologous recombination"][/caption]

Recombinant AAV is a non-pathogenic single stranded DNA-virus that has a unique and powerful capability to induce HR at rates of around 1,000 times greater than seen using simple double stranded DNA vectors. rAAV vectors are engineered to contain a single stranded DNA ‘replacement’ genome that is substantially homologous to the target gene of interest and can therefore act as a template for HR

The alternative template DNA could contain any of the full range of genetic alterations (small or large gene deletions, point-mutations, reversion of mutations to wild-type, translocations, amplifications and transgene insertions), and these will be incorporated into the cell’s genome with unparalleled precision. As the vectors do not contain any viral genes, no viral genes will be co-inserted. Though there is some potential for random integration, this is minimal so most mutations are generated without introducing unwanted and confounding genotypes and/or phenotypes. Most importantly, as these mutations are created within endogenous genes, they are subject to the correct gene-regulatory mechanisms and accurately reflect the disease events found in real patients.

rAAV has been successfully used to target both transformed cell lines and primary human cells such as keratinocytes, fibroblasts, mesenchymal stem cells, embryonic stem cells and induced pluripotent stem cells¹. rAAV vectors can be constructed using simple PCR-based methods.

A current disadvantage of using rAAV over nucleases to disrupt a gene is that alleles are targeted sequentially, so targeting multiple alleles can take additional time. Further issues are that rAAV has a limited packaging capacity (around 4.7kb) and is unable to target non-dividing cells, though similar issues surround other gene editing methods. Horizon Discovery’s gene-engineering platform technology, GENESIS, which uses rAAV vectors is increasingly being recognised as the most precise and flexible genome editing technology available.

The ability to edit the human genome in such a targeted and specific way is naturally leading to the creation of cell lines containing specific mutations relevant to human diseases, cancer in particular. Although cell lines harbouring common cancer mutations are available for research, those with less common mutations often cannot be sourced. Even if cell lines harbouring desired mutations can be found, using these to understand a gene’s function in disease or to explore susceptibility to a specific treatment can be confounded by the fact that panels of unrelated cell lines differ genetically in thousands of ways.

[caption id="attachment_30944" align="alignright" width="200" caption="Figure 3: An experiment using isogenic cell lines to demonstrate the ability to discriminate between sensitivity and resistance of particular mutations to treatment with a drug"][/caption]

Isogenic disease models, cell-line pairs that share the same genetic background except at a specific locus modified by gene-editing, will be invaluable to definitively study disease biology and profile candidate drugs against specific biomarkers throughout the entire discovery process. Systematically creating cell lines containing those mutations, amplifications and translocations identified in cancer patients will help to define which genes are actually true disease drivers rather than just random noise in genetically unstable tumours, and thus identify key targets for therapeutic intervention. In the long term these cell lines will support the generation of novel drugs tailored to those patients that harbour a given molecular target.

As increasing amounts of genomic data becomes available to researchers worldwide it is more important than ever to utilise this information in a practical manner. The possibility of readily modifying the human genome within a wide range of cell types to probe how genes affect biology is now a reality, thanks to new gene-editing techniques. By developing a broad range of human disease cell models that faithfully recapitulate predisposing or pathogenic genetic variations (SNPs and mutations) we could vastly improve our understanding of human disease. These gene-editing tools could also support the execution of a Translational Genome Project, a natural successor to the Human Genome Project and ENCODE Project.

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The authors: Eric Rhodes, CTO and Chris Torrance, CSO, Horizon Discovery Ltd