Medical uses of CRISPR-Cas9 gene editing

Further clinical trials are underway for hemoglobinopathies (including SCD and TDT), diseases reduced hemoglobin levels that compromise oxygen delivery. BRL-101s treatment of SCD/TDT also targets the BCL11A gene, disrupting BCL11A's enhancer, thereby lowering transcription and increasing HbF production. With a safety profile similar to that of the required autologous transplantation, BRL-101 enables transfusion independence and increased HBF and HBA levels (NCT06287099,NCT06287086,NCT06300723,NCT05577312) (Fu et al., 2023; Fu et al., 2022). Other hemoglobinopathy therapies directly target the HBB mutation. GPH101 edits HSPCs to reverse HBB's valine to glutamic acid change in β-thalassemia (NCT04819841) (Kanter et al., 2021).

CRISPR-Cas9 therapies combating cancers often use Chimeric Antigen Receptor T cell (CAR-T) therapy, a type of immunotherapy increasingly employing CRISPR's precision. In CAR-T therapy, patient (autologous) or donor (allogenic) T cells are edited ex-vivo to recognize and kill cancer cells. A synthetic gene is inserted that encodes a chimeric antigen receptor (CAR) containing an antigen binding domain targeting cancer cell surface proteins. In leukemias, lymphomas and myelomas, these cancer cell surface antigens include various Cluster of Differentiation (CD) genes, which create important functional proteins on the surface of white blood cells (Zhang et al., 2017).

In multiple myeloma, a B cell-derived cancer, the over-expressed B-cell Maturation Antigen (BCMA), essential for B cell maturation, survival, and proliferation, is targetted (NCT04244656) (Rinaldi et al., 2022). The mutation or overexpression of Epidermal Growth Factor Receptor (EFGR) is often seen in cancers, increasing uncontrolled cell growth, making it another important antigen target in some cancers (NCT04976218) (Sasaki et al., 2013). An important target for mesotheliomas, cancers derived from the lining of different organs, is the mesothelin gene. This is an especially attractive target because this gene's expression is limited to mesothelial cells, a cell type that is dispensible (NCT03545815,NCT03747965,NCT05812326) (Hassan et al., 2016).

Manipulating additional genes beyond the CAR insertion has improved CAR-T therapy's effectiveness and longevity. Most disrupt the T cell receptor α chain (TRAC) gene by inserting the CAR into it, which also ensures uniform CAR expression. Without the α-subunit, the T cell receptor (TCR) is not functional. This increases therapy effectiveness by reducing spontaneous activation and differentiation of the modified T cells, avoiding T cell exhaustion. In addition, the lack of the TCR protein, which normally recognizes foreign material, helps avoid graft vs. host disease (GvHD), opening up CAR-T therapy to allogenic sources, reducing costs and timelines, and standardizing treatment (Eyquem et al., 2017; Lonez and Breman, 2024; Terrett et al., 2023).

CRISPR Therapeutics's allogenic CAR-T therapies are being improved by editing additional genes beyond the CAR insertion and its disruption of TRAC. CTX110 (NCT04035434), targeting CD19⁺ cancers (B cell leukemias and lymphomas), and CTX130 (NCT04502446 and NCT04438083), targeting CD70⁺ cancers (T cell lymphomas and renal cell carcinomas), both knockout the β2M gene, a subunit of the major histocompatibility complex class 1 (MHC-I) subunit. The broken MHC-I protein prevents donor CAR-T cells from being recognized and destroyed by the patient's immune system (host vs. graft disease (HvGD) (Terrett et al., 2023; McGuirk et al., 2022).

The next-generation drug versions, CTX112 for CD19⁺ cancers (NCT05643742) and CTX131 (NCT06492304) for CD70⁺ cancers, improve on their counterparts through additional gene knockouts. Regnase-1 normally tamps down on cytokine secretion and, by extension, the immune system. The Regnase-1 knockout, therefore, keeps the immune response strong. Likewise, Transforming Growth Factor-beta (TGF-β) receptor type 2 (TFGBR2) knockouts create a CAR-T cell without a receptor to recognize the (TGF-β) produced in the tumor microenvironment that would normally inhibit the T cell. In CTX131, CD70 is also knocked out, preventing fratricide in CD70-targeting CAR-T cells. The improvements are stark. For example, CTX112 is 10X more potent than CTX110 with improvements in persistence and anti-tumor effects (Kalaitzidis et al., 2023).

Further genes been identified whose disruption in CAR-T cells can lead to therapeutic improvements (Moradi et al., 2024; Feng et al., 2024). Knockout of the Programmed Cell Death Protein 1 (PDCD-1) gene can help keep anti-tumor activity strong and avoid immune suppression and T cell exhaustion. PDCD-1 creates the PD-1 protein. When PD-1 binds its ligand (PD-L1), it acts as a brake, inactivating T cells. Tumors take advantage by overexpressing PD-L1, allowing them to inactivate immune cell that recognized the cancer, thereby evading the antitumor immune response and leading to T cell exhaustion (Munari et al., 2021; Moradi et al., 2024). Several clinical trials (NCT03545815, NCT03747965, NCT05812326) deploy PDCD-1 knockout CAR-T cells against mesothelin + breast and other solid tumors. In one case (NCT03747965), the GC008t therapy stabilized disease in four patients and achieved tumor shrinkage for two patients, though engraftment could be improved (Wang et al., 2020).

