Gene editing and CRISPR in the clinic: current and future perspectives

Abstract

Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application.

Keywords: CRISPR; clinical trial; gene activation; genome editing; transcription activator-like effector nucleases; zinc finger nuclease.

Figure 1. Genome editors can be used therapeutically in several ways, and both ex vivo and in vivo delivery for somatic genome editing have advanced to clinical trial

Ex vivo: cells can be extracted from the patient or donor modified in the laboratory and then infused into the patient. In vivo: delivery vehicles, including viral vectors and nanoparticles can be loaded with the genome editor and then injected into the patient either systemically, which results in liver editing primarily, or into the location of interest, for example, the eye.

Figure 2. Trends in genome editor use in clinical trials

Genome editing trials registered in the U.S. clinical trials database by year and selected genome editor (A), or delivery method (B) and delivery method grouped by in vivo or ex vivo use (C). Some trials did not have a clear delivery methodology (labeled as unknown). These unknown delivery methods are all ex vivo delivery making electroporation the most likely method. Data were accessed 1/01/2020.

Figure 3. Genome editing used to enhance ACT of T cells for cancer therapy

(A) Genome editing (highlighted by green arrows) is being explored to create universal donor T cells to serve as the basis for TCR and CAR T-cell engineering. Genome editing is also being explored to enhance the survival and/or efficacy or prevent self-targeting of both natural (circulating T cells and TILs) and engineered (TCR and CAR) T cells. (B) TCR engineered T cells have the addition of a second set of TCR α and TCR β genes (highlighted in red and pink) which are present in addition to the naturally occurring TCR α and TCR β genes (highlighted in blue). (C) CAR engineered T cells have a chimeric cell receptor with an scFv composed of variable heavy and light chains (V_H and V_L) of an antibody as the extracellular portion fused to intracellular T-cell signaling domains to cause T-cell activation upon interaction with the targeted cell surface marker. Genome editing is also being applied to circulating T cells collected from a patient’s blood (D) and to isolated tumor infiltrating lymphocytes (E), which utilize the native T-cell targeting to destroy tumor cells. Abbreviation: scFv, single-chain variable fragment.

Figure 4. Mechanisms of Cas9 immunity seen in experimental studies

Innate immunity is mediated by pattern recognition receptors present on the cell surface (shown in blue), in the endocytic vesicles (shown in green) and cytoplasm (shown in red) of phagocytic cells. Humoral immunity is mediated by antibodies which can neutralize Cas9 protein or delivery vehicles. Both IgG and IgM antibodies have been seen to Cas9 exposure. Cellular immunity is mediated by display of peptides from intracellular proteins on cell surface receptors that can be recognized by cytotoxic T cells mediating killing of Cas9 expressing cells.

Figure 5. Engineering hyperaccurate CRISPR systems

Several approaches have been used to improve the function of Cas9 proteins and reduce off-target effects. Cas9 function has been modified through the rational design and engineering of a higher fidelity nuclease, modifying the sgRNA for increased stability, directed evolution toward hyperaccuracy, or fusing Cas9 with programmable DNA-binding domains.

Figure 6. CRISPR/Cas9 regulators of gene expression

(A) The nuclease dead version of Cas9, dCas9, is directed to a genomic locus with an sgRNA and inhibits transcription of a target gene. (B) Fusion of the KRAB domain to dCas9 causes stronger gene repression. CRISPR-mediated gene activation (CRISPRa) involves recruiting transcriptional activators to a genomic locus using a dCas9–sgRNA scaffold. (C) The simplest CRISPRa system is dCas9 fused to VP64. More complex CRISPRa systems include (D) Sun-Tag, (E) VPR and (F) SAM, and these involve the recruitment of multiple transcriptional activators to further enhance gene expression. Abbreviations: KRAB, Krüppel-associated box domain of Kox1; SAM, Synergistic Activation Mediator.