Rolf Turk, Senior Manager, Genomics Medicine at Integrated DNA Technologies, examines how CRISPR is being used to enhance cancer therapies.
Since its discovery in 2012, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology has revolutionised the biomedicine and cell and gene therapy fields, providing a versatile tool for precise and efficient genome editing. Years of development and fast-paced research have continued to unlock its potential, expanding how CRISPR can be used to treat, detect, and prevent common diseases such as cancer and Covid-19.
For example, merging CRISPR with oncotherapies has resulted in more efficient and specific tumour-targeting capabilities of Chimeric Antigen Receptor (CAR) T-based treatments for cancer. These efforts, along with many others, are opening doors for new possibilities in translational medicine.
Better, faster, stronger: targeting cancer with precision by CRISPR-enhanced CAR T therapies
Cancer is a global public health crisis that demands powerful, innovative solutions in the battle against it. CAR T cells represent a breakthrough in personalised cancer therapy, but T cell exhaustion, susceptibility to inhibitory pathways, and cytokine-related toxicities remain bottlenecks in this immunotherapy1.
Current research is focused on designing CRISPR-enhanced CAR T cells that are resistant to immunosuppressive cytokines such as transforming growth factor-β (TGF-β)2, programmed cell death protein (PD-1)3, or other negative T cell regulators4 (CTLA-4, LAG-3, and TIM-3), thus improving anti-tumour functions.
Another strategy involves using CRISPR technology to insert cytokine-encoding DNA cassettes into specific genomic locations, temporally modulating cytokines (GM-CSF and IL-6) associated with neurotoxicity and cytokine release syndrome (CRS) during CAR T cell activation to potentiate T cell persistence and reduce toxicity.
CRISPR also addresses another limitation in CAR T therapies related to the insufficient quantity and poor quality of autologous T cells. This includes exploring CRISPR-Cas9 technology to construct allogenic universal CAR T cells, which could become the primary direction for future development. Current limitations in the development of ‘off-the- shelf’ CAR T cells from healthy donors include graft-versus-host disease (GVHD) and rejection of infused allogeneic T cells. This occurs when endogenous αβ T cell receptors (TCRs) on transferred donor lymphocytes recognise alloantigens in HLA-mismatched recipients. CRISPR technology has been used to knock down TCR, TRAC, HLA-I, B2M, and CD52 to generate without compromising CAR-mediated cytotoxicity.
Another approach to address the limited availability of CAR T therapies consists of using CRISPR technology to generate CAR T-cell therapeutics derived from inducible pluripotent stem cells (iPSCs)5. The technology can stably and reliably manipulate genes, knocking down factors such as endogenous TCR and major histocompatibility complex (MHC), creating distinct pools of allogenic T cell products with enhanced anti-tumour functionality.
The CRISPR-Cas system has also advanced CAR T therapy by enabling high-throughput screening for neoantigens and identifying novel therapeutic targets. Neoantigens, characterised by their strong immunogenicity and tumour specificity, are gaining traction as potential targets for tailored cancer immunotherapies6, prospective predictors for tumour survival prognosis, and immune checkpoint blockade responses. Incorporating in vivo pooled CRISPR screens has allowed for the interrogation of thousands of genetic perturbations in cells, leading to the determination of the involvement of molecules (PTPN2, ADAR1, or DHX37) in immune checkpoint inhibitor (ICI) responsiveness and T cell activation. Additionally, the CRISPR-Cas system can be used to create TCR T cells that can specifically recognise these neoantigens, expanding its applications beyond traditional CAR T therapy. This can be achieved by the simultaneous knockout of endogenous TCR genes and the insertion of neoantigen-specific TCRs allowing cells to recognise specific mutations.
CRISPR technology holds significant potential for advancing CAR T cell therapies for cancer by designing less toxic and more organ-specific therapies. Although adeno- associated viruses (AAVs) have been widely used for delivering CRISPR components, they can induce off-target mutagenesis and provoke immunity against the viral vector. Consequently, the CRISPR delivery system has undergone continuous refinement, incorporating non- viral approaches and modifying lipid nanoparticles (LNPs) or cell-penetrating peptides to enhance the safety and effective non-viral delivery of T cells.
For instance, virus-free approaches have been applied using CRISPR-Cas mediated homology-directed repair (HDR) to efficiently insert CARs into the TRAC locus of primary human T cells7, thereby improving their functionality. CRISPR-Cas9 technology has also been used to introduce anti-CD19 CAR cassettes into the AAVS1 safe-harbour locus to boost the killing abilities of T cells against aggressive B cell non-Hodgkin lymphoma. Another method relies on incorporating accessible plasmid-based donor templates, combined with high-fidelity Cas9-RNPs, which are introduced into primary human T cells via electroporation. This approach achieves knock-in efficiencies at multiple loci (singly or in combination) comparable to AAV-based methods.
These methods offer safer CAR T-based therapies, laying the foundation for clinical trials and the rapid development of a new generation of CAR T cells for both autologous and allogeneic use. CRISPR technology has been transforming cancer treatment by providing cytokine- resistant CAR T cell therapies, overcoming GVHD, and identifying neoantigens, thus making these therapies better, faster, and stronger, as well as safer and less immunogenic.
