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Genome Editing in Drug Discovery

Steve Rees

Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK

1.1 Introduction

The last decade has seen the development of an unprecedented number of new technologies that are being applied to transform our understanding of disease and the subsequent success of drug discovery. The dramatic advances in DNA sequencing technology and the more recent advances in other-omics technologies including transcriptomics, proteomics, and metabolomics are enabling the understanding of disease at the genetic and cellular level to identify new drug targets, and to identify new disease biomarkers to enable disease segmentation and patient stratification in the clinic. Advances in stem cell technology together with technologies that enable the creation of tissue organoids in the laboratory are allowing the creation of complex models of disease (Lancaster et al. 2013), which together with advances in imaging technology are enabling the drug discovery scientist to better understand the efficacy and safety of potential medicines in preclinical studies. The huge increases in computational power, together with advances in Artificial Intelligence and Machine Learning are allowing drug discovery scientists to extract greater knowledge from these large-omics datasets, to improve the speed and quality of chemistry design and enable the design of improved clinical studies (Vamathevan et al. 2019). Perhaps, the most impactful of the many new technologies applied in drug discovery in the last decade has been the rapid adoption of CRISPR/Cas9 throughout the drug discovery pipeline to create engineered cellular and animal models of disease to enable the study of the role of new drug targets in disease, alongside the development of CRISPR as a medicine in it's own right or as a key tool in the creation of cell therapy medicines. Taken together, these and other new technologies have impacted every drug discovery program to enable a better understanding of the role of the drug target in disease and the design of molecules more likely to be safe and efficacious in the clinic. Alongside this, a number of new therapeutic modalities are entering the clinic including antisense onligonucleotide, mRNA, protein, and gene and cell therapies which are leading to a situation where every target becomes amenable to therapeutic manipulation. Taken together, the ability of these technologies to improve our understanding of disease, to create safer medicines and to target those medicines to the patient population most likely to benefit from them is leading to an increase in the success of drug discovery. This has been seen in an increase in the number of new molecular entities approved by the FDA with over 40 new medicines being approved each year between 2011 and 2020 compared with an average of around 20 new medicines approved each year between 2001 and 2010 (Batta et al. 2020). A number of reports also describe an increase in success of drug discovery including a recent publication from colleagues in AstraZeneca. Through the implementation of a new research strategy at AstraZeneca in 2010, success from Candidate Selection to product launch has increased from 4% to 20% while 3 projects are now started in early discovery to deliver a Candidate Drug compared with 5 projects in earlier years (Morgan et al. 2018). While this represents a huge increase in drug discovery productivity, it remains the case that the majority of projects fail with the primary cause of failure in research being due to target validation and in the clinic a lack of efficacy in Phase II clinical studies. In both cases, the root cause of failure is that the hypothesis linking the drug target to disease was incorrect and significant efforts are underway in both academia and industry to continue to increase the level of confidence the drug target at the start of, and throughout a drug discovery program to further increase drug discovery success in all therapeutic areas. Throughout this book, authors will present examples of the application of CRISPR/Cas9 to identify novel drug targets, to understand the role of these targets in disease, and to create cellular and animal model systems to allow the development of new medicines more likely to succeed in the clinic. While we remain within the first decade following the discovery of the ability of CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 systems for the precise editing of mammalian genomes (Jinek et al. 2012; Cong et al. 2013; Mali et al. 2013), this technology has become embedded throughout drug discovery research (Fellmann et al. 2017). Throughout this book, authors will discuss the current use of CRISPR/Cas9 to facilitate the development of new medicines, as a medicine in its own right, and as a highly sensitive point of care diagnostic. However, we remain in the infancy of the application of this technology, and its potential to transform our understanding and treatment of disease remains huge.

1.2 Genome Engineering

Genome engineering describes the specific introduction of new genetic elements into target genes to mediate either a disruption of the gene sequence, and consequent loss of gene expression, or a change in the protein sequence of the gene being transcribed. The ability to modify gene sequences at precise locations in the genome arose in the 1990s with the development of meganucleases and Zinc Finger Nuclease (ZFN) technology (Kim et al. 1996; Bibikova et al. 2001). ZFNs are synthetic restriction enzymes created by fusing one or more zinc finger DNA-binding domains, engineered to target a specific DNA sequence, to a DNA nuclease, typically the restriction enzyme Fok1. ZFNs are able to recognize and cut a specific site in the genome to create a double-stranded DNA break that can be repaired by non-homologous end joining (NHEJ) or Homology Directed Repair (HDR) to result in gene inactivation or gene repair following the introduction of a DNA repair template. A boost to the technology came in 2009 with the discovery of transcription activator-like effector nucleases (TALENs) (Boch et al. 2009; Moscou and Bogdanove 2009). TALENs are synthetic restriction enzymes engineered to cut specific sequences of DNA. These are created by fusing a TAL effector DNA-binding domain designed to recognize the target DNA sequence with a nuclease to mediate DNA cleavage. TALEN constructs can be introduced into cells to cut the DNA at specific sequences to create a double-stranded DNA break, that following repair by NHEJ, results in the inactivation of the target gene as a consequence of the introduction of additional DNA sequences as part of the repair process. Furthermore, TALENs can be used to change specific nucleotides within a gene, or to introduce new sequences into the genome following transfection of cells with a TALEN's construct and a DNA repair template. Following HDR at the DNA cut site, the new sequence, encoded by the DNA repair template, can be introduced into the genome, albeit at a low editing efficiency. ZFNs and TALENs technologies have been used extensively in genome engineering projects in medical research and to modify plant genomes. Furthermore, Zinc Finger technology has been used by Sangamo Therapeutics and others to create genome editing medicines. Treatments for a range of diseases are in development with the most advanced project, a treatment for hemophilia currently in Phase 3 clinical studies. However, the widespread adoption of these technologies has been limited due to the requirement for expertise in protein engineering to create a ZFN or TALEN that precisely targets a specific DNA sequence, the relatively low editing efficiencies observed, and the potential for editing at multiple sites in the genome.

1.3 CRISPR/Cas9

The seminal publications in 2013 describing the ability of CRISPR/Cas9 to edit mammalian genomes have led to an explosion in the ability to make precise genetic changes within mammalian cells and animal models using a variety of CRISPR-based editing methods (Cong et al. 2013; Mali et al. 2013). Since these publications, there has been a dramatic increase in the efficiency and variety of genome editing methods available with these methods now in widespread use in the pharmaceutical industry and academia for target and drug discovery, alongside the discovery and characterization of a number of new editing enzymes and the development of forms of Cas9 with improved editing activity (Gilbert et al. 2014; Kampmann 2018).

CRISPR/Cas is part of the bacterial immune response system where its natural role is to recognize and destroy non-host nucleic acid sequences as part of the host immune defense system (Wiedenheft et al. 2012). The commonly used laboratory CRISPR system uses the Cas9 nuclease cloned from Streptococcus pyrogenes (SpCas9) although additional Cas9 enzymes have been cloned and characterized (Acharya et al. 2019). In contrast to TALENs and Zinc Finger technology, CRISPR/Cas9 is simple to use in any laboratory. It does not require protein engineering to create a nuclease able to recognize a specific site within the genome, rather targeting of the Cas9 nuclease to specific sites within the genome is mediated through the design of a specific synthetic guide RNA (sgRNA), complementary in sequence to the region of the genome to be targeted, that positions Cas9 at the target site in the genome to result in the creation of a double-stranded DNA break. Guide...