Froebel Beau R, Trujillo Alexandria J, Sullivan Jack M
Research Service, VA Western New York Healthcare System, Buffalo, New York, United States 2Department of Ophthalmology, State University of New York, University at Buffalo, Buffalo, New York, United States 3The Ross Eye Institute of University at Buffalo, Buffalo, New York, United States.
Research Service, VA Western New York Healthcare System, Buffalo, New York, United States 2Department of Ophthalmology, State University of New York, University at Buffalo, Buffalo, New York, United States 4Department of Pharmacology/Toxicology, State University of New York, University at Buffalo, Buffalo, New York, United States.
Invest Ophthalmol Vis Sci. 2017 Jul 1;58(9):3576-3591. doi: 10.1167/iovs.16-20877.
The mutation-independent strategy for hammerhead ribozyme (hhRz) or RNA interference (RNAi)-based gene therapeutics to treat autosomal dominant diseases is predicated on the hypothesis that a single therapeutic would equivalently suppress all/most of the diverse mutant mRNAs in patients with the disease phenotype. However, the hypothesis has not been formally tested. We address this through a comprehensive bioinformatics study of how mutations affect target mRNA structure accessibility for a single lead hhRz therapeutic (725GUC↓), designed against human rod rhodopsin mRNA (hRHO), for patients with hRHO mutations that cause autosomal dominant retinitis pigmentosa.
A total of 199 in silico coding region mutations (missense, nonsense, insert, deletion, indel) were made in hRHO mRNA based on Human Gene Mutation Database and Database of Single Nucleotide Polymorphisms. Each mRNA was folded with MFold, SFold, and OligoWalk algorithms and subjected to a bioinformatics model called multiparameter prediction of RNA accessibility. Predicted accessibility of each mutant over both a broad local region and the explicit lead ribozyme annealing site were compared quantitatively to wild-type hRHO mRNA.
Accessibility of the 725GUC↓ site is sensitive to some mutations. For single nucleotide missense mutations, proximity of the mutation to the hhRz annealing site increases the impact on predicted accessibility, but some distant mutations also influence accessibility.
A mutation-independent strategy appears viable in this specific context but certain mutations could significantly influence ribozyme or RNAi efficacy through impact on accessibility at the target annealing site/region. This possibility must be considered in applications of this gene therapy strategy.
基于锤头状核酶（hhRz）或RNA干扰（RNAi）的基因疗法用于治疗常染色体显性疾病的不依赖突变策略，是基于这样一种假设，即单一疗法能等效地抑制具有疾病表型患者中所有/大多数不同的突变mRNA。然而，这一假设尚未得到正式验证。我们通过一项全面的生物信息学研究来解决这个问题，该研究针对导致常染色体显性视网膜色素变性的人视紫红质mRNA（hRHO）突变患者，研究突变如何影响针对单一先导hhRz疗法（725GUC↓）的靶mRNA结构可及性。
基于人类基因突变数据库和单核苷酸多态性数据库，在hRHO mRNA中进行了总共199个计算机模拟编码区突变（错义、无义、插入、缺失、插入缺失）。每个mRNA使用MFold、SFold和OligoWalk算法进行折叠，并应用一种称为RNA可及性多参数预测的生物信息学模型。将每个突变体在广泛的局部区域和明确的先导核酶退火位点的预测可及性与野生型hRHO mRNA进行定量比较。
725GUC↓位点的可及性对某些突变敏感。对于单核苷酸错义突变，突变与hhRz退火位点的距离越近，对预测可及性的影响越大，但一些距离较远的突变也会影响可及性。
在这种特定情况下，不依赖突变的策略似乎可行，但某些突变可能通过影响靶退火位点/区域的可及性而显著影响核酶或RNAi的疗效。在应用这种基因治疗策略时必须考虑到这种可能性。
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