• by Bruce Carlson
  • May 23 2017

SHERLOCK System Brings Potential for CRISPR Gene-Editing in Diagnostics

SHERLOCK System Brings Potential for CRISPR Gene-Editing in Diagnostics

In the past few years, CRISPR technology has emerged as a revolutionary technology that is changing life science research and has the potential to dramatically change medicine.  CRISPR (clustered regularly interspaced short palindromic repeats) are DNA sequences that have a key role in a bacterial defense system, functioning as an immune system that protects bacteria and other microorganisms against viruses. Multiple CRISPR-Cas (CRISPR associated) systems have identified, each with unique Cas proteins.  Much of the focus has been on CRISPR-Cas9, but other CRISP-Cas systems are also being investigated.  CRISPR-Cas9 technology has rapidly becomean important gene editing technology used for a number of different applications including research tools (reagents), cell lines used for research, genetic knockout organisms, identification of novel therapeutic targets, and many other applications.  Until recently, these applications did not include diagnostics.  Drug development was the largest application field.   This is changing, with developments last month.  

CRISPR in Diagnostics: Large Potential

Genome editing is the process of modifying the DNA of a particular organism, by introducing breaks at specific, targeted locations in the genome. At the present time, ZFNs, TALENs and CRISPR-Cas9 are the most used and commercially developed technologies.   Kalorama Information's report on gene editing found $382 million dollar market for the technologies.   Growth could reach $2 billion in 5 years according to Kalorama's report.  These forecasts were made before diagnostic technologies were projected.  Molecular testing is already a 7 billion-dollar market with tremendous growth potential   The global market for gene editing technologies is a fast-evolving market in which innovation occurs rapidly and scientific knowledge is translated to commercial applications in fields such as biopharmaceuticals, agriculture, and industrial products. In particular, the discovery of the CRISPR-based gene editing technology has expanded and accelerated the adoption and use of gene editing tools in various fields. nstitute of Technology) and Harvard, Howard Hughes Medical Institute, and the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health published an article in Scienceentitled “Nucleic acid detection with CRISPR-Cas13a/C2c2.”  In this article, they describe a CRISPR-based diagnostic (CRISPR-Dx) which they called Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK).  This platform combines the Cas13a enzyme (which targets RNA) with isothermal amplification.  They authors used this platform to detect the Zika and Dengue virus, pathogenic bacteria, cell-free tumor DNA, and other genetic targets.

The lead author for this paper is Feng Zhang, Ph.D. (Broad Institute), one of the pioneers in CRISPR research.  The use of CRISPR technology as part of a diagnostic platform is another significant step in the CRISPR field.  However, Zhang’s laboratory is not the research group looking at the potential use of CRISPR technology for diagnostics.  For example, also in April 2017, researchers in South Korea published an article Epub ahead of print that describes the use of a CRISPR-mediated DNA FISH method for detection of MRSA (methicillin-resistant Staphylococcus aureus).  In situ hybridization represents the overwhelming majority of the molecular histology market and is used to detect disease or infection, determine risk for disease progression, and determine patient sensitivity to targeted therapeutics. The majority of the ISH products are related to cancer, cancer risk, and related proliferative disorders. Technologies used downstream of hybridization techniques such as flow cytometry, PCR, sequencing or mass spectrometry are not included. . Fluorescent in situ hybridization is used in both pre- and postnatal testing of genetic disorders as well as testing in adults. Prenatal testing is a major growth market available to FISH, but the technology has been undercut by non-invasive prenatal testing (NIPT) options such as microarray-based comparative genomic hybridization (aCGH) and NGS that do not require amniocentesis. 

At this time, the application of CRISPR technology to diagnostic testing appears to be in a relatively early stage of development.   However, the April 2017 article in Science demonstrates that CRISPR technology can be used for a broad range of applications, and the SHERLOCK platform may be an inexpensive platform that with the potential to transform areas of diagnostics.

