Novel method for homogeneous gene transfer to the inner ear

Viral vectors are widely used in gene therapy due to their efficiency. In this paper we describe a novel method for transfecting the whole inner ear of a guinea pig using adenoviral vectors. Very small perforations are made in both the cochlea and lateral semicircular canal, into which 50 microl of adenoviral suspension (8.9x10(8) plaque forming unit (PFU)/ml) is gently infused. Any excess suspension flows out through the perforation in the semicircular canal and therefore makes no contact with the central nervous system. Our method can therefore be utilized to perform homogeneous gene transfer and may eliminate any effects on other organs, such as the contralateral ear.     

A novel vestibular approach for gene transfer into the inner ear

The aim of gene transfer to the cochlea and vestibular organ is to protect the inner ear from different disorders. Although various vectors for gene delivery have been used with some success, there remains a need for a reliable transfer of genes into the inner ear without damaging cochlear function. Here, we have tested a novel application method for gene transfer into the rat inner ear in vivo using herpes simplex virus type-1(HSV-1)-based amplicon vectors. Our goal was to find an entry route into the inner ear that leaves its function intact. Besides other non-invasive and invasive application techniques, we applied the viral vector via injection through a small opening of the utriculus. Using this method, efficient -galactosidase reporter gene expression was achieved in nearly all neurons in the vestibulum and cochlea, without functional hearing deficits. At the time point of maximal expression 5 days after injection, -galactosidase activity was also observed in axonal fibres and synaptic endings close to inner and outer hair cells. Our results thus describe an efficient and reliable protocol for short-term expression of potential therapeutic genes in the neuronal compartment of the inner ear.     

Gene transfer into the mammalian inner ear using HSV-1 and vaccinia virus vectors.

The introduction of foreign genes into cells has become an effective means of achieving intracellular expression of foreign proteins, both for therapeutic purposes and for experimental manipulation. Gene delivery to the nervous system has been extensively studied, primarily using viral vectors. However, to date less work has focused on gene delivery to the inner ear, and existing studies have primarily used adenovirus and adeno-associated virus. Using two recombinant viral vectors, herpes simplex type 1 (HSV-1), and vaccinia virus, bearing the Escherichia coli lacZ gene, we tested gene delivery to the guinea pig cochlea in vivo with beta-galactosidase staining as an assay. The HSV-1 and vaccinia virus vectors were both found to infect and elicit transgene expression successfully in many cells in the guinea pig cochlea, including cells in the organ of Corti. These data demonstrate the feasibility of gene delivery to the inner ear using these two viral vectors. Such techniques may facilitate study of the auditory systems, and might be used to develop gene therapy strategies for some forms of hearing loss.     

Feasibility of inner ear gene transfer after middle ear administration of an adenovirus vector

BACKGROUND: Several groups demonstrated in animal experiments that gene transfer is a feasible tool for inner ear intervention. Various approaches for inoculation of vectors have been successfully used for inner ear gene therapy. One possible way to reduce the risk of hearing loss following the opening of the cochlea for application of the vector into the perilymphatic space is to deliver vectors through the round window. This study was designed to determine whether middle ear application of an adenoviral vector is a feasible approach to inoculate vectors and lead to transduction of cells in the inner ear. METHODS: A unilateral middle ear application of an adenoviral vector was performed in 4 guinea pigs directly on the round window membrane (RWM) and in 4 additional animals by placing a cotton patch soaked with the vector solution on the RWM. The expression of a reporter gene (lacZ) was used to localize vector-transduced cells. RESULTS: Only one out of 8 animals showed cochlear expression of the reporter gene, whereas all 8 animals showed strong lacZ expression in the middle ear mucosa, in the RWM and in the mucosa surrounding the stapes. CONCLUSION: Our results indicate that the RWM presents a close barrier, almost completely preventing the adenovirus to diffuse into the perilymphatic space. Therefore middle ear application of an adenoviral vector cannot be used to induce inner ear gene transfer. However, middle ear application of a viral vector may be useful for developing treatment for diseases of the middle ear mucosa.     

Gene transfer in human vestibular epithelia and the prospects for inner ear gene therapy.

Transfer of exogenous genetic material into the mammalian inner ear using viral vectors has been characterized over the last decade. A number of different viral vectors have been shown to transfect the varying cell types of the nonprimate mammalian inner ear. Several routes of delivery have been identified for introduction of vectors into the inner ear while minimizing injury to existing structures and at the same time ensuring widespread distribution of the agent throughout the cochlea and the rest of the inner ear.These studies raise the possibility that gene transfer may be developed as a potential strategy for treating inner ear dysfunction in humans. Furthermore, a recent report showing successful transfection of excised human vestibular epithelia offers proof of principle that viral gene transfer is a viable strategy for introduction and expression of exogenous genetic material to restore function to the inner ear. Human vestibular epithelia were harvested from patients undergoing labyrinthectomy, either for intractable Ménière's disease or vestibular schwannoma resection, and cultured for as long as 5 days. In those experiments, recombinant, multiply-deleted, replication-deficient adenoviral vectors were used to transfect and express a reporter gene as well as the functionally relevant gene, wild-type KCNQ4, a potassium channel gene that when mutated causes the autosomal dominant HL DFNA2.Here, we review the current state of viral-mediated gene transfer in the inner ear and discuss different viral vectors, routes of delivery, and potential applications of gene therapy. Emphasis is placed on experiments demonstrating viral transfection of human inner ear tissue and implications of these findings and for the future of gene therapy in the human inner ear.     

