Dual delivery of supramolecular nanoparticle-carried minicircle donor DNA with Cas9/gRNA improved HITI knock-in efficiency in X-linked juvenile retinoschisis

Abstract

X-linked retinoschisis (XLRS) is a hereditary mutation of the RS1 gene and is characterized by early-onset maculopathy with severe visual impairment. Current gene therapy utilizing CRISPR-associated protein 9 (Cas9) is ongoing; however, the optimization of nonviral/nanoparticle CRISPR/Cas9-based therapeutics for gene delivery into nondivided retinal neurons remains undetermined. Minicircles DNA, a circular DNA molecule lacking bacterial backbone sequences, has gained recognition for improving transfection efficiency and biosafety. Here, we developed a supramolecular nanoparticle (SMNP)-editing platform in which SMNPs carrying CRISPR/Cas9 integrated minicircle donor DNA (mc dDNA) to achieve highly efficient and precise gene knock-in. To increase the efficiency of RS1 gene knock-in, we replaced the conventional mc dDNA with a single flanking-Cas9/cut site. Furthermore, using homology-independent targeted integration (HITI) as an editing-nondivided cell strategy, SMNP-carried CRISPR/Cas9 could effectively facilitate the dual delivery of mc-RS1/GFP dDNA and Cas9/gRNA plasmids. Compared with the delivery of the Cas9/gRNA plasmid alone (~20 %), the delivery of mc-RS1/GFP dDNA via SMNPs had significantly higher transfection efficiency (90 %). Further flow cytometry analysis revealed that 5.99 % of the FACS-positive cells were detected in the mc-RS1/GFP dDNA group, markedly exceeding the 2.21 % with long-term expression in the conventional dDNA group. Moreover, when XLRS/iPSC-derived retinal neuron organoids were used as a patient-based disease model, compared with conventional plasmid-based delivery, robust RS1 expression with integration sustained transgene expression in XLRS/iPSC-derived retinal organoids. Collectively, these findings indicated that SMNP-mediated dual delivery of the Cas9/gRNA plasmid and mc-RS1/GFP dDNA substantially enhanced RS1-targeted integration with long-term transgene expression, providing safer and effective gene therapy for the treatment of XLRS.

Keywords: CRISPR/Cas9; Homology-independent targeted integration (HITI); Minicircle donor DNA; Supramolecular nanoparticles; X-linked juvenile retinoschisis.