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CRISPR/Cas9 — A molecular ‘scissors’ capable of cutting DNA. Image taken from Jackson Ryan,

Beyond CRISPR/Cas9 Gene Editing — What’s next for Gene Editing-based Therapeutic Approaches?

Intellia Therapeutics announced its first data on the 26th of June demonstrating the safety and in vivo efficacy of its CRISPR/Cas9-based drug candidate NTLA-2001 for Hereditary Transthyretin Amyloidosis (hATTR).

On 26th June 2021, Intellia Therapeutics became the first company to demonstrate in vivo safety and efficacy of its CRISPR/Cas9-based drug candidate NTLA-2001 for Hereditary Transthyretin Amyloidosis (hATTR) in a Phase I clinical trial¹. hATTR is a rare disease caused by mutations in the gene encoding for transthyretin (TTR), resulting in the dysfunction of various organs and tissues such as the heart and the nerves². NTLA-2001 exhibited dose-dependent serum TTR reduction, with percentages ranging from 80–96% reduction in the high dosage group (0.3mg/kg)³. Furthermore, the treatment was generally well-tolerated, with no serious adverse effects (AE) being noted³. However, any potential off-target edits in the patients remains to be seen, although no detectable edits were detected in vitro with the pharmacologic concentration of single-guide RNA (sgRNA)³. This is the first trial which demonstrates CRISPR/Cas9-based in vivo gene editing in humans, and provides proof-of-concept that it might eventually become a viable therapeutic strategy. Another noteworthy observation from the study is that the max reduction of serum TTR reduction in the high dosage group (~96%), represents an increase in efficacy over other first-in-class gene silencing therapies such as Patisiran. In its Phase III APOLLO Study, Patisiran demonstrated an 81% median reduction in serum TTR levels, although this was in a significantly larger number of patients. Intellia is now looking to follow up on these initial findings with higher doses of NTLA-2001, which might lead to a robust reduction in serum TTR levels across more patients.

The discovery of CRISPR/Cas9 as a potential tool for gene editing in eukaryotic cells has sparked a wave of renewed interest in the field and its related applications. While still in the early stages, the data is indeed promising and bodes well for the future of in vivo gene editing as a therapeutic approach for certain conditions. Intellia and other major players in the field such as CRISPR Therapeutics and Editas Medicine will no doubt be looking for similarly positive results in their other CRISPR-based drug candidates. However, other than being used to disrupt genes in vivo, what other CRISPR-based approaches are being developed for therapeutic applications?

NTLA-2001 utilizes the basic concept of the CRISPR/Cas9 machinery to treat TTR —it recognizes the defective gene, and ‘cuts’ it. Apart from in vivo therapy, CRISPR is also being used to edit cells out of the body (ex vivo) so that they can be used as treatment. Immuno-oncology is a particular focus of this approach, with CRISPR being used to engineer allogeneic, i.e. ‘off-the-shelf’, CAR-T cells. Chimeric antigen receptor (CAR)-T cells are immune cells which are usually derived from the patient themselves, and then genetically engineered to target and destroy cancer cells. While this approach has proved effective in certain patients, some drawbacks still remain. CAR-T cells take a few weeks to manufacture, and a patient’s condition may worsen during that period. Furthermore, the CAR-T cells produced may have low potency due to the patient’s condition, denting their therapeutic efficacy. Candidates such CRISPR Therapeutics’ CTX110 and Intellia Therapeutics’ NTLA-5001 seek to use CRISPR as a method of both inserting the CAR gene construct into extracted T cells, as well as disrupting components such as major histocompatibility complex I (MHC I) to prevent unwanted immunological reactions when introduced into patients. In theory, this would allow for the creation of allogeneic CAR-T cells that can be stored and used when necessary. The CRISPR-mediated insertion of the CAR gene construct would be more efficient and scalable compared to other current options, while the disruption of MHC I and MHC II complexes would help to prevent rejection by the patient’s own immune cells. These ‘3rd Generation’ CAR-T cells could also be engineered in other ways by CRISPR to make them more potent, whether by knocking out negative regulators such as PD-1, or making them resistant to immunosuppressive factors such as TGF-β. CAR-T cells can thus be manufactured from healthy donors and engineered for optimum efficacy before being administered to patients appropriately. Although the promising efficacy of CTX110 was overshadowed by the death of one patient during its Phase I CARBON trial in 2020¹⁰, further advances will hopefully help make CRISPR-generated allogeneic CAR-T cells for therapy a reality some day.

