In vivo CRISPR gene editing in patients with herpetic stromal keratitis

Abstract

In vivo CRISPR gene therapy holds large clinical potential, but the safety and efficacy remain largely unknown. Here, we injected a single dose of herpes simplex virus 1 (HSV-1)-targeting CRISPR formulation in the cornea of three patients with severe refractory herpetic stromal keratitis (HSK) during corneal transplantation. Our study is an investigator-initiated, open-label, single-arm, non-randomized interventional trial at a single center (NCT04560790). We found neither detectable CRISPR-induced off-target cleavages by GUIDE-seq nor systemic adverse events for 18 months on average in all three patients. The HSV-1 remained undetectable during the study. Our preliminary clinical results suggest that in vivo gene editing targeting the HSV-1 genome holds acceptable safety as a potential therapy for HSK.

Keywords: CRISPR-Cas9; herpes simplex virus; herpetic stromal keratitis; in vivo gene editing; lentiviral particle.

Graphical abstract

Figure 1

The antiviral mechanism of CRISPR in human corneas HELP is a lentiviral viral particle carrying SpCas9 mRNA and a gRNA-expression cassette. The SpCas9 protein translated from mRNA directly and the gRNA expressed from the reverse transcribed viral DNA assemble into ribonucleoproteins (RNPs), which cleave the HSV-1 genome at UL8 and UL29 loci for degradation. A total of 0.2 mL (2.4 μg of p24) of HELP formulation was injected into the recipient graft bed in six to eight locations by using a 27G ophthalmic syringe after corneal transplantation.

Figure 2

Preclinical results of HELP eliminating HSV-1 in tissue culture of a healthy cornea (A) Flowchart for evaluating the antiviral effects of HELP in HSV-1-infected healthy human cornea. (B) Schematic illustration of the HSV-1 genome and gRNA loci. TRL, terminal repeat long; UL, unique long; IRL, internal repeat long; IRS, internal repeat short; US, unique short; TRS, terminal repeat short. (C) qPCR analysis of HSV-1 genome (fold change); p < 0.0001. (D) HSV-1 titer of supernatants from human corneal cultures measured by plaque assay; mock versus ACV and HELP, p < 0.0001, respectively. (E) Immunohistochemistry analysis of HSV-1 dissemination in a human cornea; scale bar, 20 μm. (F) The profile of HELP-induced mutations in HSV-1 UL8 and UL29 loci in a human cornea. Frequencies >0.035% and 0.07% in UL8 and UL29 loci are shown, respectively. The minus symbol indicates a deletion and the plus symbol indicates an insertion. PAM, protospacer adjacent motif; WT, wild type. Data and error bars represent mean ± SEM; n.s., not significant. One-way ANOVA with Dunnett’s multiple comparisons test was performed.

Figure 3

The main clinical results of CRISPR-treated HSK patients (A) The time scheme and corresponding anterior segment photographs of three participants. (B) The changes of visual acuity in each participant during follow-ups, wherein last visit means 14 months, 18 months, and 21 months for patients 1, 2, and 3, respectively. (C) Potential off-target loci of HELP in the human genome identified by the GUIDE-seq. (D) Deep-sequencing results on the potential off-target sites in the patients’ corneal epithelial cells at 1 month post HELP treatment. (E) Deep-sequencing results on the potential off-target sites in 293T cells. The genome was isolated 48 h post HELP transduction. OT, off-target site. Data and error bars represent mean ± SEM; n.s., non-significant; unpaired two-tailed Student’s t tests.