Gene editing for patients with beta-haemoglobinopathies

Beta-haemoglobinopathies, the most common of which are sickle cell disease (SCD) and β-thalassaemia (BT), represent a collection of inherited monogenic recessive disorders characterised by faulty or reduced production of beta-globin chains, respectively. These conditions are associated with significant morbidity and mortality rates and are notably prevalent in the Mediterranean populations, Southern and Southeastern Asia, the Middle East, Africa and the Pacific Islands. They stand out as the most prevalent genetic disorders worldwide, with an estimated annual birth incidence surpassing 300,000 children.

In SCD, a single base substitution in the β-globin chain results in a missense mutation, replacing glutamic acid with valine at the sixth amino acid position. This alteration prompts the sickle haemoglobin to polymerise, distorting red blood cells into the characteristic sickle shape. These misshapen cells can block small blood vessels, resulting in compromised oxygen delivery to tissues and consequential complications such as pain crises, breathing difficulties and organ damage. On the other hand, BT is associated with inadequate β-globin production, which leads to an excess of unpaired α-globin chains precipitating in erythroid precursors. Thus maturation is impaired, resulting in precursor cell death and ineffective erythrocyte production. The ensuing significant anaemia and expansion of erythroid precursors contribute to secondary issues in bones and other organs. Despite available treatments for both diseases, severe symptoms and complications may still exist even with intervention. Bone marrow transplantation is a potential cure that relies on finding a healthy, compatible donor, limiting its feasibility to only a fraction of patients. It is also associated with risks of transplant-related mortality, graft-versus-host disease (GVHD) and graft rejection (Ref Reference Motta, Ghiaccio, Cosentino and Breda38). The majority of individuals with SCD or BT depend on regular frequent, often lifelong blood transfusions as a critical part of their management. This is typically combined with iron chelation therapy (ICT) to prevent excess iron from transfused red blood cells from accumulating in the body and damaging vital organs such as the heart and liver (Refs Reference Yousuf, Akter, Wasek, Sinha, Ahmad and Haque39, Reference Tanhehco40). Among the major limitations of these approaches are the scarcity of blood products, which leads to a lack of adequate and safe blood transfusions, as well as low accessibility to ICT, treatment toxicity, adverse events (including alloimmunisation, transfusion-related reactions and infections) and high costs (Ref Reference Njeim, Naouss, Bou-Fakhredin, Haddad and Taher41). These limitations underscore the need for curative therapies, including foetal haemoglobin (HbF) induction via gene editing, as a feasible and efficient approach with the potential to provide long-term solutions for beta-haemoglobinopathies.

The transition from foetal to adult haemoglobin and suppression of HbF during human development have long captured interest. HbF is a type of haemoglobin produced by feotuses in the womb but absent in children and adults, remaining unaffected by sickle cell mutation. Clinical observations have consistently indicated that enhanced HbF production mitigates the severity of SCD and BT. In individuals with SCD, symptoms typically emerge in infancy as HbF levels naturally decline. Accordingly, asymptomatic SCD until after infancy was attributed to elevated HbF levels initially based on clinical observations (Ref Reference Watson42). This concept gained support from the study of rare patients with compound heterozygosity for SCD and hereditary persistence of HbF mutations, who exhibited predominantly asymptomatic profiles. Subsequent larger epidemiological studies in SCD confirmed that elevated HbF levels substantially and quantitatively alleviate clinical severity while reducing mortality (Refs 43–Reference Castro, Brambilla, Thorington, Reindorf, Scott and Gillette46). Similar patterns emerged in patients with BT. Observations in rare BT cases with increased HbF production revealed a milder clinical course; infants manifested symptoms only after the decline in HbF expression in the months following birth (Refs Reference Weatherall43, Reference Weatherall47). Larger epidemiological studies within thalassaemia populations consistently confirmed these findings (Refs Reference Premawardhena, Fisher, Olivieri, de Silva, Arambepola and Perera48–Reference Galanello, Sanna, Perseu, Sollaino, Satta and Lai50).

The clinical induction of HbF production thus held great promise in alleviating the severe symptoms associated with SCD and BT (Ref Reference Sankaran and Orkin51). Although non-specific pharmacological inducers displayed some success at inducing HbF, more effective and targeted approaches were required in the clinical setting (Ref Reference Bou-Fakhredin, De Franceschi, Motta, Cappellini and Taher52). The most advanced approach to filling this gap followed an innovative route by elevating HbF levels via genome engineering rather than restoring healthy adult haemoglobin (Figure 4). The initial phase of treatment involves the collection of CD34+ haematopoietic stem cells (HSCs) directly from the patient’s bloodstream, followed by genome modification to activate the HbF gene. The patient then receives chemotherapy to eliminate ailment-triggering blood stem cells, making way for the edited cells. Lastly, the genome-edited stem cells are reintroduced into the patient’s bloodstream through intravenous (IV) administration. The goal is for these edited cells to establish themselves in the bone marrow and create a fresh population of blood stem cells that exclusively produce HbF-expressing erythrocytes. This ex vivo genome editing approach ensures that the genome-editing tools specifically interact with the intended target cells and mitigates the risk of persistent CRISPR components in the body, thus reducing the chances of unintended edits or immune reactions (Ref Reference Henderson53).

Figure 4. CRISPR-based gene editing strategies to correct beta haemoglobinopathies such as sickle cell disease (SCD) and beta-thalassaemia (BT). CASGEVY, developed by Vertex and CRISPR Therapeutics, entails the genetic modification of a patient’s own HSPCs via CRISPR/Cas9 and SPY101 single guide RNA (Ref 54). This modification aims to disrupt the GATA1 transcription factor binding domain of the B-cell lymphoma/leukaemia 11A (BCL11A) gene erythroid enhancer through ex vivo editing. BCL11A, a known suppressor of foetal haemoglobin (HbF) expression, presents a target for intervention. Consequently, this disruption leads to a significantly increased HbF expression, effectively correcting the deficient production of adult beta haemoglobin (Panel A). Another notable approach (EDIT-301) developed by Editas Medicine involves targeting the promoters of the γ-globin genes [HBG1 (Aγ) / HBG2 (Gγ)], introducing distinct sequence alterations to interfere with BCL11A binding sites, leading to enhanced production of HbF (Panel B) (Ref 55). This alteration is accomplished by employing the AsCas12a protein, which is well-known for its superior efficiency and specificity in gene editing. The CRISPR base editors are also the subject of intense interest, with two primary methods developed by the BEAM Therapeutics for addressing haemoglobinopathies (Panel C). The first one, BEAM-101, involves performing an A-G transition in the BCL11A binding regions located in the promoter regions of gamma-globin genes to prevent the binding of BCLA11A, thereby increasing gamma-globin expression (Ref 56). The preclinical BEAM-102, the latter of the two, involves converting adenine to guanine at the specific point in the mutant beta-globin gene responsible for sickle cell formation (Ref 57). Due to this process, the haemoglobin produced, known as Haemoglobin Makassar, inhibits the formation of sickle cells. Other clinical trials, such as those involving the replacement of the mutated beta-globin gene through CRISPR-Cas9 knock-in (CRISPR_SCD001) and the correction of mutations in HBB to restore normal haemoglobin expression (GPH101), are omitted for clarity.

The initial CRISPR-based clinical trial entailing the use of CRISPR to reawaken HbF production for SCD and transfusion-dependent β-thalassaemia (TDT) received support from Vertex Pharmaceuticals (Boston, Massachusetts) and CRISPR Therapeutics (Zug, Switzerland) (Ref 58). In this strategy, the erythroid-specific enhancer region of the BCL11A gene, which prevents the production of HbF, is targeted and cut in both strands by Cas9 (Figure 4a). Once disrupted, this gene can no longer block HbF production, allowing it to display a therapeutic effect by boosting oxygen supply to tissues.

Despite not directly addressing the mutations responsible for SCD or BT, this treatment modality proved functional as a practical cure for both conditions. In November 2023, the U.K. Medicines and Healthcare Products Regulatory Agency (MHRA) approved this one-time IV treatment of CRISPR-edited cellular therapy under the commercial name of Casgevy for conditional marketing authorisation. The treatment is aimed for use in SCD patients 12 years of age and older with recurrent vaso-occlusive crises or TDT patients eligible for HSC transplantation, for whom an HLA-matched HSC donor is not available. In the trial for SCD, 29 out of 45 participants were followed long enough to announce reliable results; 28 of these patients no longer suffered from the vaso-occlusive crises characteristic of the disease at least 1 year following treatment. The same regimen was tested for TDT, and out of the 54 people who received the treatment, 42 participated for sufficient duration to draw reliable conclusions. Among these patients, transfusions were unnecessary for at least 1 year for 39 individuals, while three patients experienced a 70% reduction in transfusion requirement (Ref Reference Wong59).

Casgevy received FDA approval for SCD in December 2023, followed by European Medicines Agency (EMA) approval in February 2024. According to the FDA, it is the first FDA-approved treatment to employ a novel genome editing technology, marking a groundbreaking advancement in the field of gene therapy. The results of this single-arm multicenter trial of safety and efficacy testing in adolescent and adult SCD patients were announced by the agency as follows: Casgevy treatment was administered to 44 patients, and out of the 31 individuals who were monitored for an adequate period to assess their condition, 29 achieved relief from vaso-occlusive crises lasting at least 12 consecutive months. The FDA’s report also stated that there were no instances of graft failure or rejection. Low platelet and leukocyte levels, nausea, abdominal pain, mouth sores, musculoskeletal pain, headache, itching and febrile neutropenia were presented as the most common side effects.