Another consideration for CRISPR-based therapeutic approaches is that many diseases cannot be cured by disrupting genes alone. In order to truly perform gene editing, we require a method which actually allows us to edit the genome itself, as opposed to merely cutting it up. This is where newer methods such as base and prime editing come in. Both technologies are still CRISPR-based, but still only at a very preliminary stage. Rather than merely trying to cut up and disrupt defective genes, base and prime editing attempt to edit the DNA sequence, correcting any unwanted ‘mistakes’. Base editing, as its name implies, attempts to correct single bases within the DNA sequence. The base editing machinery introduces single nucleotide variants (SNVs) into cells, essentially converting one DNA base to another¹¹. For example, the first DNA base editors developed to perform genome editing without causing a double stranded break (DSB) were capable of converting a C-G base pair to a T-A base pair¹². Prime editing was developed later on by the same group that made the first base editors under Prof. David Liu of Harvard University. Rather than convert single bases, prime editors are capable of directly targeting and editing a specific sequence in the genome. Prime editing has been demonstrated to be able to perform targeted insertions/deletions (indels), and all 12 types of point mutations in various cell types¹³. Most importantly, base and prime editing are able to perform these edits without causing a DSB or needing a donor DNA template, factors which can cause issues with the repair process.

This is not to say that base and prime editing are inherently superior to the currently used CRISPR/Cas9 machinery. Both these technologies remain relatively nascent, with only early studies being done. Indeed, they are likely to be complementary, each potentially addressing their own niche. For example, base editors provide higher editing efficiency and precision as compared to prime editors if only point mutations are necessary. However, unlike base editors, prime editors possess greater targeting flexibility and can be used to ‘seek’ out specific sequences for editing without having to worry about similar bases nearby, or require a protospacer adjacent motif (PAM) exactly 15 bases upstream of the edit site¹⁴. If there is a need to directly knock out or insert a gene as quickly and efficiently as possible, such as in the generation of allogeneic CAR-T cells, the current CRISPR/Cas9 machinery being used by CRISPR Therapeutics, Intellia Therapeutics, and Editas Medicine still represents one of the most straightforward approaches available. Ultimately, different diseases will require us to understand their underlying causes so that the appropriate therapeutic approach can be chosen.

Following this positive breakthrough by Intellia Therapeutics with NTLA-2001, I am hopeful that we will see more breakthroughs in the field, leading to safer and effective cures for other diseases in the future. Prof. Jennifer Doudna, the 2020 Nobel Laureate in Chemistry for her discovery of CRISPR’s potential in gene editing, has also recently commented on Intellia’s success, highlighting that it is “one of the fastest rollouts, I think, of technology from the fundamental, initial science to an actual application”. Indeed, only ten years have passed since her group’s seminal paper on CRISPR/Cas9¹⁵, and we are already seeing successful results in early stage in vivo clinical trials. The convergence of various technological platforms such as modern high-speed internet, machine learning and AI algorithms, greater data processing capacity and power, has been central to this high rate of growth, giving scientists the tools to study biological processes at a higher resolution than ever before. In fields such as genomics and immunology, these improved technologies will be critical in allowing us to better understand their intricacies. Hopefully, this increased understanding will continue to drive discovery and innovation in the biological sciences, helping to provide better therapeutic solutions for people around the world.

  1. Press release: Intellia and Regeneron Announce Landmark Clinical Data Showing Deep Reduction in Disease-Causing Protein After Single Infusion of NTLA-2001, an Investigational CRISPR Therapy for Transthyretin (ATTR) Amyloidosis. Intellia Therapeutics Inc., 2021.
  2. Luigetti et al., Diagnosis and Treatment of Hereditary Transthyretin Amyloidosis (hATTR) Polyneuropathy: Current Perspectives on Improving Patient Care. Therapeutics and Clinical Risk Management, 2020.
  3. 2021 PNS Annual Meeting: Gillmore et al., In vivo CRISPR/Cas9 Editing of the TTR Gene by NTLA-2001 in Patients with Transthyretin Amyloidosis. Peripheral Nerve Society, 2021.
  4. Adams et al., Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis. The New England Journal of Medicine, 2018.
  5. CAR T cells: Engineering Immune Cells to Treat Their Cancers. The National Cancer Institute, 2021.
  6. Immuno-oncology: CRISPR/Cas9 will drive the next generation of immuno-oncology cell therapy. CRISPR Therapeutics, 2021.
  7. Ex Vivo Therapies: CRISPR/Cas9 Improves Engineered Cell Therapies for Cancer. Intellia Therapeutics, 2021.
  8. Atsavapranee et al., Delivery technologies for T cell gene editing: Applications in cancer immunotherapy. EBioMedicine, 2021,
  9. Salas-Mckee et al., CRISPR/Cas9-based genome editing in the era of CAR T cell immunotherapy. Human Vaccines and Immunotherapeutics, 2019.
  10. News release: CRISPR Therapeutics Reports Positive Top-Line Results from Its Phase 1 CARBON Trial of CTX110™ in Relapsed or Refractory CD19+ B-cell Malignancies. CRISPR Therapeutics, 2020.
  11. Porto et al., Base editing: advances and therapeutic opportunities. Nature Reviews Drug Discovery, 2020.
  12. Komor et al., Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 2016.
  13. Anzalone et al., Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 2019.
  14. Begley, You had questions for David Liu about CRISPR, prime editing, and advice to young scientists. He has answers. STAT News, 2019.
  15. Jinek et al., A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 2012.



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