Harnessing the potential of gene editing technology using CRlSPR in inflammatory bowel disease

Viktor Limanskiy, Arpita Vyas, Lakshmi Shankar Chaturvedi, Dinesh Vyas

Abstrac t The molecular scalpel of clustered regularly interspersed short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) technology may be sharp enough to begin cutting the genes implicated in inflammatory bowel disease(IBD) and consequently decrease the 6.3 billion dollar annual financial healthcare burden in the treatment of IBD. For the past few years CRISPR technology has drastically revolutionized DNA engineering and biomedical research field. We are beginning to see its application in gene manipulation of sickle cell disease,human immunodeficiency virus resistant embryologic twin gene modification and IBD genes such as Gatm (Glycine amidinotransferase, mitochondrial),nucleotide-binding oligomerization domain-containing protein 2, KRT12 and other genes implicated in adaptive immune convergence pathways have been subjected to gene editing, however there are very few publications. Furthermore,since Crohn's disease and ulcerative colitis have shared disease susceptibility and share genetic gene profile, it is paramount and is more advantageous to use CRISPR technology to maximize impact. Although, currently CRISPR does have its limitations due to limited number of specific Cas enzymes, off-target activity,protospacer adjacent motifs and crossfire between different target sites. However,these limitations have given researchers further insight on how to augment and manipulate enzymes to enable precise gene excision and limit crossfire between target sites.

Key words: Clustered regularly interspersed short palindromic repeats; Inflammatory bowel disease; Crohn's disease; Ulcerative colitis; Gene excision; Gene editing; Gene therapy; Financial impact of inflammatory bowel disease on healthcare; Clustered regularly interspersed short palindromic repeats crossfire

INTRODUCTION

Clustered regularly intersp ersed short p alind romic rep eats/CRISPR-associated protein 9 (CRISPR/Cas9) technology has drastically revolutionized DNA engineering and biomed ical research in the past few years. It has enabled researchers to model disease and advance therapeutic intervention techniques in a pragmatic and clinically efficacious w ay. The Figure 1 below d emonstrates the exp onential grow th in publications and interest in CRISPR technology over the past few years. Abreast is Figure 2 d emonstrating use of CRISPR technology in inflammatory bow el d isease(IBD). Strong interest in CRISPR has driven gene therapy publications dow n in the past few years as seen in Figure 3. Application of CRISPR is gaining momentum and is seen in sickle cell disease gene modification of hematopoietic stem cells, embryonic stem cells, gene-based therapy for HIV-1 infected individuals and other disease gene modification[1-6]. It is a new era in biomedical research for use of CRISPR technology in IBD (Figure 4). These advancements in biomedical research w ith the use of CRISPR w ill enable researchers to begin treating IBD and other gene mod ifying d iseases,thereby relieving the healthcare financial burden of IBD.

IBD affects an estimated 1.5 million people in North America[7,8]. These p atients have considerable morbidity and the associated financial burden on healthcare system is estimated to be 6.3 billion d ollars annually w ith tw o third s of that d ue to hospitalizations and pharmaceutical therapy alone[9-11]. This does not take into account the financial implications of work and disability of patients and impact on daily life.Εven though gene therap y remains exp ensive in any d isease treatment d ue to pharmaceutical patents, consid erable thought should be taken to propel CRISPR use in IBD because the use of this novel technology is less taxing than previously used epigenetic modification[12].

Prior to the d iscovery of CRISPR/Cas9 enzyme, RNA sequencing[7,8], mod ular protein recognition DNA such as zinc finger nuclease or TAL effector nuclease w ere used in epigenetic modification and disease modeling, which are complex and require very sp ecific p rotein engineering and p rogramming[13]. How ever, w ith the revolutionary CRISPR technology, researchers now may ed it genomes in a much simpler and precise way as modeled by bacteria[14,15].

CRISPR/Cas9 was found in Streptococcus pyogenes, which uses this mechanism as defense against invading viruses[14]. Upon infection by a virus the bacteria stores the viral DNA sequence in betw een sections of regularly intersp aced p alind romic rep eated segments that are closely associated w ith genes that encod e CRISPRassociated p roteins. In ord er for the system to confer und erlying efficacy and sp ecificity, the CRISPR and the viral DNA is converted to tw o RNA moieties:Tracr RNA and cr RNA, the tracr RNA confers enzymatic activity and the cr RNA determines substrate specificity[13,16]. Once the moieties bind together, they traverse the cell seeking any genetic material that matches the cr RNA. Upon sequence matching,the tracr RNA part of Cas enzyme clips the target DNA in specific nucleotide bases disabling its replication. This incredibly precise system can be utilized in therapeutic ad vances and programed to target any sequence in the cell. This novel mechanism will be assessed for potential application and targeting of genes implicated in IBD.

Figure 1 Number of CRlSPR publications by year.