Intriguingly, Editas Medicine, Inc. is currently conducting two phase 1/2 trials for individuals with severe SCD (RUBY trial) and TDT (EdiTHAL trial), employing a CRISPR system featuring AsCas12a protein (EDIT-301: renizgamglogene autogedtemcel: reni-cel) (Figure 4b) (Ref 60). The method involves genomic modification of the γ-globin gene promoters [HBG1 (Aγ) / HBG2 (Gγ)] to interfere with the BCL11A binding sites to reactivate γ-globin expression, thus increasing HbF production in autologous HSCs. The study marks the first instance of Cas12 being utilised in a clinical trial. A detailed update by the company on 11 December 2023, included the safety and efficacy data in 11 patients enrolled in RUBY and 6 in EdiTHAL. All treated patients in the RUBY trial were reported to be free of vaso-occlusive crises since the infusion, which induced an early and substantial increase in total and foetal haemoglobin. Normal haemoglobin levels and an HbF level of >40% were reported in 6 patients throughout 5–18 months of follow-up. A similar early and substantial rise in the total and foetal haemoglobin levels was also evident in the efficacy results reported for the EdiTHAL trial; importantly, the total haemoglobin increased above the transfusion dependence threshold (9 g/dL). As of October 2024, the company announced that 28 patients received the drug in the RUBY trial, which was well tolerated, at a median of 9.5 months follow-up. Eleven patients had over one year of follow-up. Twenty-seven of the patients were reported to be free of vaso-occlusive events, with early normalisation of total haemoglobin. Mean total haemoglobin increased from 9.8 g/dL at baseline to 13.8 g/dL at month 6 (n = 18) (Ref 61). New safety and efficacy data for the EdiTHAL trial presented at the 66th American Society of Hematology (ASH) Annual Meeting and Exposition revealed that the 7 patients, who were at a median (range) of 10.5 (6.3–15.1) months post-infusion with two patients having over 1-year follow-up, had total haemoglobin levels remaining above the transfusion-independence threshold of 9.0 g/dL. This level increased to 12.5 (1.5) g/dL by month 6. Overall, all 7 patients were announced to be transfusion independent for a range of 5.8–14.5 months following the last red blood cell transfusion at 0.7–2.2 months post-reni-cel infusion. The company reveals the safety profile as consistent with myeloablative conditioning with busulfan, with no serious adverse effects reported related to the drug (Ref Reference Frangoul, Hanna, Walters, Chang, Jaskolka and Kim62).

Furthermore, Beam Therapeutics initiated their phase 1/2 trial (BEACON) for a base editing therapy targeting severe SCD in the United States in November 2022, and the dosing of the first patient was announced in January 2024 (Ref 56). The therapeutic named BEAM-101 used in this trial involves an A-G transition in the BCL11A binding site within the promoter regions of the γ-globin genes, which the company claims to have several advantages over other therapeutics, as a ‘next-generation form of CRISPR’. The most significant benefit of the approach seems to lie in its action mechanism, excluding a double-strand cut in DNA but instead involving precise, single-letter changes mimicking single nucleotide polymorphisms involved in the hereditary persistence of foetal haemoglobin (HPFH). Undesired chromosomal abnormalities and genotoxic stress are claimed to be prevented via this modality. The company revealed clinical data from 7 patients in the 66th American Society of Hematology (ASH) Annual Meeting and Exposition in December 2024, stating that >60% HbF induction and <40% Haemoglobin S (HbS) reduction along with resolution of anaemia were achieved in all 7 patients (Ref 63).

Another base-editing strategy in SCD used a custom ABE (ABE8e-NRCH) that converts the sickle cell allele to the HBB^G Makassar allele, a non-pathogenic variant reported in individuals living in the Makassar region of Indonesia (Ref Reference Newby, Yen, Woodard, Mayuranathan, Lazzarotto and Li57). mRNA encoding the BE with a targeting gRNA was delivered ex vivo into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD. The researchers reported an 80% conversion of HBB^S to HBB^G. Durable gene editing was evident with 68% frequency of HBB^G and fivefold-decreased hypoxia-induced sickling of bone marrow reticulocytes 16 weeks following transplantation of the edited human HSPCs into immunodeficient mice, demonstrating preclinical therapeutic relevance. Beam Therapeutics is now testing the HbG-Makassar direct editing strategy via its preclinical drug BEAM-102 (Figure 4c).

One other advancement towards the treatment of SCD is the replacement of the mutated β-globin gene through CRISPR-Cas9 knock-in in a planned phase 1/2 trial in subjects ≥12 years old to 35 years old with SCD, via a single infusion of sickle allele-modified CD34+ HSPCs (CRISPR_SCD001). Also, nulabeglogene autogedtemcel, formerly known as GPH101, has been announced as the first CRISPR-based therapy candidate aiming to correct the HBB point mutation to restore normal haemoglobin expression. The phase 1/2 CEDAR trial was initiated to assess GPH101 regarding safety, efficacy and pharmacodynamics in adults and adolescents with severe SCD. In 2022, a single participant was dosed in a phase 1/2 trial, employing a combination of electroporation to deliver the CRISPR proteins into the cell and a viral vector to introduce a DNA ‘template’ for copying the new gene variant into the cell. In early January 2023, the company disclosed that the initial participant exhibited prolonged decreased blood cell counts (pancytopenia), necessitating continual blood transfusions and other therapies. Thus, discontinuance of the programme was announced in February 2023 to seek a partnership agreement for the external development of the drug (Ref Reference Philippidis64).

Beta haemoglobinopathies are among the diseases that will benefit a great deal from gene editing approaches, as even partial correction of related mutations with a suitable strategy may provide adequate levels of functional haemoglobin production and mitigate disease severity. One of the primary limitations of the CRISPR-Cas9 HDR system for disease correction is its relatively low efficiency in quiescent cells and the formation of large unintended deletions and chromosome-level changes resulting from the DSBs. Additionally, indels in the coding region of the β-globin locus could result in severe β0-thalassaemia phenotypes. Disruption of HbF repressors or upregulation of HbF expression via the introduction of HPFH-like mutations through base editing approaches are attractive strategies to compensate for the deficient beta globin, along with those to correct beta-thalassaemia point mutations (Ref Reference Prasad, Devaraju, George, Ravi, Paul and Mahalingam65). Researchers point out that uncontrolled mixtures of Cas9-mediated indels and other challenges, such as an adaptive immune response against Cas9 protein and activation of the p53 pathway in human stem cells, may lead to a reduction in CRISPR/Cas9 editing efficiency in clinical applications, hindering HSPC proliferation and engraftment (Refs Reference Mayuranathan, Newby, Feng, Yao, Mayberry and Lazzarotto66, Reference Frati and Miccio67). Base editing and PE techniques eliminate such consequences to a great extent, as strong alternative approaches that do not rely on DSBs like Cas9 nucleases (Ref Reference Christakopoulos, Telange, Yen and Weiss68).

Rearming of T-Cells via gene editing against cancer

T cells are an important group of lymphocytes pivotal to the immune system, playing a key role in anticancer immunity. They navigate the body to eliminate foreign or harmful cells and recruit other immune cells for assistance. Their functions are mediated through diverse specialised T-cell receptors that distinguish between safe and threatening cells (Ref Reference Cherry, Gherardin and Sikder69). Chimeric antigen receptor (CAR) T-cells are promising new genetically engineered cell-based drugs against cancer.

Many approaches involving CAR-T therapies are autologous, where T cells extracted from a patient’s blood are reinfused to the patient after being genetically modified and multiplied. It’s an effective yet costly and time-intensive treatment, with bottlenecks in the manufacturing process. Thus, a primary focus is the development of allogeneic CAR T-cells, sourced from a healthy donor and modified to specifically attack cancer cells while avoiding detection by the recipient’s immune system. These edited cells are subsequently multiplied into substantial quantities, enabling widespread administration to numerous recipients as needed. Reduced costs and shorter preparation times are major advantages of allogeneic products, as well as providing robust high-quality cells for on-demand cancer immunotherapy (Ref Reference Dimitri, Herbst and Fraietta70).

CRISPR Therapeutics is currently investigating the effects of allogeneic CRISPR-modified CAR-T cell variants (Figure 5). The company’s first allogeneic T-cell products, CTX110 (targeting CD19+ malignancies) and CTX130 (targeting CD70+ malignancies), were announced to have favourable results in B- and T-cell lymphoma and renal cell carcinoma (NCT04035434, NCT04502446, and NCT04438083, respectively). CD19 is a protein that is frequently present in leukaemia and lymphoma cells. CD70 is a protein commonly overexpressed in cancer cells of various solid and haematological origins. CTX130 was tested in relapsed/refractory T- or B-cell malignancies under the COBALT-LYM trial and relapsed/refractory renal cell carcinoma under the COBALT-RCC trial. The drug received FDA Orphan Drug and Regenerative Medicine Advanced Therapy (RMAT) designations. These two treatments were, however, also associated with T cell exhaustion leading to loss of response and reduced efficacy, particularly in high tumour burden patients. Thus new edits via CRISPR/Cas9 technology were included in the ‘next-generation’ CAR T cell programmes, applied under the names of CTX112 and CTX131. The company describes three distinct modifications in healthy donor T lymphocytes in preparation for these treatments. Firstly, aiming to block the host-versus-graft disease (HVGD), class 1 major histocompatibility complex (MHC I) is eliminated by knocking out the β2 M subunit (Ref Reference Kagoya, Guo, Yeung, Saso, Anczurowski and Wang71). This increases persistence and the chance for durable remissions. Secondly, CRISPR/Cas9 eliminates the existing TCRs, aiming to reduce the risk for GVHD. Lastly, CRISPR/Cas9 technology is used to insert the CAR construct into the TCR alpha constant (TRAC) locus to improve safety and consistency.

