Translational Neuroscience Facility & Department of Physiology, School of Medical Sciences, UNSW Sydney, NSW, Australia.
The Graduate School of Biomedical Engineering, UNSW Sydney, NSW, Australia.
Hear Res. 2019 Sep 1;380:137-149. doi: 10.1016/j.heares.2019.06.002. Epub 2019 Jun 21.
This Review outlines the development of DNA-based therapeutics for treatment of hearing loss, and in particular, considers the potential to utilize the properties of recombinant neurotrophins to improve cochlear auditory (spiral ganglion) neuron survival and repair. This potential to reduce spiral ganglion neuron death and indeed re-grow the auditory nerve fibres has been the subject of considerable pre-clinical evaluation over decades with the view of improving the neural interface with cochlear implants. This provides the context for discussion about the development of a novel means of using cochlear implant electrode arrays for gene electrotransfer. Mesenchymal cells which line the cochlear perilymphatic compartment can be selectively transfected with (naked) plasmid DNA using array - based gene electrotransfer, termed 'close-field electroporation'. This technology is able to drive expression of brain derived neurotrophic factor (BDNF) in the deafened guinea pig model, causing re-growth of the spiral ganglion peripheral neurites towards the mesenchymla cells, and hence into close proximity with cochlear implant electrodes within scala tympani. This was associated with functional enhancement of the cochlear implant neural interface (lower neural recruitment thresholds and expanded dynamic range, measured using electrically - evoked auditory brainstem responses). The basis for the efficiency of close-field electroporation arises from the compression of the electric field in proximity to the ganged cochlear implant electrodes. The regions close to the array with highest field strength corresponded closely to the distribution of bioreporter cells (adherent human embryonic kidney (HEK293)) expressing green fluorescent reporter protein (GFP) following gene electrotransfer. The optimization of the gene electrotransfer parameters using this cell-based model correlated closely with in vitro and in vivo cochlear gene delivery outcomes. The migration of the cochlear implant electrode array-based gene electrotransfer platform towards a clinical trial for neurotrophin-based enhancement of cochlear implants is supported by availability of a novel regulatory compliant mini-plasmid DNA backbone (pFAR4; plasmid Free of Antibiotic Resistance v.4) which could be used to package a 'humanized' neurotrophin expression cassette. A reporter cassette packaged into pFAR4 produced prominent GFP expression in the guinea pig basal turn perilymphatic scalae. More broadly, close-field gene electrotransfer may lend itself to a spectrum of potential DNA therapeutics applications benefitting from titratable, localised, delivery of naked DNA, for gene augmentation, targeted gene regulation, or gene substitution strategies.
本文综述了基于 DNA 的治疗方法在治疗听力损失方面的发展，特别是考虑利用重组神经营养因子的特性来改善耳蜗听觉（螺旋神经节）神经元的存活和修复。几十年来，人们一直在进行大量的临床前评估，以期改善与耳蜗植入物的神经接口，从而减少螺旋神经节神经元的死亡，甚至使听神经纤维再生。这为讨论利用新型耳蜗植入电极阵列进行基因电转移提供了背景。可以使用基于阵列的基因电转移（称为“近场电穿孔”），选择性地将（裸露）质粒 DNA 转染到排列在耳蜗外淋巴间隙的间质细胞中。这项技术能够在耳聋豚鼠模型中表达脑源性神经营养因子 (BDNF)，使螺旋神经节外周神经突向间质细胞生长，从而与耳蜗植入电极在鼓阶内接近。这与耳蜗植入物神经接口的功能增强有关（通过电诱发听觉脑干反应测量，较低的神经募集阈值和扩展的动态范围）。近场电穿孔效率的基础源于靠近排列的耳蜗植入电极的电场压缩。具有最强场强的阵列附近区域与表达绿色荧光报告蛋白 (GFP) 的生物报告细胞（贴壁人胚肾 (HEK293)）的分布非常吻合，这些细胞在基因电转移后表达 GFP。使用这种基于细胞的模型优化基因电转移参数与体外和体内耳蜗基因传递结果密切相关。新型合规的小型质粒 DNA 骨架（pFAR4；抗生素抗性 v.4 无抗生素的质粒）的可用性为基于神经营养因子的耳蜗植入物增强的基于电极阵列的基因电转移平台向临床试验的推进提供了支持，该小型质粒 DNA 骨架可用于包装“人源化”神经营养因子表达盒。包装到 pFAR4 中的报告基因盒在豚鼠基底回外淋巴腔中产生了明显的 GFP 表达。更广泛地说，近场基因电转移可能适用于一系列潜在的 DNA 治疗应用，这些应用受益于可滴定的、局部的裸露 DNA 递送，用于基因增强、靶向基因调节或基因替代策略。
