Targeted-Deletion of a Tiny Sequence via Prime Editing to Restore SMN Expression

Abstract

Spinal muscular atrophy (SMA) is a devastating autosomal recessive motor neuron disease associated with mutations in the survival motor neuron 1 (SMN1) gene, the leading genetic cause of infant mortality. A nearly identical copy gene (SMN2) is retained in almost all patients with SMA. However, SMN2 fails to prevent disease development because of its alternative splicing, leading to a lack of exon 7 in the majority of SMN2 transcripts and yielding an unstable truncated protein. Several splicing regulatory elements, including intronic splicing silencer-N1 (ISS-N1) of SMN2 have been described. In this study, targeted-deletion of ISS-N1 was achieved using prime editing (PE) in SMA patient-specific induced pluripotent stem cells (SMA-iPSCs) with a high efficiency of 7/24. FL-SMN expression was restored in the targeted-deletion iPS clones and their derived motor neurons (iMNs). Notably, the apoptosis of the iMNs, caused by the loss of SMN protein that leads to the hyperactivity of endoplasmic reticulum (ER) stress, was alleviated in targeted-deletion iPSCs derived-iMNs. Thus, this is the first study to demonstrate that the targeted-deletion of ISS-N1 via PE for restoring FL-SMN expression holds therapeutic promise for SMA.

Identification of the Efficient Enhancer Elements in FVIII-Padua for Gene Therapy Study of Hemophilia A

“…To explore the enhancer effect of C400 and its application for universal targeted gene therapy for HA in different genetic backgrounds, we nucleofected F8 expression vectors into the rDNA loci of HA-iPSCs and normal human iPSCs (B6-iPSCs), respectively. Both iPSCs were previously established [26,27]. After selection using G418, single clones were selected for identification.…”

“…(1) Cell lines were purchased from Procell Life Science & Technology (Wuhan, China) and cultured in DMEM/HG (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and split every 3-4 days. (2) iPSCs, HA-iPSCs [26], and B6-iPSCs (also referred to as hiPSCs) [27] have been generated. All iPSCs were cultured on Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) -coated well plates (Corning, NY, USA) in mTesR plus medium (StemCell Technologies, Vancouver, BC, Canada).…”

Gene editing innovations and their applications in cardiomyopathy research

“…Its editing flexibility and ability to correct indels and introduce large deletions, which base editing cannot achieve, make it a highly effective method. Although relatively new to the cardiac research field, prime editing has already demonstrated efficiency in generating cell and animal models of DMD ( Zhou et al, 2022c ; Happi Mbakam et al, 2022a ,b; Chemello et al, 2021 ), which could eventually help identify new drug targets or potential therapies for cardiac disease. Compared to other gene editing technologies, PE has the advantage of reduced off-target effects, making it a highly desirable therapeutic option.…”

Recent Research Trends in Stem Cells Using CRISPR/Cas-Based Genome Editing Methods

“…Most patients with SMA are characterized by exon 7 skipping in the SMN2 transcript, producing unstable truncated proteins. Zhou et al ( 93 ) inser-ted full-length SMN into SMA patient-derived iPSCs by inducing a targeted 9bp-deletion in the intronic splicing silencer-N1 of SMN2 with a prime editor. In the motor neurons derived from rescued iPSCs, apoptosis was reduced by restoring SMN protein.…”