Figure 5. Distinctive design of allogeneic CAR T-cells modified using CRISPR technology. CTX110 is a chimeric antigen receptor T-cell (CAR-T) therapy developed by CRISPR Therapeutics (Ref Reference Kagoya, Guo, Yeung, Saso, Anczurowski and Wang71). It is designed to target and treat cancers by modifying a patient’s T cells to recognise and attack cancer cells expressing the CD19 antigen. CTX110 uses CRISPR gene editing technology to precisely modify T cells to express a synthetic receptor (CAR) that targets CD19, allowing the modified T cells to recognise and destroy cancer cells expressing this antigen. CTX110 is currently being investigated in clinical trials for the treatment of various haematologic malignancies, including non-Hodgkin lymphoma and chronic lymphocytic leukaemia. CTX120 and CTX130 employ a similar CRISPR-edited allogeneic T cell framework, differing in their CAR targets and, in the case of CTX130, incorporating additional editing.

Additional teams have achieved remarkable outcomes by targeting CD19 in the context of challenging and aggressive B-cell non-Hodgkin lymphomas. Caribou Biosciences applied a promising technology in their treatment approach; in addition to directing their cells toward CD19, they incorporated a second genetic alteration, a ‘knockout’ deactivating the programmed death-1 (PD-1) gene, often used by the cancer cells for their advantage to evade the immune system (Ref Reference Rupp, Schumann, Roybal, Gate, Ye and Lim72). This approach is designed to enhance antitumour activity by restricting premature CAR T-cell exhaustion. The strategy utilises Cas9 chRDNA guides to make the necessary edits. It is a technology defined by the company as a CRISPR hybrid RNA–DNA (chRDNA), aiming to improve CRISPR genome-editing precision through the highly reduced affinity of the chRDNA guide to the off-target sequences. Mismatches between the chRDNA guide and off-target sites significantly reduce the stable binding of the Cas complex, thereby hindering cleavage by the Cas nuclease. As of July 2023, Caribou Biosciences shared their long-term follow-up results for their product CB-010 under the ANTLER Phase 1 clinical trial (Ref 73). Notably, the treatment demonstrated a generally well-tolerated and safe profile. In this dose-escalation study involving 16 patients, a 94% overall response rate was reported, with 69% of the patients (11 of 16) displaying a complete response (CR). Seven of the 16 patients achieved CR for over 6 months, with the longest CR announced as 24 months. A related abstract presented for the 2024 ASCO Annual Meeting also stated a manageable safety profile and promising efficacy in patients with refractory/resistant B-NHL, with the dose escalation phase being completed (Ref Reference Hu, Nastoupil, Holmes, Hamdan, Kanate and Farooq74). The company also pursues Phase 1 trials involving allogeneic anti-BMCA CAR-T cell therapy for relapsed or refractory multiple myeloma (CB-011) and allogeneic anti-CLL-1 CAR-T cell therapy against relapsed or refractory acute myeloid leukaemia (CB-012), where a Cas12a chRDNA genome-editing technology is used. CB-010 holds RMAT, Fast Track and Orphan Drug FDA Designations, whereas CB-011 and CB-012 hold Fast Track and Orphan Drug FDA Designations. Both trials are currently recruiting patients.

Intriguingly, in a recent strategy involving the generation of off-the-shelf allogeneic CAR T cells, lentiviral-mediated expression of a CAR targeting CD7 (CAR7) was obtained on healthy donor T cells, followed by base editing for the inactivation of three genes encoding the CD52 and CD7 receptors along with the β chain of the αβ T-cell receptor (Ref Reference Chiesa, Georgiadis, Syed, Zhan, Etuk and Gkazi75). These modifications were carried out to prevent lymphodepleting serum therapy, CAR7 T-cell fratricide, and GVHD, respectively. The safety of these edited T cells was investigated in 3 children with relapsed leukaemia. The first patient was a 13-year-old girl with relapsed T-cell ALL following allogeneic stem-cell transplantation. Molecular remission within 28 days was reported after the single-dose base-edited CAR7 treatment (BE-CAR7). A nonmyeloablative allogeneic stem-cell transplantation from the patient’s original donor followed this process, leading to ongoing leukemic remission. BE-CAR7 cells were effective in the other two patients in the same trial, although one developed progressive lung complications related to cytokine release syndrome along with fatal fungal complications. The third patient received allogeneic stem-cell transplantation during remission. These results indicated the anticipated risks related to immunotherapy-related complications in this phase I study, where cytokine release syndrome, multilineage cytopenia, and also opportunistic infections were reported as serious adverse effects.

In another recent phase I trial, a simultaneous knockout of the endogenous TRAC (encoding TCRα) and TRBC (encoding TCRβ) genes was performed via CRISPR-Cas9 genome editing, along with two chains of a neoantigen-specific TCR (neoTCR) acquired from patients’ circulating T cells inserted into the TRAC locus. The trial, sponsored by PACT Pharma, involved 16 patients with different refractory metastatic solid cancers, including melanoma, urothelial carcinoma, head and neck squamous cell carcinoma, non-small cell lung carcinoma and colorectal, ovarian, prostate and hormone receptor-positive and triple-negative breast cancers. This approach is unique in assessing the genetic makeup of an individual’s tumour and then utilising CRISPR technology to customise the patient’s T cells to specifically target the individual disease. Each participant received up to three distinct engineered T cells. Five patients displayed stable disease; a high percentage of neoTCR transgenic T cells were reported in the periphery, and there was a decrease in some target lesions; thus, the therapy was considered likely to have had an effect. All patients were reported to display the expected side effects associated with lymphodepleting chemotherapy (Ref Reference Foy, Jacoby, Bota, Hunter, Pan and Stawiski76).

Overall, CAR-T cell therapy emerged as a strong treatment strategy for malignant tumours. Yet the survival and persistence of CAR T-cells are often impaired due to their terminally differentiated phenotype and exhausted status. CRISPR/Cas9 technology has been used in various trials to reduce exhaustion, generate a memory phenotype, and look for new targets to improve anti-cancer potential, providing an effective strategy to efficiently promote the proliferation and persistence of CAR T-cells in vivo (Ref Reference Wei, Chen and Wang77). Yet challenges such as off-target effects and Cas9 protein-mediated immunogenicity limit the application of the CRISPR/Cas system to CAR T-cells. Rational designs of sgRNAs by bioinformatics tools, use of alternative Cas nucleases and adjustment of delivery systems are a few measures to avoid the off-target effects. Strategies such as epitope masking are among the solutions to the Cas9 protein-related immunogenicity in the in vivo CRISPR/Cas9 editing (Ref Reference Chew78). Overall, CAR T-cell therapies face other challenges such as the emergence of T-cell malignancies, including CAR-positive lymphoma. T-cell lymphomas are especially notable in this clinical context due to concerns that CAR T-cell vector integration may contribute to cancer development. Researchers emphasise the infrequent occurrence of second tumours in CAR T-cell applications, while still acknowledging it as a significant concern (Refs 79–Reference Ghilardi, Fraietta, Gerson, Van Deerlin, Morrissette and Caponetti82). Incorporating CRISPR into these therapies may improve the approach by utilising a more precise strategy than conventional vector integration.

Patients undergoing CAR T-cell therapy often encounter cytokine release syndrome (CRS), a severe adverse event triggered by systemic levels of pro-inflammatory cytokines such as interleukin-6 (IL-6), tumour necrosis factor (TNF), and interferon-gamma (IFN-γ) (Ref Reference Santomasso, Bachier, Westin, Rezvani and Shpall83). The condition is characterised by life-threatening risks such as severe fever, hypoxia, and organ damage. CAR’s engagement with its target antigen, initial cytokine release from activated CAR T-cells, and subsequent activation of bystander immune cells contribute to the pathophysiology of CRS. This leads to the release of a broad spectrum of cytokines from both CAR T-cells and native immune cells, accompanied by the expansion of CAR T-cells. CRISPR-Cas9 editing may also be very useful in addressing this problem, as in the approach where the technology was used to modify CAR T-cells with a GM-CSF genetic knockout, decreasing the production of proinflammatory cytokines and chemokines (Ref Reference Sterner, Sakemura, Cox, Yang, Khadka and Forsman84).

Genetic engineering of photoreceptors for genetic blindness

Leber Congenital Amaurosis (LCA) is among the earliest and most severe forms of inherited retinal dystrophies (IRDs), responsible for 20% of early childhood blindness (Ref Reference Huang, Yang, Yang, Hou and Chen85). The disease is characterised by degeneration and/or dysfunction of photoreceptors and eventual death of retinal cells. The most prevalent form of the disease, LCA10, occurs due to mutations in the centrosomal protein of 290 kDa (CEP290) gene. The CEP290 protein plays an important role in cilium assembly and ciliary protein trafficking, localised in the connecting cilium as a multi-protein complex required for structural and functional integrity. Thus, when individuals with LCA10 are exposed to light, these compromised cells are unable to effectively transmit all the necessary signals to the brain, resulting in loss of vision. The CRISPR-based approach for LCA10 treatment aims to address this issue by modifying the defective photoreceptor gene, prompting it to produce a complete and functional protein instead of the defective, truncated version. The goal is to edit a sufficient number of cells to generate healthy protein for the patients to regain their lost vision.