CRISPR DNA engineering has ad vanced quickly and is evolving rap id ly. It has already been used in diseases such as sickle cell, beta-thalassemia or HIV resistant human embryos[1,2,6]. Given its exponential application in disease variants, it may suggest that it's an optimal time to further our understanding of IBD pathogenesis and treatment. Primarily IBD refers to two major categories of chronic relapsing inflammatory intestinal disorders: Ulcerative colitis (UC) and Crohn's disease (CD).With the ad vent of genetic research both diseases have seen notable success culminating in the discovery of over 160 susceptible genes, among w hich, many potentially may be targeted by the CRISPR technology[17,18]. Around one-third of these loci/genes described, confer susceptibility to both CD and UC, which may make targeting and gene editing a viable option[18,19]. The genetic architecture of IBD has shed much light on central themes in IBD and the level of cellular process that the pathogenesis emerges. A putative CD-susceptibility locus has been mapped to chromosome 16 around locus D16S409 and D16S419 which may shed more light etiology of IBD[20]. In CD a common genetic theme is seen in defective processing of intracellular bacteria, autophagy and innate immunity. In UC, genetic evidence demonstrates genes that are responsible for proper barrier function are important in preventing UC. However, when analyzing genetic data in more detail, CD and UC have shared d isease susceptibility and shared genetic gene profile that may be targeted[18]. Table 1 demonstrates common genes implicated and their strength of role in IBD[18].

Recent research has identified and linked a gene, Gatm, to IBD using CRISPR/Cas9 gene-editing technology. When the Gatm gene is activated, it induces synthesis of creatine which helps in intestinal mucosal barrier, which helps protect the intestinal wall against inflammation that's caused by bacteria[21]. When inducing a frame-shift mutation in the Gatm gene via CRISPR/Cas9 there w as signs of inflammatory response in the intestinal wall. This demonstrated its important role in mucosal barrier protection and potential manipulation of this gene using CRISPR. Protection from inflammation by an intact barrier is vital to decrease immune response, which is suppressed or increased by enhancers in the immune activation cascade. In the inflammatory response pathway, IL2RA plays a role in signaling T cells to hamper or increase the response. If the enhancers that switch on IL2RA are defective the T cells won't suppress inflammation and chronic inflammation is associated with 15%-20%of all human malignancies[8,22]. Inflammation also results in autoimmune disorders such as IBD and inflammatory-induced colon cancer mediated by NF-k B pathway[22-24].Previously it has been demonstrated that single nucleotide polymorphisms -SNPs'mutation of IL2RA leads to improper activation of T cells and subsequently resulting in autoimmune disord ers. These SNP's may be targeted by CRISPR/Cas9 and repaired with non-homologous end-joining repair. This has been demonstrated in KRT12 mutations-specific targeting of SNP's as well[25]. Recent advancements in CRISPR/Cas9 specificity and potency of targeted genes demonstrate that SNP's or genes that have point mutations may be targeted and editing may be attempted.

Figure 2 Publications on inflammatory bowel disease using CRlSPR.

POTENTIAL THERAPEUTIC TIMING

CD and UC in general have three stages of d isease p rogression, mild-mod erate;mod erate-severe; and severe. Currently there are no stud ies ind icating potential therap eutic timing w hen to target affected genes using CRISPR in IBD, how ever several multicenter trials conducted administering human recombinant IL-10 during active mild/mod erate stage of CD or d uring refractory CD as w ell as p atients undergoing curative ileal or ileocolonic resection[26,27]. How ever, results did not show significant clinical improvement or higher remission rates secondary to too low IL-10 dose and ad verse effects of med ication. In addition, IL-10 alone failed to effectively supp ress variety of d ysregulated pro-inflammatory cytokines[26,27]. In later stages of d isease p rocess, significant d ysregulation of p ro-inflammatory cytokines and red und ant p athw ays occur, such as NF-k B receiving activation from d ifferent pathways[28]thus single target impact is futile. Given that CRISPR can simultaneously multiplex several genes, it w ill aid researchers to d evise approp riate intervention timing[29]. We also suggest early intervention is optimal to prevent progression of disease and reduce complications. It is imperative to cond uct studies to best identify role of CRISPR in various stages of disease.

CURRENT STATUS

Currently, CRISPR is applied in many fields of scientific study. In biotechnology it is used to mod ify Maize genome in protop lasts. In d rug d evelopment, it is used to understand mod es of drug resistance and drug-target interactions. In epigenetics, it has taken the p lace of zinc finger nuclease and TALΕN in ep igenetic mod ification because the ind el frequency is more superior[6]. Since the CRISPR debut, researchers are improving and enhancing the specificity and accuracy of the Cas9. Currently the Cas9 not only cuts the DNA, but can be altered to perform d esired functions. The Cas9 protein has a deaminase region that may be altered to increase highly specific alternation of genome sequence, w hich w ill allow for broader sp ecific DNA bases manipulation[13]. It can also promote gene transcription using enzyme by deactivating the endonuclease activity and add transcriptional activator to increase transcription.The Cas9 can silence domains that recruit factors so that genes are blocked and they are not transcribed. In general targeting studies, Cas9 can be tagged with fluorescent dye so genes can be follow ed. Furthermore, Cas9 can be multiplexed w ith multiple guide RNAs to generate multiple breaks in order to cut out large sequences of DNA in one experiment[29]. This limits time and repetitiveness of experiments conducted and time is of an essence in this race to invent even-more versatile or efficient variations of this p ow erful enzyme, w hich greatly simplifies the ed iting of DNA. Furthermore,very recently successful attempts w ere made to edit CCR5 gene in human embryos to enable resistance to HIV[30]. Although, ethics and imp lications of such stud ies are currently w id ely d ebated. Also, recently KRAS oncogenic alleles w ere mod ified leading to decreased cancer cell growth w ithout disturbing w ild type alleles[31]. Since major oncogenic mutations occur on cod on-12 of KRAS exon-2, d irect targeting of oncogenic KRAS single-nucleotid e missense substitution c.35G＞T mutation using CRISPR/Cas9 system inhibited cancer cell grow th and is dependent on efficient target cell transduction[31].

