Therapeutic gene correction for Lesch-Nyhan syndrome using CRISPR-mediated base and prime editing

Abstract

Lesch-Nyhan syndrome (LNS) is inherited as an X-linked recessive genetic disorder caused by mutations in hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1). Patients with LNS show various clinical phenotypes, including hyperuricemia, gout, devastating behavioral abnormality, intellectual disability, and self-harm. Although uric acid overproduction can be modulated with the xanthine oxidase inhibitor allopurinol, there exists no treatment for behavioral and neurological manifestations of LNS. In the current study, CRISPR-mediated base editors (BEs) and prime editors (PEs) were utilized to generate LNS-associated disease models and correct the disease models for therapeutic approach. Cytosine BEs (CBEs) were used to induce c.430C>T and c.508C>T mutations in HAP1 cells, and then adenine BEs (ABEs) were used to correct these mutations without DNA cleavage. PEs induced a c.333_334ins(A) mutation, identified in a Korean patient with LNS, in HAP1 cells, which was corrected in turn by PEs. Furthermore, improved PEs corrected the same mutation in LNS patient-derived fibroblasts by up to 14% without any unwanted mutations. These results suggest that CRISPR-mediated BEs and PEs would be suggested as a potential therapeutic strategy of this extremely rare, devastating genetic disease.

Keywords: HPRT1, CRISPR-Cas; LNS; Lesch-Nyhan syndrome; MT: RNA/DNA Editing; base editing; gene correction; prime editing.

Graphical abstract

Figure 1

Analysis of LNS-associated HPRT1 variants that are targetable by BEs and PEs (A) Classification of mutation types of HPRT1 variants derived from the LNS-associated genetic variants database (http://www.lesch-nyhan.org/). (B) The proportion of HPRT1 variants that are targetable by CRISPR-mediated CBEs, ABEs, and PEs.

Figure 2

Introduction and correction of patient-derived HPRT1 mutations using CBEs and ABEs (A) Schematic overview of BE-meditated disease modeling and gene correction in human cells. (B) Target sequences of CBEs and ABEs for LNS-associated HPRT1 variants, c.430C>T (p.Q144∗) and c.508C>T (p.R170∗). The spacer sequences are indicated by boxes, and the target C:G pairs of CBEs and A:T pairs of ABEs are shown in blue and red, respectively. PAM sequences of each target site are shown in bold. (C) Heatmaps of CBE-mediated base editing frequencies for disease modeling of c.430C>T (top panel) and c.508C>T (bottom panel) in HEK293T/17 cells. Data are shown as means from two biologically independent samples. (D) Heatmaps of ABE-meditated base editing frequencies for correction of c.430C>T (top panel) and c.508C>T (bottom panel) in HEK293T/17 cells. Data are shown as the mean of three biologically independent samples. (E) Sanger sequencing results of endogenous c.430C>T (p.Q144∗) and c.508C>T (p.R170∗) target sites in mutated and corrected HAP1 cells. The red boxes indicate nucleotides converted by CBEs and ABEs. (F) Western blotting analysis of HPRT protein expression in mutated and corrected HAP1 cells. GAPDH was used as an internal control. (G) Crystal violet staining of mutated and corrected HAP1 cells selected in media containing 6-TG or HAT. (H) Results of IMP assay for HPRT activity in mutated and corrected HAP1 cells.

Figure 3

Introduction and correction of patient-derived HPRT1 mutation using PE (A) Schematic overview of PE-mediated disease modeling and gene correction in human cells. The patient-derived HPRT1 mutation, c.333_334ins(A), is representatively described. (B) Target sequences of PEs for the LNS-associated HPRT1 mutation, c.333_334ins(A) (p.G112Rfs∗10). The representative spacer-#1 sequence and PAM sequence are indicated in box and bold, respectively. The inserted adenine is highlighted in red. (C) Prime editing frequencies for introducing c.333_334ins(A) with pegRNAs containing variable lengths of PBS and RTT. The red arrow indicates the pegRNA used in subsequent experiments. (D) Prime editing frequencies of PE2, PE3, and PE3b to induce c.333_334ins(A) mutation. (E) Prime editing frequencies for correcting c.333_334ins(A) with pegRNAs containing variable lengths of PBS and RTT. The red arrow indicates the pegRNA used in subsequent experiments. (F) Prime editing frequencies with PE2 and PE3b to correct c.333_334ins(A) mutation. (G) Sanger sequencing results of endogenous c.333_334ins(A) (p.G112Rfs∗10) target sites in mutated and corrected HAP1 cells. The red arrow indicates the adenine inserted for disease modeling and gene correction by PEs. (H) Western blotting analysis of HPRT protein expression in mutated and corrected HAP1 cells. GAPDH was used as an internal control. (I) Crystal violet staining of PE-mediated mutated and corrected HAP1 cells selected with media containing 6-TG or HAT. (J) Results of IMP assay for HPRT activity in mutated and corrected HAP1 cells. Data are means from two or three biologically independent samples, and error bars indicate the standard error of the mean.

Figure 4

PE-mediated gene correction of c.333_334ins(A) mutations in patient-derived fibroblasts (A) Renal ultrasound results of a patient with LNS with HPRT1 c.333_334ins(A) mutation. (B) Family pedigree of the patient with LNS with HPRT1 c.333_334ins(A) mutation. Fibroblasts were obtained from the patient with LNS as indicated with the red arrow. (C) Sequencing analysis of patient-derived fibroblasts to confirm the HPRT1 c.333_334ins(A) mutation. (D) Prime editing frequencies of various types of improved PEs and pegRNAs (top) for correcting the HPRT1 c.333_334ins(A) mutation. The red arrow indicates the pegRNA used in the subsequent experiment. Representative results of high-throughput sequencing of patient-derived fibroblasts treated with PE5max and tevopreQ pegRNA to correct the HPRT1 c.333_334ins(A) mutation (bottom) are shown. (E) Crystal violet staining of PE-mediated corrected fibroblasts with media containing 6-TG or HAT. Data are means from two biologically independent samples, and error bars indicate the standard error of the mean. (F) Western blotting analysis of HPRT protein expression in patient-derived fibroblast cells and HPRT1 c.333_334ins(A)-corrected patient-derived fibroblasts selected with HAT medium. GAPDH was used as an internal control.