EDIT-101 represents an experimental medicine based on CRISPR/Cas9 editing, aimed at eliminating the abnormal splice donor site induced by the c.2991+1655A>G IVS26 mutation in CEP290 (Figure 6) (Ref 86). To reinstate normal CEP290 expression, an upstream sgRNA guides the initial Cas9 cleavage to a location preceding the IVS26 mutation, while a downstream sgRNA directs the second Cas9 cleavage to a site situated beyond the mutation. The resulting cleavage ends undergo direct ligation through the NHEJ process, and thus the intronic fragment flanking the IVS26 mutation is removed (Ref Reference Ruan, Barry, Yu, Lukason, Cheng and Scaria87). The mRNA processing machinery subsequently eliminates the truncated intron 26 during RNA splicing. EDIT-101 is delivered through a subretinal injection to precisely target and convey the gene editing machinery directly to photoreceptor cells (Ref Reference Maeder, Stefanidakis, Wilson, Baral, Barrera and Bounoutas88).

Figure 6. A gene-editing approach for genetic blindness. EDIT-101 is a novel gene therapy developed by Editas Medicine, aimed at treating Leber congenital amaurosis 10 (LCA10), a rare genetic form of blindness (Ref Reference Maeder, Stefanidakis, Wilson, Baral, Barrera and Bounoutas88). It utilises CRISPR-Cas9 gene editing technology to correct mutations in the CEP290 gene, responsible for the LCA10 phenotype. An AAV5 vector was used to deliver the Staphylococcus aureus Cas9 (SaCas9) and CEP290-specific guide RNAs (gRNAs) to photoreceptor cells by subretinal injection. By targeting and repairing the faulty genetic sequence, EDIT-101 aims to restore vision in affected individuals. The therapy is administered through intraocular injection, directly into the eye, allowing it to target retinal cells. U6: human U6 polymerase III promoter; 323: gRNA; CEP290–323; 64: gRNA CEP290–64; hGRK1: human G protein-coupled receptor kinase 1 promoter; SV40 SD/SA: simian virus 40-splice donor and splice acceptor containing intronic sequence.

The first in vivo CRISPR therapy trial, conducted in the United States and sponsored by Editas Medicine, targeted the LCA10 (Ref 86). Commencing in March 2020 with the first patient receiving treatment, successive dosing of limited cohorts was extended until July 2022. Editas initially administered low-dose treatments to adult cohorts before progressing to high-dose adult cohorts and a paediatric cohort. This sequential approach aimed to mitigate potential hazardous side effects throughout the trial, especially concerning the paediatric group. The subretinal administration involved treating one eye, while the other eye served as a control for assessing vision in the treated eye. According to Editas’ official statements, no severe adverse events or dose-limiting toxicities surfaced during the trial. Evaluating treatment efficacy posed a greater challenge than ensuring safety in these cases. Directly gauging the percentage of edited cells or detecting unintended edits in participants proved difficult. Due to the substantial reduction in vision, conventional line-by-line letter reading tests were impractical. Instead, alternative assessments such as mobility tests (e.g., navigating obstacles) and light detection capabilities were employed (Refs Reference Henderson53, 86).

During their phase 1/2 trial named BRILLIANCE for testing EDIT-101, Editas disclosed that merely 3 among 14 patients had shown ‘clinically meaningful’ improvements in their vision by November 2022 (Ref 86). Notably, two of these responsive individuals harboured mutations in both copies of the pertinent gene, hinting at the potential effectiveness of the treatment within this specific subset of the LCA10 population. This particular subgroup, existing within an already rare condition, comprises only approximately 300 individuals in the US. Owing to the exceedingly limited patient pool for this costly drug, Editas paused enrolment in the BRILLIANCE trial, yet keeping the possibility of resuming the efforts in the future should a suitable partner for this undertaking be identified. Their update in June 2023 stated that 8/14 participants expressed improved vision-related quality of life (QoL) (Ref Reference Pierce, Aleman, Ashimatey, Kim, Rashid, Myers and Pennesi89). Recently, the group published the latest status of the BRILLIANCE phase 1–2 study in early May 2024 in the New England Journal of Medicine (Ref Reference Pierce, Aleman, Jayasundera, Ashimatey, Kim and Rashid90). According to the report, 12 adults (17–36 years of age) and 2 children (9 and 14 years of age) were injected with varying doses (low, intermediate, high) of EDIT-101. No serious adverse effects related to the treatment or procedure, or dose-limiting toxic reactions were reported. Briefly, 6 participants displayed a meaningful improvement from baseline in cone-mediated vision; meaningful progress from baseline in the best-corrected visual acuity was reported in 9 participants; and improvement from baseline in the vision-related QoL score was evident in 6 participants.

Thus CRISPR holds promise for potentially treating genetic blindness by targeting and correcting mutations associated with the condition. While initial safety data for EDIT-101 may be promising, uncertainties persist regarding its long-term safety. Delivery of this treatment via a viral vector implies sustained expression of CRISPR-Cas components within the eye, thereby increasing the risk of unintended DNA alterations and potential immune reactions to the viral vector or the Cas protein over an extended period. Monitoring these patient volunteers over several years will be imperative to assess their long-term outcomes, as the potential for unintended genetic changes, such as off-target effects or genomic instability, underscores the need for extended monitoring to assess risks that may not manifest immediately but could have significant consequences over time.

Currently, there is no direct method available to evaluate the percentage of edited cells or identify unintended edits. Evaluation of editing efficiency can only be inferred based on observed improvements in vision among patient volunteers. Researchers actively monitor individuals who have received treatment to determine the stability, progression, or regression of vision improvements over time. Variability in editing outcomes could impact the overall efficacy of the treatment, particularly in a condition like LCA10, where the restoration of function in retinal cells requires high accuracy and uniformity. Furthermore, the researchers acknowledge that the results of their study support the safety of the treatment to the extent that it can be assessed in a small number of patients, as it sets limitations to the interpretation of the data and presents challenges for drawing robust conclusions (Ref Reference Pierce, Aleman, Jayasundera, Ashimatey, Kim and Rashid90). Addressing these critical aspects will allow future studies to build a more comprehensive understanding of the risks and benefits associated with genome editing in clinical applications for LCA10.

Genetic modification of stem cell-derived pancreatic cells for diabetes

Type 1 diabetes (T1D) is characterised by autoimmune destruction of pancreatic beta cells and consequent inadequate levels of insulin secretion. Vigilant management of blood sugar and insulin levels throughout a lifetime is necessary. Common serious complications of T1D encompass kidney damage, nerve pain, vascular and cardiac issues, vision impairment and limb amputation. Pancreatic islet transplantation has proven effective in treating individuals with unstable, high-risk T1D. However, this procedure is associated with a scarce supply of donor organs and the complexities of obtaining consistent and reliable islet preparations. Although ongoing clinical trials indicate substantial benefits from pancreatic cell transplantation, recipients of conventional transplants necessitate continual immune system suppression to avert rejection. The use of immunosuppressant drugs poses serious risks, including an elevated susceptibility to infections and cancers. A successful replacement therapy using stem cell-derived islets has the potential to overcome these challenges, offering a solution that could serve a larger number of people to constitute an effective alternative to the other treatment methods (Ref Reference Shapiro, Thompson, Donner, Bellin, Hsueh and Pettus91).

Results from a phase 1/2 open-label trial, the first of its kind conducted in humans, offer compelling evidence that pluripotent stem cell-derived pancreatic endoderm cells (PEC-01) transplanted into individuals diagnosed with T1D transform into islet cells capable of releasing insulin and c-peptide in a manner that mimics natural physiological regulation (Ref Reference Shapiro, Thompson, Donner, Bellin, Hsueh and Pettus91). Study participants were administered immunosuppressive medications to support the growth of these cells and prevent rejection by the body’s immune system of the implanted VC-02™ macro-encapsulation devices (Figure 7). This system holds refinements for increased engraftment and insulin production via direct vascularisation by the host vasculature, compared to the earlier VC-01 immuno-isolating units which depended on semipermeable membranes that were cell impermeant (Ref Reference Jones and Persaud92). In this research, which involved 17 subjects aged between 22 and 57, all diagnosed with T1D, PEC-01 cells were subcutaneously implanted into VC-02 units facilitating direct vascularisation. Early clinical results reveal that following the implantation and successful engraftment, the PEC-01 pancreatic progenitor cells undergo maturation into human endocrine islet tissue. Throughout the clinical trials conducted thus far, ViaCyte’s product candidates have exhibited strong tolerability with minimal side effects related to the product. Both histological evidence and measurements of c-peptide (insulin) production confirm the intended functionality of PEC-01 cells following engraftment.

Figure 7. Schematic representation of VC-02 Macroencapsulation Device (Ref Reference Shapiro, Thompson, Donner, Bellin, Hsueh and Pettus91). The VC-02 macroencapsulation device is designed to encapsulate and protect insulin-producing cells for transplantation into individuals with type 1 diabetes (T1D). The encapsulation provided by the VC-02 device helps to maintain the viability and function of the transplanted cells. This can lead to more stable and consistent insulin production, which aids in better controlling blood sugar levels in individuals with T1D. By providing immune protection, the VC-02 device may reduce or eliminate the need for immunosuppressive drugs, typically required to prevent rejection in traditional islet cell transplantation.