Companies Involved in CRISPR

Sangamo Biosciences is the leader in the ZFN technology, having developed a pipeline of ZFP Therapeutics, which includes five clinical-stage drug candidates and multiple candidates in preclinical development. Sangamo has also licensed its ZFN platform to Dow AgroSciences and Sigma-Aldrich for the development of agricultural and research applications, respectively.  The first application of this technology for human therapeutics, SB-728-T, is a ZFN-modified autologous T-cell drug candidate currently undergoing Phase II clinical trials for the treatment of HIV/AIDS. In this therapeutic approach, ZFNs are delivered into T-cells to inactivate the gene encoding the CCR5 receptor in T-cells. CCR5 is the major co-receptor used by HIV to infect immune system cells.   Besides SB-728-T, the company is evaluating another ZFN-based compound for the treatment of HIV/AIDS, namely, the SB-728-HSCP compound, which is a CCR5-inactivated hematopoietic stem cell product currently undergoing Phase I clinical trials.  Sangamo Biosciences has also developed the In Vitro Protein Replacement Platform (IVPRP), which uses the albumin gene locus on liver cells to edit the genome in vivo with ZFNs, and insert desired therapeutic genes. As a result, the patient is able to produce the correct levels of the proteins deficient in certain diseases, after just a single treatment.
Based on this platform, the company has developed several therapeutic approaches for monogenic disorders, which are currently undergoing Phase I clinical trials.  

Cellectis has pioneered the allogeneic CAR-T cells therapy for use in immuno-oncology. In allogeneic therapies, T-cells are obtained from healthy donors, and not from the patient, which enables their manufacturing and use as off-the-shelf therapeutics. Cellectis’ UCARTs (Universal Chimeric Antigen Receptor T-cells) are engineered using a TALEN-based technology in which TALEN mRNAs are introduced into cells via the proprietary Pulse Agile electroporation technology.   The company’s lead product, UCART19, is an allogeneic TALEN-engineered T cell expressing anti-CD19 CAR. UCART19 was acquired in November 2015 by Servier; subsequently, Servier and Pfizer entered into a global development and commercialization collaboration to continue the clinical development of this drug candidate. UCART19 is currently undergoing a Phase I clinical study in the UK for the treatment of acute lymphoblastic leukemia.

In addition to these clinical stage compounds developed using gene editing technologies, various other companies, including Blue Bird Biotechnology, CRISPR Therapeutics, Editas Medicine, Intellia Therapeutics, Precision Biosciences, and Poseida Therapeutics are involved in numerous research programs that investigate the use of gene editing technologies for the development of human therapeutics.  BlueBird Biotechnologies is investigating the use of MegaTALs and homing endonucleases gene editing technologies in discovery research programs across various therapeutic areas, such as cancer and severe rare genetic disorders. In 2014, the company acquired Pregenen, a company that developed homing endonucleases and MegaTAL technologies using the LAGLIDADG family of homing endonucleases.  CRISPR Therapeutics is developing various in vivo and ex vivo CRISPR-engineered therapies for diseases such as hemophilia, severe combined immunodeficiency, cancer, cystic fibrosis, Duchenne muscular dystrophy, sickle cell disease, and beta thalassemia. The company expects that therapies for blood disorders will be the first to reach clinical trials by the end of 2017. CRISPR Therapeutics has also established research collaborations with Bayer and Vertex Pharmaceuticals.

Editas Medicine is involved in the development of in vivo and ex vivo CRISPR therapeutics for a variety of diseases, including cancer, eye diseases, non-malignant hematological diseases, cystic fibrosis, Duchenne muscular dystrophy, and antitrypsin deficiency. The company aims to initiate clinical trials in 2017 for its Leber Congenital Amaurosis type 10 (a genetic form of progressive blindness) drug candidate. Editas also collaborates with Juno Therapeutics to develop oncology CAR-T therapeutics engineered using Editas’ genome editing technologies.

Like Editas and CRISPR Therapeutics, Intellia Therapeutics is also pursuing the development of various in vivo and ex vivo CRISPR-engineered therapies. The company targets areas such as the liver disease transthyretin amyloidosis, antitrypsin deficiency, cancer, hepatitis B virus, hematopoietic stem cells, auto-immune and inflammatory diseases, and central nervous system. Intellia has established partnerships with Novartis and Regeneron Pharmaceuticals in areas such as immuno-oncology and transthyretin amyloidosis.  Precision Biosciences entered into a research collaboration with Baxalta Incorporated to develop allogeneic CAR-T therapies for various cancers using the ARCUS technology. The two companies plan to initiate clinical trials by the end of 2017.  Transposagen’s spin out company Poseida Therapeutics is currently developing in preclinical stages two CAR-T product candidates based on the Foot-print Free gene editing method, which combine CRISPR-Cas9 and the piggyBac transposase technology.