One autologous clinical trial (NCT05566223) uses CRISPR-Cas9 to knockout the CISH (Cytokine-induced SH2 protein) gene in tumor infiltrating lymphocytes (TILs), a type of T cell that penetrates solid tumors. CISH limits T cell activation and signaling, so its disruption keeps anti-tumor responses high. This therapy treats non-small cell lung cancer (NSCLC), which accounts for ∼85% of diagnosed lung cancers, with lung cancers being the leading cause of cancer-related deaths globally (Gridelli et al., 2015).

CRISPR-Cas9 is also used to alter CAR-T cells to cope with concurrent monoclonal antibody treatment. One autologous therapy (NCT05662904) treats acute lymphoblastic leukemia (ALL) by inactivating the CD33 gene in the patient's HSPCs to make them immune to the CD33-specific antibody-drug conjugate Gemtuzumab-ozogamicin (GO), allowing escalation of GO doses (Godwin et al., 2017). In another study, PBLTT52CAR19 targets CD19⁺ pediatric B cell ALL (NCT04557436). The disruption of the CD52 gene allowed the concurrent use of Alemtuzumab (Drugs.com, 2024), an anti-CD52 monoclonal therapy. Four of six patients showed CAR-T cell proliferation, achieved remission, and then received allogenic stem cell transplantation for a more permanent therapy (Ottaviano et al., 2022).

CRISPR-cas9 editing can also introduce safety switches into cancer therapies to avoid serious side effects, including Cytokinin Release Syndrome (CRS) and immune cell-associated neurotoxicity syndrome (ICANS) (Xiao et al., 2021). CT125A is an autologous CAR-T cell therapy that targets CD5⁺ hematologic malignancies, including T cell-derived leukemias and lymphomas (NCT04767308). The endogenous CD5 gene was disrupted using CRISPR-Cas9 to avoid fratricide. A safety switch was added to the CAR-T cells by editing a truncated epidermal growth factor receptor (tEGFR) into the genome. The resulting receptor, though not functional, was still recognized by Cetuximab, a monoclonal antibody therapy, killing the CAR-T cells when administered to the patient. Clinical outcomes were mixed. One patient went into complete remission but died of sepsis and multi-organ dysfunction. The other two patients achieved partial remission but one relapsed. As expected, the therapy caused CRS, but cetuximab administration eliminated most (but not all) CAR-T cells, limiting toxicity. Nevertheless, this study showed that safety switches can be viable strategies for limiting patient exposure to therapies with dangerous side effects (Lin et al., 2024).

Improvements over CARs are being tested, including STAR (Synthetic TCR and Antigen Receptor) T cell therapy. STAR-T therapy uses a construct that mimics TCRs, increasing sensitivity to the cancer-presented antigens, which is especially important in solid tumors with low antigen density (Huang et al., 2024). Two related studies (NCT05631912:autologous and NCT06321289: allogenic) are trialing CD19-targeting STAR-T therapy for B cell non-Hodgkin's lymphoma. Additional knockouts of TRAC, PDCD-1, human leukocyte antigen (HLA)-A/B, and Class II Transactivator (CIITA) strengthened the intervention. In addition to reducing the immune suppression, delaying T cell exhaustion, and increasing anti-tumor activity with the TRAC and PDCD-1 knockouts, knockouts of HLA-A/B and CIITA, which are subunits of MHCI and MHCII proteins, respectively, reduce the recognition of allogenic STAR-T cells as foreign, thereby reducing the risk of GvHD.

These are some of many promising CRISPR-Cas9-based cancer therapies and strategies. The number of antigen targets is expanding, additional constructs are improving on CARs, and therapies are becoming more sophisticated with additional gene edits to improve longevity and safety and keep immune and anti-tumor functions high.

CRISPR-Cas9 therapies can also fight infectious disease, either by targeting host or pathogen genes. Two clinical trials explore unique methods to treat Acquired Immunodeficiency Syndrome (AIDs), caused by human immunodeficiency virus I (HIV-1). These therapies target the host CC chemokine receptor 5 (CCR5) gene, which is one of the co-receptors that HIV-1 uses to enter the host's CD4⁺ lymphocytes, thereby destroying a critical part of the host's immune function. A frameshifting 32-nt deletion in CCR5 occurs naturally in a small proportion of the human population. This CCR5-\upDelta 32 mutation, when homozygous, prevents HIV-1 from entering the cell, allowing infected individuals ("HIV controllers") to live with the virus (Oppermann, 2004; Carrington et al., 1997).

One allogenic study (NCT03164135) used CRISPR-Cas9 to modify donor HSPCs, ablating the CCR5 receptor to make the immune cells resistant to HIV-1. This study was designed for HIV patients who also had a hematologic malignancy that required stem cell transplantation, creating an opportunity to simultaneously test CCR5 ablation with minimal additional risk to the patient. One HIV-positive patient in this study had ALL. Transplantation and long-term engraftment was achieved. However, CCR5 was disrupted in only ∼5% of lymphocytes (Xu et al., 2019).