Taking this strategy further, in February 2022, CRISPR Therapeutics and ViaCyte performed the first-in-human transplant of the CRISPR-edited, stem cell-derived pancreatic cells for T1D treatment (CTX210A). In this innovative approach, CRISPR technology is utilised to modify immune-related genes within pancreatic cells derived from pluripotent stem cells, rendering them impervious to the patient’s immune system (Ref 93). The ultimate goal is to furnish patients with robust, new pancreatic cells capable of managing or potentially curing T1D without the need for chronic immunosuppression. CRISPR Therapeutics and ViaCyte sponsored this phase 1 trial, marking the initial application of CRISPR for treating an endocrine disease. Shortly after the initial dosing of the first patient in spring 2022, Vertex Pharmaceuticals acquired ViaCyte. Yet on 8 January 2024, Vertex announced it would cut ties to the T1D stem cell therapy by CRISPR Therapeutics. It appears that CRISPR Therapeutics is now planning to hold a phase I/II trial of the now-called CTX211 as the next-generation drug candidate, which they define as an ‘investigational allogeneic, gene-edited, immune-evasive, stem cell-derived beta-cell replacement therapy’ in their pipeline.

This method could provide patients with the advantages of transplantation, even potentially curing T1D, without encountering the risks and side effects linked to immunosuppressive drugs. CRISPR/Cas9 technology is often utilised in the hypoimmunogenic induced pluripotent stem cell (iPSC) line development, as a future potential universal source of ‘off-the-shelf’ cells to be used in allogeneic cell therapy (Ref Reference Karpov, Sosnovtseva, Pylina, Bastrich, Petrova and Kovalev94). Although providing easy and successful modification of the target cells, the requirement of several alterations for the process brings along a high probability of off-target effects, which remain to be addressed thoroughly in clinical studies (Ref Reference AlJanahi, Lazzarotto, Chen, Shin, Cordes and Fan95). Overall, the pivotal outcome of these trials will be whether the edited cells can effectively evade detection by the immune system, a critical factor in determining the success of the treatment.

Gene editing tools against HIV

Human Immunodeficiency Virus (HIV) is a viral infection that attacks the immune system by infecting CD4 (helper) T lymphocytes. Reproducing within the CD4 cells, HIV leads to cell death and the release of more viruses to infect and eliminate other helper T cells. If left untreated, HIV can progress to acquired immunodeficiency syndrome (AIDS), causing severe immune system damage and leaving individuals susceptible to common infections that can lead to serious illness or death. Those with AIDS also face increased vulnerability to rare infections and cancers uncommon in individuals with healthy immune systems.

Excision Biotherapeutic’s EBT-101 is a unique experimental in vivo CRISPR-based treatment developed to provide a one-time intravenous infusion for the treatment of HIV infections (Ref Reference Thomas, Cradick, Thompson, Fine, Kaminski and Hayden96). Using an adeno-associated virus-9 (AAV9), EBT-101 transports CRISPR-Cas9 and dual guide RNAs, employing a multiplex editing technique that targets three specific locations within the HIV genome. This enables the removal of significant segments of the HIV genome, reducing the likelihood of viral escape. This is the first instance of a CRISPR-based therapy administered for infectious disease, as well as being the first to target a retrovirus. Researchers employed COTANA (CRISPR-Off-Target Nomination and Analysis) to steer CRISPR-Cas9 editing, creating sets of gRNAs that precisely target HIV without bearing significant resemblance to locations in the human genome. A subsequent analysis using multiplex amplicon sequencing demonstrated the effective removal of a substantial portion of the viral genome without any unintentional insertions or deletions in the genomic DNA.

Sponsored by Excision Biotherapeutics, this trial was granted Fast Track Designation by the FDA in July 2023. As a phase 1/2 trial, its objectives included assessing safety and side effects, determining the correct dosage, and evaluating the treatment’s efficacy in excising the virus from CD4 cells in individuals living with HIV Type I, constituting nearly 95% of the prevalence worldwide (Ref 97). The first participant was dosed in September 2022. In October 2023, at the European Society of Gene and Cell Therapy Congress (ESGCT), the company presented favourable safety and biodistribution findings for up to 48 weeks, based on the results from three patients dosed safely, experiencing no adverse events or dose-limiting toxic effects.

Before the clinical trial, EBT-101 displayed curative potential in mice and macaque monkeys, as announced by Excision Biotherapeutics in a press release. However, it was a concern that the percentage of latently infected cells in humans would be much lower than that modelled in cell cultures and animal subjects (up to 100%), as a factor to reduce the efficacy of the eradication process (Ref Reference Maslennikova and Mazurov98). The clinical data from this trial, presented recently at the American Society of Gene and Cell Therapy (ASGCT) meeting in May 2024, in fact, revealed that HIV viral suppression was not maintained at the initial dose tested, possibly because EBT-101 failed to reach all the cells with latent HIV in the 5 patients dosed. What is planned next throughout the course is yet to be announced (Ref Reference Cohrt99).

In a novel approach to combat HIV infection, mature primary B cells from mice and humans were edited in vitro using CRISPR/Cas9 to express mature neutralising antibodies (bNAbs) from the endogenous immunoglobulin heavy chain (Igh) locus (Ref Reference Hartweger, McGuire, Horning, Taylor, Dosenovic and Yost100). The modified B cells retained their capacity to take part in humoral immune responses. Wild-type mice that received these edited B cells and were immunised with the corresponding antigen exhibited HIV-1-neutralising bNAb titers sufficient to protect against infection, facilitating humoral immune responses that might be challenging to achieve through conventional immunisation methods. This is a promising application where advanced gene editing is combined with immunology, creating a cutting-edge strategy in the fight against HIV infection.

Although effective gene editing approaches raise hope in the combat against HIV, challenges remain, such as HIV-1’s high mutation rate, besides the common issues such as the off-target effects, immunogenicity, and delivery of the large CRISPR/Cas9 complex. The high specificity required for safely targeting HIV without compromising host cell integrity remains a significant technical barrier. The need to effectively target a sufficient number of cells to eliminate the disease makes this task more complex than treating conditions such as blood disorders. This incomplete targeting can allow residual viral reservoirs to persist, potentially leading to viral rebound if treatment is halted. Furthermore, the in vivo CRISPR editing strategies against HIV infection lead to the prolonged presence and widespread distribution of genome-editing components, heightening the risk of unwanted edits and immune reactions. Participants in related trials will undergo long-term monitoring to assess any potential health effects associated with unintended DNA alterations (Refs Reference Bhowmik and Chaubey101, Reference Hussein, Molina, Berkhout and Herrera-Carrillo102). Refining the specificity of guide RNAs and minimising off-target activity through advanced editing technologies will be crucial for translating this approach into a safe and effective therapy for HIV.

Lipid nanoparticle-mediated targeted delivery of genome editing tools against protein folding disease

Transthyretin (TTR) is a transport protein found in both plasma and cerebrospinal fluid, dedicated to transporting the thyroid hormone thyroxine (T4) and retinol to the liver. TTR is released by the liver into the bloodstream and to the cerebrospinal fluid by the choroid plexus. Transthyretin amyloidosis, also known as ATTR amyloidosis, is an uncommon, progressive and fatal disease. Hereditary ATTR amyloidosis (ATTRv amyloidosis) arises when mutations in the TTR gene are present from birth, causing the liver to produce structurally abnormal TTR proteins tending to misfold (Ref Reference Gertz, Mauermann, Grogan and Coelho103). These faulty proteins accumulate as amyloid deposits throughout the body, resulting in severe complications affecting various tissues such as the heart, nerves, and the digestive system. ATTRv amyloidosis commonly presents as polyneuropathy (ATTRv-PN) causing nerve damage or cardiomyopathy (ATTRv-CM) leading to heart failure. NTLA-2001 is an in vivo gene-editing tool targeting ATTR amyloidosis leveraging the CRISPR/Cas9 technology to reduce serum TTR concentrations (Figure 8) (Ref 104). It is the first investigative CRISPR therapy candidate designed for systemic administration, delivered intravenously as a single-dose treatment to execute gene editing within the human body. Intellia’s exclusive non-viral platform employs lipid nanoparticles (LNPs) for the targeted delivery of a two-part genome editing system to the liver. This system includes customised gRNA designed for the disease-associated gene and messenger RNA encoding the Cas9 enzyme responsible for precise editing. Extensive preclinical data display a significant and enduring reduction in TTR levels following in vivo inactivation of the target gene (Ref Reference Khoshandam, Soltaninejad, Mousazadeh, Hamidieh and Hosseinkhani105).