Figure 3 Total publications using gene therapy.

LIMITATIONS

The excitement generated by the new CRISPR technology in the science community is reflected by exponential publications and application in various fields of study,including agriculture, drug development and epigenetic control but its revolutionary genome editing method is far from perfect. The CRISPR is an RNA based DNA recognition system. It is d ependent on a guide molecule composed of RNA to recognize a sequence in the DNA that has specific molecular features. The problem is that the original Cas9 can only land on genome segments that have a trio of ‘NGG' (N being any nucleotid e) nucleotide base pairs and cut only limited fraction of the genome. The human genome contains 3.2 billion-bases and only one-sixteenth of the genome where Cas9 can land. This limits the specific target genes of interest and leads to “off-target” mutations however to limit these effects a highly specific guide RNA sequence is selected[15,32]. Also, recently researchers modified the enzyme and developed an xCas9s that has a broader range of three-base landing pads, referred to as protospacer adjacent motifs or PAMs. It works best with NGN sequence, which occurs in one-fourth of the genome. It allows for researches to perform gene knockout studies, which help them, determine what gene is implicated in disease. However,since majority of diseases are associated with “point mutations”, it is difficult to target and repair a mutated gene. Not only that, once the enzyme clips the desired DNA gene, the repair mechanism of the cell is wobbly and during repairs it tends to insert or delete DNA bases. Furthermore, Cas9 enzyme only cuts DNA and only 2% of the genome codes directly from DNA to protein and 98% of genome is regulatory gene sequences. This poses a challenge for precisely modifying RNA. Although, very recently a new enzyme w as characterized called Cp f1, w hich w as found in Staphylococcus aureus, and is capable of cleaving both DNA and RNA[33]. This will allow targeting of RNA gain-of-function mutations such as NOD2 or other mutations that can be edited. Furthermore, Cpf1 is also smaller which makes transfection much easier[33].

Although some of these limitations of the Cas9 are quickly becoming insightful opportunities and researchers are drastically improving it by altering its capabilities.The new enzymes are more precise than the original Cas9 and now there is xCas9,d Cas9 and d Cas13 w hich are capable of editing specific base pairs. For example,d Cas13 can convert base A to I in RNA and I is a universal base[34]. Such manipulation of bases is a very ap pealing target for therapies, p articularly inflammation.Furthermore, because RNA is around in the cell for a short period of time before it is degraded repeated administration of RNA base editors would need to be given[34].Also, sequencing RNA is problematic and laborious[35]. On the other hand this may seem disadvantageous but w orking with RNA may limit some off target genome mutations. In addition to off target genome mutations, crossfire between different cells may occur. Intended target may be either gain of function mutation or loss of function mutation, either way, altering alleles may have their own detrimental effect.For example, as indicated in Figure 5, NOD2 loss of function leads to Crohn's and gain of function causes end othelial to mesenchymal transition of glomerular endothelial cells causing d iabetic nephropathy[36]. How ever, some of these cross reactions may not be as detrimental. In Figure 6, altering ATG16L1 allele to T300A ATG16L1 only incases risk for CD type, but not disease onset[37]. Figures 5-7 below demonstrate positive and negative effects of altering main genes implicated in IBD;NOD2, STAT3 ATG16L1, IL23R genes using CRISPR technology[37-41].

Figure 4 Publications on inflammatory bowel disease using gene therapy.

CONCLUSION

Desp ite CRISPR/Cas technology limitations, as new innovative techniques such as anti-CRISPR proteins and new Cas proteins are developed to advance precise DNA editing, application of this revolutionary mechanism is at its prime time to hone in on genes implicated in IBD. Implementation of CRISPR in IBD research w ill lead to better outcomes and may decrease financial burden on the health care system.

Table 1 Genes to target for optimal impact using CRlSPR

Figure 5 Positive and negative effects of CRlSPR when targeting NOD2 gene.

Figure 6 Positive and negative effects of CRlSPR when targeting gene ATG16L1. UC: Ulcerative colitis; CRC: Colorectal cancer.

Figure 7 Positive and negative effects of CRlSPR when targeting STAT3 and lL23R genes. UC: Ulcerative colitis.