CRISPR-powered diagnostics: nucleic-acid based testing for diseases and future outbreaks
The Covid-19 pandemic, which affected over 660 million people worldwide, has unveiled the potential of CRISPR technology in detecting nucleic acid- based biomarkers of common infectious diseases7. Nucleic acid-based diagnostics, which typically require PCR reagents and laboratory equipment, are crucial for identifying, treating, and preventing common infectious diseases. CRISPR diagnostics have successfully detected various pathogens, including RNA viruses (eg. parvovirus B19, Flaviviridae, Ebola, and Coronaviridae), DNA viruses (e.g., Herpesviridae, Polyomaviridae, and Papillomaviridae), bacteria (e.g., Mycobacterium tuberculosis, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, and Salmonella enteritidis), and parasites like Plasmodium species that cause malaria. In the context of non- infectious diseases, CRISPR diagnostics have been developed to detect RNA species, such as human CXCL9 mRNA, for kidney transplant rejection.
While type II (Cas9) is mainly used for gene-editing applications, CRISPR detecting tools primarily rely on type V (Cas12) and type VI (Cas13) collateral activity. These enzymes cleave untargeted single-stranded DNA (ssDNA; Cas12) or single-stranded RNA (ssRNA; Cas13)
in solution, enabling nucleic acid sensing, signal amplification, and a variety of readouts through the incorporation of functionalised reporter nucleic acids that are cleaved by collateral activity.
In some aspects, Cas12 and Cas13 are considered superior to Cas9. Cas12 protein creates staggered double-strand breaks (DSBs) and promotes the HDR mechanism instead of utilizing both non-homologous ends joining (NHEJ) and HDR. Although wild-type Cas12a has variable editing efficiency, novel Cas12a mutants have high levels of editing comparable to Cas9. Furthermore, the modular single- stranded structure of Cas13 enables significant scalability and allows for the production of various guide RNA libraries. It also enables the downregulation of gene expression by directly knocking out the cytoplasmic mRNA transcripts. Finally, the recently discovered mutants, dCas13 and Cas13x, efficiently target different effectors to specific RNAs to induce specific mutations.
The evolution of diagnostics for nucleic acid detection employing CRISPR with endonucleases Cas12 and Cas13 has led to the development of various detection systems, such as STOP, CASdetect, SHERLOCK, CREST, SHINE, CARVER, CONAN, and FELUDA. These systems have been used to detect SARS-CoV-2, allowing for rapid, sensitive, and specific diagnoses. Overall, the Covid-19 pandemic has propelled the use of CRISPR to detect nucleic acid-based biomarkers of common infectious diseases, offering rapid and accessible diagnostic tools.
The future of CRISPR
Despite the rapid acceleration of CRISPR, several limitations must be addressed to fully harness the technology from bench to bedside. One limitation is the possibility of off-target effects associated with creating double-strand breaks (DSBs). To address these issues, scientists are developing alternative CRISPR-based approaches that avoid DSBs, such as base editing and prime editing. Base editors, cytosine base editors (CBEs), and adenine base editors (ABEs) enable the precise introduction of targeted point mutations without the need for DSBs or donor DNA templates. Another approach to mitigate DSBs consists of developing high- fidelity Cas9 variants (HiFi Cas9), which limit off-target effects while maintaining high on-target activity, offering a promising solution for therapeutic genome- editing applications.
Similarly, prime editors avoid the creation of DSBs by using a catalytically-impaired Cas9 protein fused to a reverse transcriptase enzyme and a specially designed prime editing guide RNA (pegRNA). This system allows for precisely installing various genetic modifications, including insertions, deletions, and all 12 possible base-to-base conversions – without creating DSBs. Finally, to reduce risks of off-target effects and avoid the formation of DSBs, there is a strong inclination towards comprehensive mining and characterisation of new nucleases and the development of proprietary nucleases.
There are also challenges with efficiently delivering CRISPR components into specific cells without causing toxic effects, particularly in vivo. The most common methods use viral vectors such as lentiviruses, AAVs, or non-viral lipid-based nanoparticles (LNPs). While AAVs show limited packaging capacity and may be costly and time-consuming, the delivery of LNPs to tissues other than the liver is particularly complicated. Alternative approaches have been sought to overcome these limitations, including alternative nanocarriers, such as polymeric nanoparticles or exosomes, or physical methods, including electroporation, microinjection, and hydrodynamic injection.
To achieve cell-specific delivery, many systems rely on using cell-specific DNA aptamers (e.g., AS1411), which can be synthesized and screened against a range of targets. Another approach consists of using selective organ targeting (SORT), which incorporates changes in the molar compositions of lipid nanoparticle carriers with an additional ‘SORT’ molecule to alter their internal charge and achieve cell-specific delivery. This system has been reported to be compatible with CRISPR-Cas mRNA/gRNA and RNP complexes. Another promising approach consists of conjugating antibodies, such as an anti-CD38, to the LNPs for targeted delivery into specific tissues. The system shows potential as a therapy against multiple melanomas.
Although we have seen accelerated development and application of CRISPR in life sciences, further advancements in this technology may resemble more of a marathon than a sprint. Clinicians, patients, policymakers, regulatory agencies, and industries will need to work together closely to address the most relevant issues, from minimising off-target effects and implementing precise delivery methods, to make CRISPR accessible for the treatment, detection, and prevention of common diseases.
DDW Volume 24 – Issue 3, Summer 2023
References
- https://pubmed.ncbi.nlm.nih. gov/36193597/
- https://academic.oup.com/bfg/ article/19/3/175/5707552
- https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC7227242/
- https://pubmed.ncbi.nlm.nih. gov/35303871/
- https://pubmed.ncbi.nlm.nih. gov/31243643/
- https://pubmed.ncbi.nlm.nih. gov/36604431/
- https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC8787289/
About the author:
Rolf Turk is Senior Manager, Genomics Medicine at Integrated DNA Technologies, a genomics solutions provider whose mission is to accelerate the pace of genomics.