Figure 8. The mechanism of in vivo gene editing for Transthyretin Amyloidosis (Ref Reference Gillmore, Gane, Taubel, Kao, Fontana and Maitland106). NTLA-2001 employs a lipid nanoparticle (LNP) as its carrier system. The active ingredients of NTLA-2001 consist of a human-optimised messenger RNA (mRNA) molecule encoding the Streptococcus pyogenes (Spy) Cas9 protein and a single guide RNA (sgRNA) molecule targeting the human gene responsible for transthyretin (TTR) production. After NTLA-2001 is administered intravenously and enters the bloodstream, the LNP becomes opsonised by apolipoprotein E (ApoE) and is then transported through the systemic circulation directly to the liver. The NTLA-2001 lipid nanoparticle (LNP) is absorbed by hepatocytes via the surface LDL receptors and undergoes endocytosis. Subsequent to the breakdown of the LNP and the disruption of the endosomal membrane, the active constituents, namely the TTR-specific single guide RNA (sgRNA) and the messenger RNA (mRNA) encoding Cas9, are liberated into the cytoplasm. The Cas9 mRNA is then translated via the standard ribosomal process, leading to the generation of the Cas9 endonuclease enzyme. The TTR-specific sgRNA engages with the Cas9 endonuclease, thereby forming a CRISPR–Cas9 ribonucleoprotein complex. The Cas9 ribonucleoprotein complex is targeted for nuclear import, and it subsequently enters the nucleus. The 20-nucleotide sequence at the 5′ end of the sgRNA binds to the target DNA, enabling the CRISPR-Cas9 complex to access the gene and induce precise DNA cleavage at the TTR sequence through conformational changes and nuclease domain activation. Endogenous DNA repair mechanisms then join the cut ends, potentially causing insertions or deletions of bases (indels). The formation of an indel may lead to reduced levels of functional mRNA for the target gene due to missense or nonsense mutations, ultimately resulting in decreased production of the target protein.

Conducted by Intellia and spanning sites in the EU, UK and New Zealand, the trial initiated dosing its first participants in late 2020 and is bifurcated into two arms (Ref Reference Gillmore, Gane, Taubel, Kao, Fontana and Maitland106). One arm focuses on patients presenting neuropathy symptoms, while the other targets those with symptoms of cardiomyopathy. Across both arms, data has been collected from 27 participants receiving varying doses. Remarkably, even at the lowest treatment dosage, a substantial reduction (>85%) was reported in toxic protein levels in participants’ bloodstreams, with those at the highest dose experiencing a reduction exceeding 90%. Sustained reduction in TTR protein has been observed over time for all patients, including those for whom a year of findings has been disclosed. Given the correlation between TTR protein levels and disease severity, researchers hold optimistic expectations for participant outcomes. Although some infusion-related side effects were observed, they were temporary and of a non-severe nature (Refs Reference Henderson53, Reference Gillmore, Gane, Taubel, Kao, Fontana and Maitland106). The treatment’s FDA clearance to start a pivotal Phase 3 trial of NTLA-2001 came in October 2023. In November 2023, the company shared updated data from over 60 patients included in the Phase I study; deep and durable serum TTR reduction was evident via a single dose of NTLA-2001, including the initial 29 patients, followed up for 12 months or longer. The drug was generally well tolerated across both arms of trials. The company announced a redosing in June 2024 with a press release, stating a 90% median reduction in serum TTR levels at day 28 in three patients who received the lowest dose in the previous Phase 1 dose-escalation. The company specifies that the MAGNITUDE trial (NCT06128629), which is currently recruiting, will be conducted as a randomised, double-blind, placebo-controlled study to evaluate the safety and efficacy of the drug in 765 patients.

Although an effective strategy, additional research on the long-term safety and effectiveness of NTLA-2001, especially in higher-risk patients, is crucial. This includes continued monitoring to determine if knocking out the TTR gene with this approach leads to a sustained reduction of TTR levels over an extended period. Assessing the suitability of this technology for other eligible diseases will also be significant (Ref Reference Kotit107).

Gene disruption technology to stop inflammatory disease

In hereditary angioedema (HAE), individuals experience severe episodes of inflammation resulting in swelling, typically affecting the arms and legs, face, intestines, or airway. While intestinal swelling may lead to intense pain, nausea, and vomiting, swelling in the airway may present a life-threatening risk (Ref Reference Busse108). HAE attacks typically begin during childhood, and if left untreated, tend to reoccur every 1–2 weeks, each episode lasting for 3–4 days. It is a rare disease affecting approximately 1 in every 50,000 to 1 in every 100,000 individuals. Three distinct categories of HAE are acknowledged, and Types I and II are linked to genetic mutations that affect the production of the C1 inhibitor protein (C1-INH), a serine protease inhibitor that plays a critical role in regulating the kallikrein-kinin system (Ref Reference Wilkerson and Moellman109). Type I HAE is caused by mutations in the SERPING1 gene, leading to reduced levels of functional C1-INH protein. Type II HAE is also caused by SERPING1 mutations but results in normal or elevated levels of a dysfunctional C1-INH protein. HAE with normal C1 inhibitor (HAE-nC1-INH) is a form of HAE where the levels and function of C1-INH are normal. Unlike Type I and Type II HAE, which are caused by mutations in the SERPING1 gene affecting C1-INH, HAE-nC1-INH is associated with mutations in other genes that disrupt the regulation of bradykinin or related pathways. This includes subtypes associated with mutations in genes such as FXII, PLG, or ANGPT1, or cases without identified genetic mutations (Ref Reference Wedner110).

In individuals with a healthy immune system, precise coordination of proteins regulates inflammation, enabling the body to react effectively to threats and injuries. The C1 inhibitor protein plays a pivotal role in suppressing inflammation. However, when C1 inhibitor protein levels are reduced, as in HAE, the bradykinin protein accumulates in the bloodstream. Excess bradykinin, in turn, causes fluid to escape from blood vessels into the body tissues, initiating HAE swelling attacks. Current treatment options include daily oral medications or administration via IV infusions or injections, sometimes needed as frequently as twice a week. Despite regular administration, individuals with HAE may still encounter occasional attacks. Similar to hATTR, angioedema can be acquired but also may be inherited (Ref Reference Ghazi and Grant111).

NTLA-2002 is a CRISPR drug candidate developed by Intellia Therapeutics for HAE, intended to target the KLKB1 gene in liver cells to reduce kallikrein protein production (Ref Reference Kaiser112). The excessive activity of kallikrein results in the overproduction of bradykinin, causing recurrent, severe and potentially life-threatening swelling attacks in HAE. Reduced bradykinin levels provided via lowered kallikrein activity correlate with decreased inflammation and swelling. Administered through a single IV dose, the objective is gene disruption to halt the progression of the disease. Throughout the process, DSB damage is generated in the KLKB1 target gene, and further mutations are initiated as the cell attempts to repair the damage without a corrected template. Severe damage in the gene ultimately may lead to cessation of protein production. In this trial, CRISPR-Cas9 reagents are delivered via LNPs to edit cells in the liver, leveraging the natural tendency of LNPs to accumulate in the liver, thus ensuring precise targeting. The NTLA-2002 therapy shows promise for Type I and II HAE but has limited applicability for HAE with normal C1-INH (e.g., HAE-FXII), as the applicability of this drug to nC1-INH HAE depends on whether kallikrein overproduction plays a significant role in the pathophysiology. Some patients with nC1-INH may not benefit if their swelling episodes are not driven by the kallikrein-bradykinin pathway.

In New Zealand, a range of three doses was administered to 10 participants, and the extended follow-up data has reached over 2 years in the earliest patients dosed. According to Intellia Therapeutics’ update on 2 June 2024, the majority of the patients remained attack-free for over 18 months or longer, with the longest attack-free interval reported as over 26 months for an individual patient post-application. Plasma kallikrein reduction was 60% for the low dose (25 mg), 88% for the medium dose (50 mg), and 95% for the high dose (75 mg) NTLA-2002 application. The treatment has shown good tolerance across all dosage levels, with no severe adverse events (Ref 113). Intellia has recently (22 January 2025) announced the dosing of the first subject in their Phase III trial of NTLA-2002. Termed ‘HAELO’, this randomised, double-blind, placebo-controlled study aims to determine the safety and efficacy of the drug in 60 adults with Type 1 or Type II HAE. The five regulatory designations received by the drug at this time are listed as Orphan Drug (September 2022) and RMAT (March 2023) Designations by the FDA, the Innovation Passport by the UK Medicines and Healthcare products Regulatory Agency (MHRA) (January 2023), Priority Medicines (PRIME) Designation by the European Medicines Agency (October 2023), and Orphan Drug Designation by the European Commission (November 2023) (Ref 114).

While early results from these trials are encouraging, several challenges remain, including the conclusions yet to be driven from a small number of patients. The recently initiated Phase III trial, involving 60 patients, will provide safer conclusions to be drawn in this regard. Other risks include potential off-target effects and immune response to delivery methods like LNPs, which could impact efficacy or cause inflammation. The long-term safety of sustained kallikrein reduction is also unclear. Manufacturing scalability is another concern due to the complexity of mass-producing CRISPR components like Cas9 and guide RNA, coupled with high costs that may limit accessibility. Delivery poses challenges in ensuring precise targeting to the liver and addressing variability in patient factors such as liver health and genetics. Further optimisation is needed to improve safety, delivery, affordability, and broader applicability (Ref Reference Longhurst, Lindsay, Petersen, Fijen, Gurugama and Maag115).

Bacteriophage therapy involving CRISPR-Cas3 for chronic infection

Urinary tract infections (UTIs) are prevalent complications leading to more than 8 million healthcare provider visits annually. The primary culprit is typically E. coli, a common faecal bacterium. UTIs often present with symptoms such as a burning sensation during urination and frequent drives to urinate (Ref Reference Flores-Mireles, Walker, Caparon and Hultgren116). In addition to causing discomfort, these infections can become a concern if they progress to affect the kidneys or if bacteria manage to enter the bloodstream. While most UTIs respond well to a brief antibiotic course, there are instances where antibiotics prove ineffective or the infection persists, referred to as chronic UTIs (Ref Reference Sabih and Leslie117).

