Advancing CRISPR genome editing into gene therapy clinical trials: progress and future prospects

Abstract

Genome editing has recently evolved from a theoretical concept to a powerful and versatile set of tools. The discovery and implementation of CRISPR-Cas9 technology have propelled the field further into a new era. This RNA-guided system allows for specific modification of target genes, offering high accuracy and efficiency. Encouraging results are being announced in clinical trials employed in conditions like sickle cell disease (SCD) and transfusion-dependent beta-thalassaemia (TDT). The path finally led the way to the recent FDA approval of the first gene therapy drug utilising the CRISPR/Cas9 system to edit autologous CD34+ haematopoietic stem cells in SCD patients (Casgevy). Ongoing research explores the potential of CRISPR technology for cancer therapies, HIV treatment and other complex diseases. Despite its remarkable potential, CRISPR technology faces challenges such as off-target effects, suboptimal delivery systems, long-term safety concerns, scalability, ethical dilemmas and potential repercussions of genetic alterations, particularly in the case of germline editing. Here, we examine the transformative role of CRISPR technologies, including base editing and prime editing approaches, in modifying the genetic and epigenetic codes in the human genome and provide a comprehensive focus, particularly on relevant clinical applications, to unlock the full potential and challenges of gene editing.

Keywords: CRISPR/Cas; gene editing; gene therapy; genetic diseases; medical genetics.

Figure 1.

The structure and mechanism of action of the most commonly used programmable nucleases (Ref 3). (a) Zinc-Finger Nucleases (ZFNs). (b) Transcription Activator–Like Effector Nucleases (TALENs). (c) Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-Associated protein 9 (CRISPR-Cas9).

Figure 2.

The potential applications of CRISPR-Cas systems for editing genomes and base editing technology (Refs 14, 15). Panel (a): CRISPR-Cas9 functions via a guide RNA molecule to target specific DNA sequences and a Cas9 protein to cleave the DNA at those target sites. This process allows for precise genome editing by either inducing DNA repair mechanisms to create mutations or by facilitating the insertion of new genetic material at the targeted location. Genome modification through CRISPR-Cas systems relies on the two primary pathways for repairing double-strand breaks (DSBs). Indel mutations and gene deletions result from the predominant nonhomologous end-joining (NHEJ) repair pathway. On the other hand, gene insertion, correction, and replacement occur through the homology-directed repair (HDR) pathway, utilising a DNA donor template. Panel (b): Base Editing Technology. The mechanism of the Cytosine Base Editor (CBE) is outlined, with key components labelled in text boxes. In the presence of the optional uracil glycosylase inhibitor (UGI), the U•G intermediate is safeguarded against excision by uracil DNA glycosylase (UDG), enhancing the efficiency of the final base-edited DNA outcome. The nickase version of Cas9 (Cas9n) induces a nick on the top strand (indicated by the blue arrow), while the cytidine deaminase transforms cytosine into uracil. The comprehensive conversion of a C•G to T•A base pair is accomplished through the specified steps. The mechanism of Adenine Base Editor (ABE) mirrors that of CBE, with the distinction that the UGI domain is not included in the ABE architecture. ABE-mediated editing leads to the conversion of an A•T to G•C base pair through an inosine-containing intermediate. Key elements include guide RNA (gRNA), protospacer adjacent motif (PAM), target A (desired base substrate for ABE), and target C (desired base substrate for CBE). The PAM sequence is representatively shown as 3 bp. dsDNA: double-stranded DNA, ssODN: single-stranded oligodeoxynucleotide.

Figure 3.

Strategies for gene modification therapies in humans. This figure illustrates two key approaches for therapeutic gene editing, as ex vivo and in vivo. Ex vivo gene editing involves the isolation and modification of patient cells using CRISPR/vector technology within a controlled in vitro environment. The genetically modified cells undergo proliferation before being transplanted back into the patient. In contrast, in vivo gene editing directly administers therapeutic genes using viral or non-viral vectors through intravenous or intraocular injection. The depicted gene editing methods include ribonucleoprotein (RNP), non-viral vectors (nanoparticles and plasmids), and viral vectors (adenovirus, lentivirus, and adeno-associated virus), showcasing the diverse strategies employed in the pursuit of targeted gene modifications.

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.

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 71). 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.

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 88). 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.

Figure 7.

Schematic representation of VC-02 Macroencapsulation Device (Ref 91). 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.

Figure 8.

The mechanism of in vivo gene editing for Transthyretin Amyloidosis (Ref 106). 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.

Figure 9.

Mechanism of Prime Editing Approach (Ref 140). 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.