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Formulation and efficacy of ECO/pRHO-ABCA4-SV40 nanoparticles for nonviral gene therapy of Stargardt disease in a mouse model.

ABSTRACT: It is still a challenge to develop gene replacement therapy for retinal disorders caused by mutations in large genes, such as Stargardt disease (STGD). STGD is caused by mutations in ABCA4 gene. Previously, we have developed an effective non-viral gene therapy using self-assembled nanoparticles of a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid containing rhodopsin promoter (pRHO-ABCA4). In this study, we modified the ABCA4 plasmid with simian virus 40 enhancer (SV40, pRHO-ABCA4-SV40) for enhanced gene expression. We also prepared and assessed the formulations of ECO/pDNA nanoparticles using sucrose or sorbitol as a stablilizer to develop consistent and stable formulations. Results demonstrated that ECO formed stable nanoparticles with pRHO-ABCA4-SV40 in the presence of sucrose, but not with sorbitol. The transfection efficiency in vitro increased significantly after introduction of SV40 enhancer for plasmid pCMV-ABCA4-SV40 with a CMV promoter. Sucrose didn't affect the transfection efficiency, while sorbitol resulted in a fluctuation of the in vitro transfection efficiency. Subretinal gene therapy in Abca4^-/- mice using ECO/pRHO-ABCA4 and ECO/pRHO-ABCA4-SV40 nanoparticles induced 36% and 29% reduction in A2E accumulation respectively. Therefore, the ECO/pABCA4 based nanoparticles are promising for non-viral gene therapy for Stargardt disease and can be expended for applications in a variety of visual dystrophies with mutated large genes.

SUBMITTER: Sun D

PROVIDER: S-EPMC9066847 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Formulation and efficacy of ECO/pRHO-ABCA4-SV40 nanoparticles for nonviral gene therapy of Stargardt disease in a mouse model.

Sun Da D Sun Wenyu W Gao Song-Qi SQ Wei Cheng C Naderi Amirreza A Schilb Andrew L AL Scheidt Josef J Lee Sangjoon S Kern Timothy S TS Palczewski Krzysztof K Lu Zheng-Rong ZR

Journal of controlled release : official journal of the Controlled Release Society 20201221

It is still a challenge to develop gene replacement therapy for retinal disorders caused by mutations in large genes, such as Stargardt disease (STGD). STGD is caused by mutations in ABCA4 gene. Previously, we have developed an effective non-viral gene therapy using self-assembled nanoparticles of a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid containing rhodopsin promoter (pRHO-ABCA4). In this study, we modified the ABCA4 plasmid with simian virus 40 enhancer (SV ...[more]

PMID: 33358976

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Project description:BackgroundABCA4, the gene implicated in Stargardt disease (STGD1), contains 50 exons, of which 17 contain multiples of three nucleotides. The impact of in-frame exon skipping is yet to be determined. Antisense oligonucleotides (AONs) have been investigated in Usher syndrome-associated genes to induce skipping of in-frame exons carrying severe variants and mitigate their disease-linked effect. Upon the identification of a STGD1 proband carrying a novel exon 17 canonical splice site variant, the activity of ABCA4 lacking 22 amino acids encoded by exon 17 was examined, followed by design of AONs able to induce exon 17 skipping.MethodsA STGD1 proband was compound heterozygous for the splice variant c.2653+1G>A, that was predicted to result in in-frame skipping of exon 17, and a null variant [c.735T>G, p.(Tyr245*)]. Clinical characteristics of this proband were studied using multi-modal imaging and complete ophthalmological examination. The aberrant splicing of c.2653+1G>A was investigated in vitro in HEK293T cells with wild-type and mutant midigenes. The residual activity of the mutant ABCA4 protein lacking Asp864-Gly885 encoded by exon 17 was analyzed with all-trans-retinal-activated ATPase activity assay, along with its subcellular localization. To induce exon 17 skipping, the effect of 40 AONs was examined in vitro in WT WERI-Rb-1 cells and 3D human retinal organoids.ResultsLate onset STGD1 in the proband suggests that c.2653+1G>A does not have a fully deleterious effect. The in vitro splice assay confirmed that this variant leads to ABCA4 transcripts without exon 17. ABCA4 Asp864_Gly863del was stable and retained 58% all-trans-retinal-activated ATPase activity compared to WT ABCA4. This sequence is located in an unstructured linker region between transmembrane domain 6 and nucleotide-binding domain-1 of ABCA4. AONs were designed to possibly reduce pathogenicity of severe variants harbored in exon 17. The best AON achieved 59% of exon 17 skipping in retinal organoids.ConclusionsExon 17 deletion in ABCA4 does not result in the absence of protein activity and does not cause a severe STGD1 phenotype when in trans with a null allele. By applying AONs, the effect of severe variants in exon 17 can potentially be ameliorated by exon skipping, thus generating partial ABCA4 activity in STGD1 patients.

Dataset Information

Formulation and efficacy of ECO/pRHO-ABCA4-SV40 nanoparticles for nonviral gene therapy of Stargardt disease in a mouse model.

Publications

Formulation and efficacy of ECO/pRHO-ABCA4-SV40 nanoparticles for nonviral gene therapy of Stargardt disease in a mouse model.

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