Bacteriophages, commonly called phages, are viruses that attack, infect and replicate in bacteria. Their typical mode of action involves injecting genetic material into bacteria and utilising them as factories to generate more phages. Ultimately, the bacteria may undergo bursting, releasing additional copies of the phage. Phages are currently being explored for their potential use against bacterial infections, gaining increased attention in response to the escalating threat of antibiotic resistance. Although the concept dates back about a century, the advent of antibiotics like penicillin and challenges in patenting phages impeded its therapeutic development.

Over the past few decades, phages have been utilised in ‘compassionate treatment’, which involves the use of an unapproved drug or therapy to treat severely ill individuals when no other treatment options are available (Ref Reference McCallin, Sacher, Zheng and Chan118). Differing degrees of success were reported in around 25 documented instances in the last 20 years, although clinical trials are required to evaluate safety and efficacy (Ref Reference McCallin, Sacher, Zheng and Chan118). Phages may offer a distinct advantage of targeting specific types of bacteria, while antibiotics can harm healthy bacteria without discrimination. Thus, phage therapy has the potential for more specific and accurate interventions.

The clinical trial ELIMINATE conducted by Kim et al. utilises CRISPR technology to develop phage therapy against uncomplicated UTIs, as the first rigorously controlled trial in the field (NCT05488340) (Ref Reference Kim, Sanchez, Kime and ousterout119). In this innovative strategy involving the CRISPR-enhanced six-bacteriophage cocktail drug LBP-EC01, bacteriophages are modified to boost their effectiveness against E. coli via a CRISPR-Cas3 system incorporated into their genome for DNA-targeting activity. Experimental findings from animal models with urinary tract and other infections demonstrate that CRISPR-mediated modifications significantly enhance the phages’ ability to eliminate E. coli (Ref Reference Jin, Chen, Zhao, Hu, Wang and Liu120). LBP-EC01 is carefully designed to target the genomes of three E. coli strains responsible for greater than 80% of UTIs, regardless of the antibiotic drug resistance status of the bacteria (Refs Reference Kim, Sanchez, Kime and ousterout119, Reference Kim, Sanchez, Penke, Tuson, Kime and McKee121).

During the phase 1 trial, Locus Biosciences administered the treatment directly to the bladder through a catheter. In February 2021, a Phase 1b trial was completed in the United States, confirming the innovative therapy’s safety and tolerability without any drug-related adverse effects (Ref 122). In 2022, Locus initiated the enrolment of participants for a phase 2/3 trial to test the preliminary efficacy of the drug, with the dosing of the first participant officially announced in September 2022. The aim is to recruit around 800 participants from the United States and the European Union (Ref Reference Anderson123). An update published by the researchers in the Lancet Infectious Diseases in December 2024 reported outcomes from the Part 1 dose regimen selection portion of a 2-part trial examining LBP-EC01, from 39 patients between the ages of 18 and 70, enrolled between August 2022 and August 2023 (Refs Reference Kim, Sanchez, Penke, Tuson, Kime and McKee121, Reference Kim, Sanchez, Penke, Tuson, Kime and McKee124). The trial was held in 6 private clinical sites in the USA. A treatment regimen involving 2 days of intraurethral LBP-ECO1 and 3 days of concurrent LBP-ECO1 intravenous administration along with the oral application of trimethoprim-sulfamethoxazole (TMP-SMX) twice a day was reported to be well-tolerated. Consistent pharmacokinetic profiles in blood and urine were specified in the report, with the treatment providing a fast and durable reduction of E. coli and the clinical symptoms eliminated in evaluable patients.

Although phage therapy is considered safe so far, evaluating possible side effects of phage accumulation will need more studies. Bacteria may possess various mechanisms for evading killing by the phages, though it is argued that bacterial mechanisms used for evasion of killing by bacteriophages may also have an overall reducing effect on their virulence and fitness in the patient. This may be a means of making these treatments successful even in the presence of resistance and even ‘steering’ these bacteria back to states of antibiotic susceptibility (Refs Reference Stacey, De Soir and Jones125, Reference Zulk, Patras and Maresso126).

Editing genes for cardiovascular disease through genetic interruption

Increased low-density lipoprotein cholesterol (LDL-C) has been strongly associated with cardiovascular diseases (CVDs) by many epidemiological and interventional studies as a major risk factor (Ref Reference Jung, Kong, Ro, Ryu and Shin127). The influence of genetics on cholesterol levels is evident in individuals with mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene, leading to familial hypercholesterolaemia (FH). FH is a hereditary condition characterised by dangerously high cholesterol levels irrespective of diet and exercise. As a result, plaque accumulates in the arteries, leading to reduced blood flow or blockage (Ref Reference Arnold and Koenig128). In 2022, Verve Therapeutics initiated a trial targeting patients who are heterozygous for a high-risk subtype of FH (HeFH) with established atherosclerotic cardiovascular disease (ASCVD) and uncontrolled levels of LDL-C, using a lipid nanoparticle (LNP)-delivered base editor system (NCT05398029) (Ref 129). In this specific trial, the in vivo liver base editing medicine (VERVE-101) is designed to introduce a single-letter change in the PCSK9 gene to turn off the disease-causing gene permanently (Ref Reference Oostveen, Khera, Kathiresan, Stroes, Fitzgerald and Harms130).

The initial participant in the phase 1b clinical trial Heart-1 received the treatment in July 2022, only 6 years after Harvard University researchers invented base editing (Refs Reference Komor, Kim, Packer, Zuris and Liu25, 129). Two more participants were dosed by October 2022, and no serious side effects have been noted (Ref 131). Meanwhile, the FDA has placed a clinical hold on the Investigational New Drug (IND) application for VERVE-101, thereby delaying the initiation of a clinical trial for this therapeutic. The FDA’s directive was rooted in the need for Verve to furnish additional preclinical data concerning potency differences between human and non-human cells, potential risks associated with editing germline cells, as well as off-target studies in non-hepatocyte cell types, and available clinical data from the ongoing trial. Having met the requirements, the FDA lifted the clinical hold on VERVE-101 in October 2023 (Ref Reference Armstrong132).

The company reported significant trial findings, indicating a time-averaged reduction in blood PCSK9 levels ranging from 39% to 84% across different doses. Patients in the two higher-dose groups experienced treatment-related adverse effects, including infusion reactions (which were transient and ranged from mild to moderate), temporary asymptomatic increases in liver transaminases, below the upper normal limits of bilirubin, and serious cardiovascular events in those with severe underlying ASCVD. Thirteen patients were dosed in total, with the 3 additional patients dosed in April 2024. In 2 patients with the longest follow-up in the higher-dose cohorts, LDL-C reduction was maintained for 270 days, with the follow-up ongoing. However, Verve has decided to pause enrolment in the trial due to VERVE-101-associated laboratory abnormalities to conduct an investigation (Ref 133). The Clinical Trial Applications (CTAs) in the UK and New Zealand and the Investigational New Drug Application (IND) in the US were announced to be active.

Verve Therapeutics also reports a second PCSK9 gene editor, VERVE-102, developed similarly for PCSK9 inactivation like VERVE-101, but to be delivered using their proprietary GalNAc-LNP technology. This system allows access to the LNPs to deliver the drug to liver cells via either the asialoglycoprotein receptor (ASGPR) or the low-density lipoprotein receptor (LDLR). It comprises an adenine base editor-expressing messenger RNA and an optimised RNA targeting PCSK9. The drug is currently tested in the Heart-2 open-label Phase 1b clinical trial in two patient populations: adults with heterozygous familial hypercholesterolaemia (HeFH) and adults with premature coronary artery disease (CAD). In May 2024, the company announced the dosing of the first patient in the Heart-2 Phase 1b clinical trial where VERVE-102 is being evaluated. As of October 2024, the company already reported 7 participants dosed in the Heart-2 clinical trial across two cohorts.

Verve Therapeutics’ VERVE-201, on the other hand, is an investigational CRISPR base editing tool targeting the inactivation of the ANGPTL3 gene in liver cells via alteration of a single DNA base, thus turning off its production by the liver to reduce LDL-C and triglyceride levels. Preclinical data reveals on-target precise and potent editing in primary human hepatocytes, Ldlr^−/− and wild-type mice, and non-human primates, displaying its potential in treating severe or complete LDLR deficiency that is evident in homozygous FH (HoFH). The first participant in their clinical trial for VERVE-201 was recently announced to be dosed in November 2024 (Ref 134). The challenge with LNP-mediated delivery to the liver is a major problem for patients with HoFH due to complete deficiency in the LDLR in these patients. This problem is overcome by the use of GalNAc-lipid nanoparticles to enable non-LDLR-dependent hepatic delivery (Refs 135, Reference Kasiewicz, Biswas, Beach, Ren, Dutta and Mazzola136).

New technology in progress

Prime editing and CRISPR-Cas effectors as next-generation CRISPR technologies

Emerging technologies in genome editing are pushing the boundaries of genetic engineering, eliminating or reducing several limits associated with the therapeutic applicability of the conventional CRISPR technology, offering more precise, versatile and efficient tools for manipulating genetic material. Among these innovations are prime editing (PE) and approaches involving CRISPR-Cas effectors such as Cas12 and Cas13, each representing a significant step forward in their respective fields.