Another AIDS therapy, EBT-101, uses CRISPR-Cas9 to disrupt the HIV-1 genome in aviremic patients (patients with latent infections and no detectable blood virus levels (NCT05144386). Initial results met safety benchmarks and temporarily suppressed viral reservoirs (Johnson, 2024).

Persistent human papillomavirus (HPV) infection, the major cause of cervical cancer, is also being targeted by CRISPR-Cas9 therapies. The viral E6 and E7 oncoproteins inactivate host tumor suppressor genes promoting uncontrolled cell growth (Narisawa-Saito and Kiyono, 2007). Although small interfering RNA targeting of these oncogenes may temporarily inhibit HPV, it does not destroy the viral genes (Hu et al., 2015). Gene editing by administration of a CRISPR-Cas9 E6/E7-targeting plasmid in a gel reduced E6/E7 DNA and expression, initiated cell death, and prevented tumor growth (NCT03057912) (Hu et al., 2014).

The SARS-CoV-2 virus, which causes COVID-19, is targeted in a study that uses CRISPR-Cas9 to ablate the host PDCD1 and ACE2 receptor genes in CD8⁺ virus-reactive memory T cells (NCT04990557). PDCD-1 was knocked out because its upregulation during COVID-19 infection, even in patients with mild symptoms, promotes T-cell exhaustion. Knocking out the ACE2 receptor removes SARS-CoV-2's main entry path into the modified T cell (Scialo et al., 2020).

CRISPR-Cas9 therapies are also beginning to target bacterial pathogens. One study uses CRISPR-Cas9 to disrupt virulence and β-lactam antibiotic resistance genes in Enterobacteriaceae genomes (NCT05850871). Because antibiotic resistance genes are horizontally transferred between species, including pathogens that cause different diseases, individual therapies could potentially target multiple pathogens and diseases.

CRISPR-Cas9 therapies work well for eye diseases because they can be injected directly into the relevant eye tissue. In Intraocular Hypertensive Primary Open Angle Glaucoma (POAC), increased intraocular pressure damages the optic nerve, leading to blindness (Quigley et al., 1983). Dominant mutations in the cytosketetal myocilin (MYOC) gene, which is expressed in the trabecular meshwork where intraocular pressure is regulated, can cause POAC. The BD113 therapy is delivered in a virus-like particle (VLP) by eye injection to knockdown or knockout the mutated MYOC gene, reducing the amount of mutated protein (NCT06465537).

Another VLP therapy (BD111) is injected into the cornea to treat recalcitrant herpes stromal keratitis, which can cause infectious blindness (NCT04560790). The therapy uses CRISPR-Cas9 to disrupt the herpes simplex virus type 1 (HSV-1) genome. No HSV-1 was detected in follow-ups, averaging 18 months (Wei et al., 2023).

Reinitis pigmentosa results in rod cell loss, leading to night blindness, and the gradual loss of cone cells, leading to tunnel vision or blindness. The therapy (ZVS203e) is administered by subretinal injection and fixes a causal rhodopsin (RHO) gene mutation to create a functional protein that is activated under low light conditions (NCT05805007) (Nathans and Hogness, 1984; National Library of Medicine, 2025).

Another CRISPR-Cas9 therapy (EDIT-101) targets Leber Congenital Amaurosis 10 (LCA10) (NCT03872479). A homozygous mutation in the centrosomal protein 290 (CEP290) gene causes retinal degeneration leading to blindness or severe vision loss at birth or shortly thereafter (den Hollander et al., 2006). The mutation causes an additional splice site that forms a cryptic (additional) exon. Initial clinical trial results established safety and 75% of participants showed improved vision.

Hemophilia B is a bleeding disorder caused by a mutated coagulation Factor IX (FIX) gene that results in insufficient FIX (Kurachi and Kurachi, 2000). CRISPR-Cas9-based therapies insert wildtype FIX gene into liver and B cells, enabling clotting factor production (NCT06379789,NCT06611436).

Hereditary Angioedema (HAE) results in debilitating or fatal swelling under the skin. Treatments target kallikrein, a protease encoded by the KLB1 gene, which causes swelling when overproduced in blood plasma (Longhurst et al., 2022; Banerji et al., 2017). NTLA-2002 is a CRISPR-Cas9-based therapy that disrupts KLB1 in liver cells, reducing plasma kallikrein levels (Longhurst et al., 2022). Initial results established safety and showed a reduction in plasma kallikrein levels (NCT05120830,NCT06634420).

Transthyretin amyloidosis is a disease resulting from accumulation of misfolded proteins from a mutated or wildtype transthyretin (TTR) gene, into harmful amyloid fibril deposits, leading to polyneuropathy and/or cardiomyopathy Ruberg and Berk (2012). NTLA-2001 (NCT04601051), delivered in vivo through lipid nanoparticles, showing that systemic in vivo gene editing is possible (Gillmore et al., 2021).