PE is a breakthrough technology that goes beyond traditional CRISPR-Cas9 by offering unprecedented precision in genome editing (Ref Reference Anzalone, Randolph, Davis, Sousa, Koblan and Levy140). It is the first precise genome-editing approach, allowing all 12 possible base-to-base conversions, plus insertions or deletions, with minimised off-target effects (Figure 9). It directly rewrites the target DNA sequence without relying on DSBs or donor DNA templates and functions without the need for a precisely positioned PAM sequence for nucleotide targeting, offering more flexible and precise editing (Ref Reference Chen and Liu141). The PE guide RNA (pegRNA) not only guides Cas9 to the target DNA but also provides the necessary template for the insertion, deletion, or conversion of specific DNA sequences. Since the report of the initial version, several new generations and variants of PE have been developed to enhance efficiency through modification of the involved Cas9 and RT enzymes, the pegRNA/sgRNA combination, the structure of the pegRNA, and host protein expression regulation via epigenetic mechanisms (Ref Reference Hosseini, Mallick, Makinen and Yla-Herttuala142). Despite its precision, at its early stage of development, PE still faces challenges in terms of efficiency and delivery, and a universal PE mechanism needs to be optimised. G1 state was shown to be the most suitable step for cell modification for the PE process. Adjusting the endogenous host factors to make the cells permissive for this editing is listed among the future challenges. Other challenges also remain, such as establishing an optimised universal vector for the delivery of the large PE complexes along with the long pegRNAs and regulatory elements and managing immunity, particularly against the pathogen-associated molecular patterns (PAMPs) possessed by the components of the PE machinery (Ref Reference Hosseini, Mallick, Makinen and Yla-Herttuala142). Overall, PE is still a newer technology, relatively in its infancy, that may require additional optimisation and expertise to be transferred fully and effectively into the clinic (Ref Reference Anzalone, Randolph, Davis, Sousa, Koblan and Levy140). The first clinical trial application involving a prime editor received FDA clearance in April 2024 (Refs Reference Godbout and Tremblay143, 144). The study is held by Prime Medicine, Inc., and is structured as an open-label, single-arm, multicenter Phase 1/2 study testing the efficacy and safety of the transplantation of ex vivo-modified prime-edited autologous CD34+ stem cells (PM359) in autosomal recessive Chronic Granulomatous Disease (CGD) caused by NCF1 (Neutrophil Cytosolic Factor 1) gene mutations (NCT06559176). The company is developing PE-based strategies for several other diseases, including X-linked CGD, Wilson’s disease, and cystic fibrosis, as specified in their pipeline.

Figure 9. Mechanism of Prime Editing Approach (Ref Reference Anzalone, Randolph, Davis, Sousa, Koblan and Levy140). Cell transfection involves introducing both the pegRNA and the fusion protein for genomic editing. This is typically achieved by delivering vectors into the cells. Once inside, the fusion protein initiates genomic editing by cleaving the target DNA sequence, revealing a 3′-hydroxyl group. This group serves as the starting point (primer) for the reverse transcription of the RT template section of the pegRNA. This process gives rise to an intermediate structure that branches out, featuring two DNA flaps: a 3′ flap containing the freshly synthesised (edited) sequence and a 5′ flap holding the unnecessary, unedited DNA sequence. Subsequently, structure-specific endonucleases or 5′ exonucleases cleave the 5′ flap. This sequential process facilitates the ligation of the 3′ flap, resulting in a heteroduplex DNA comprised of one edited strand and one unedited strand. The reannealed double-stranded DNA exhibits nucleotide mismatches at the editing site. To rectify these mismatches, cells utilise the inherent mismatch repair mechanism, which leads to two potential outcomes: (i) the information in the edited strand is replicated into the complementary strand, thus permanently incorporating the edit; (ii) the original nucleotides are reintegrated into the edited strand, effectively excluding the edit.

In the realm of genome engineering, the term ‘CRISPR’ or ‘CRISPR-Cas’ is commonly employed as a broad reference encompassing various systems such as CRISPR-Cas9, Cas12, Cas13 and others. These systems are programmable to target specific genetic code stretches, enabling precise DNA editing and serving diverse purposes, including the development of new diagnostic tools with over 200 engineered variants currently present. Cas12 effectors (also known as Cpf1) exhibiting a variety of sizes, PAM requirements, substrate recognition patterns and interference mechanisms were classified as a unique type V CRISPR–Cas system following the discovery of the Cas12a nuclease as an alternative to Cas9 (Ref Reference Wu, Adiego-Perez and van der Oost145). More than a dozen distinct Cas12 subtypes have reported since (Ref Reference Teng, Cui, Feng, Guo, Xu and Gao146). They share many features with Cas9 but have some key distinctions, such as the DNA-cutting mechanism. Unlike Cas9, which makes a blunt cut across both strands of DNA, Cas12 generates staggered (sticky) ends, which can facilitate more precise integration of foreign DNA into the genome. This characteristic is particularly useful for certain types of genome-editing applications, such as gene knock-ins, where inserting a gene is more efficient with staggered cuts. Also, Cas12 recognises a different PAM than Cas9; while Cas9 typically requires a 5′-NGG-3′ PAM sequence, Cas12 recognises a 5′-TTTV-3′ PAM, where V is any base except for T. This expands the range of targetable genomic sequences, offering additional flexibility where Cas9 may not work as effectively. Cas12 exhibits higher specificity for its target DNA compared to Cas9, which can reduce off-target effects. Additionally, Cas12 has a collateral cleavage activity; once it cuts its target DNA, it can cleave single-stranded DNA non-specifically, which could have potential applications in diagnostics and biosensing. The Doudna lab employed Cas12a’s non-specific single-stranded DNA degradation to establish the DNA Endonuclease Targeted CRISPR Trans Reporter method, referred to as DETECTR (Ref Reference Chen, Ma, Harrington, Da Costa, Tian and Palefsky147). It leverages the indiscriminate cleavage and degradation of nearby ssRNA and single-stranded DNA (ssDNA), which triggers the cleavage and activation of a reporter. The observable signal produced by this reporter can be assessed and measured, allowing for the identification and quantification of the presence of DNA, RNA, or a specific mutation. In summary, CRISPR-Cas12 expands the range of editable genomic sites and offers distinct advantages for precise DNA insertion and lower off-target effects, making it a promising tool for genome engineering, gene therapy and synthetic biology, already utilised in various clinical trials (Table 1). While constituting a breakthrough in human gene editing, the immunogenicity of the Cas effectors remains a problem to be solved via advanced protein engineering and/or improved delivery systems (Refs Reference Chew78, Reference Wu, Adiego-Perez and van der Oost145).

CRISPR-Cas13 is a single-strand RNA-targeting genome-editing tool, distinguishing itself from other CRISPR systems like Cas9 and Cas12, which target DNA (Ref Reference Huang, Fang, Zhou, Gong and Xiang148). It can be programmed to work on specific RNA molecules for degradation or modification without modifying the genomic DNA, for induction of temporary changes to RNA or when DNA editing may be challenging. By enabling RNA-specific editing, CRISPR-Cas13 adds a new dimension to genetic engineering, allowing post-transcriptional alteration of gene expression to explore gene regulation mechanisms, developing RNA-based therapies and improving diagnostics. Through its in vitro collateral activity, Cas13 not only specifically cleaves its target RNA but also indiscriminately degrades any nearby RNA, which makes it useful for diagnostic applications to develop quick and highly sensitive nucleic acid detection methods (Refs Reference Gootenberg, Abudayyeh, Lee, Essletzbichler, Dy and Joung149, Reference Bot, van der Oost and Geijsen150). This has been used in various platforms for rapid and sensitive detection of RNA pathogens, such as viruses. The Zhang lab recently introduced the Specific High Sensitivity Enzymatic Reporter UnLOCKING methods, known as SHERLOCK and SHERLOCKv2, for in vitro precise diagnostics which aim to provide quick, multiplexed ultra-sensitive detection of RNA or DNA in relevant samples (Refs Reference Gootenberg, Abudayyeh, Lee, Essletzbichler, Dy and Joung149, Reference Kellner, Koob, Gootenberg, Abudayyeh and Zhang151). SHERLOCK uses the Type VI CRISPR system (Cas13a), while SHERLOCKv2 utilises types III, V and VI (Csm6, Cas12a and Cas 13, respectively) for improved efficiency in a single reaction to detect four different DNA or RNA fragments (Ref Reference Zahra, Shahid, Shamim, Khan and Arshad152). Furthermore, Cas13-mediated approaches are suitable for use in various treatment approaches. A CRISPR/Cas13-mediated RNA targeting therapy (HG202) against neovascular age-related macular degeneration (nAMD) is currently recruiting patients for an early phase 1 study (SIGHT-1; NCT06031727). Perturbation of vascular endothelial growth factor (VEGF) is given as the primary cause of nAMD, where overexpression of VEGF results in the abnormal growth of choroidal neovascularisation (CNV). HG202 employs a single AAV vector to partially reduce VEGFA expression to inhibit CNV formation in AMD patients. Besides the great potential, unforeseen risks and effects remain in relation to the collateral activity of Cas13. Other challenges include the requirement for optimisation of delivery systems and potential immunotoxicity and off-target effects in vivo with long-term, constitutive expression of Cas13 proteins (Ref Reference Zhu, Zhou, Wen, Qiao, Li and Yao153).

An overview of the challenges associated with CRISPR technologies

While genome editing tools like CRISPR-Cas have revolutionised our ability to target and modify specific genomic sequences, their application is not without challenges, as we also specified in relevant sections throughout the text. Overall, reducing off-target effects, refining delivery systems for better efficiency and accuracy, and enhancing the safety of applications are issues that still need to be addressed, along with ethical considerations. Also, possible variability in editing efficiencies and the complexities of certain genomic regions mean that not all sequences can be easily or reliably manipulated.