内耳疾病基因治疗方法的研究

目的:探讨一种高效、安全和简便的内耳疾病基因转移和导入方法。方法:将30只健康豚鼠随机分为A、B、C 3组,每组10只。用Ad—GFP报告基因分别由圆窗、耳蜗底回和脑脊液三种途径导入30只豚鼠内耳,在术前和术后测定听性脑干反应(ABR)阈值和畸变产物耳声发射(DPOAE)振幅改变。再行耳蜗铺片,观察AdGFP在内耳组织中的转染表达和耳蜗毛细胞的形态结构。结果:三种途径导入Ad—GFP在耳蜗螺旋神经节、血管纹和Corti器上均有转染和表达。在术后3 d的表达产物最高,7 d后减弱,10 d后更弱。圆窗和耳蜗底回钻孔导入手术可引起蜗内出血,凝血块淤积在耳蜗顶回的中阶,造成顶回毛细胞损害。而脑脊液给药途径无此现象。ABR阈值在各组动物手术前后无明显改变,而圆窗和耳蜗底回给药途径造成DPOAE幅度明显下降,脑脊液给药组无DPOAE改变。结论:应用腺病毒载体进行基因转移通过圆窗和耳蜗底回钻孔途径可引起蜗内出血,影响DPOAE幅度,而应用脑脊液给药途径则相对安全和方便。     

Superparamagnetic nanoparticles as vectors for inner ear treatments: driving and toxicity evaluation

Conclusion Super paramagnetic nanoparticles (MNP) are a promising vector to achieve controlled drug delivery into the cochlea.

Objective The goal of the study was to evaluate the toxicological risk of MNP upon the inner ear.

Methods Fe₃O₄-MNP displacement was studied in various catheter materials, shape, and solvent with a local magnetic field. EC5V cells (derived from the inner ear) were cultured with MNP (100 and 500?nm) at various concentrations or without MNP. Cell survival was assessed with a flow cytometry analysis. Localization of MNP within the cells was studied with confocal microscopy. In vivo, a single intra-cochlear administration of 200?nm MNP (3?×?10¹⁰MNP/mL, n?=?8; 1.5?×?10¹² MNP/mL, n?=?6) or saline (n?=?14) was performed in guinea pigs. Hearing thresholds were assessed with auditory brainstem responses at Day 7.

Results MNP could be concentrated at different locations of the catheter with sequential activation of solenoids. MNP were internalized in the cytoplasm, but not in the nuclei nor in endosomes at 48?h. After 48?h of incubation, no difference for cell survival between the groups was observed, whatever the MNP concentration. A size effect was observed with less survival in the 100?nm group. In guinea pigs at day 7, hearing threshold shift was not different in the three groups.     

Viral-mediated gene transfer to study the molecular physiology of the Mammalian inner ear

Several classes of viral vectors including adenovirus, adeno-associated virus, herpes simplex virus, lentivirus and vaccinia virus have been reported to infect cells of the inner ears of mammals and may be useful for protein manipulation and therapeutic purposes. We have screened a few of these for use as vectors to mediate gene transfer into the sensory hair cells of organotypic cultures from the neonatal mouse cochlea and utricle. Recombinant, replication-deficient adenovirus has emerged as a useful vector for several reasons: ease of vector generation at high titer; efficient hair cell specific infection; robust expression of reporter genes and minimal toxicity. Previously, we characterized adenovirus infected hair cells using a vector that carried the gene for green fluorescent protein (GFP). We screened GFP-positive cells electrophysiologically and found that although hair cells survive adenoviral vector infection, their mechanosensitivity was compromised. Until recently this has limited the scope of adenovirus application to the problems of inner ear physiology and pathophysiology. However, a modified adenoviral vector, now available, has been reported to have reduced ototoxicity in vivo. The modifications include the deletion of the adenoviral genes E1, E3, the viral polymerase, and the preterminal protein. We are currently working to characterize viral-mediated gene transfer into hair cells of the cultured mouse utricle using this new modified adenoviral vector. We have found that hair cells infected with the modified vector have intact hair bundles and robust mechanotransduction.     

三种内耳抗原与自身免疫性内耳病的相关性研究

目的探讨3种纯化的内耳抗原与自身免疫性内耳病的关系,并确定其在耳蜗的表达.方法以粗制内耳抗原的3种亚组份(31000、42000～45000和60000蛋白)作为抗原,分别免疫动物(B、C和D组),观察听阈、血清IgG水平和内耳形态学的改变,并应用免疫组织化学方法确定其在耳蜗的表达.A组为对照组,以不含抗原的聚丙烯酰胺凝胶匀浆液代替内耳抗原.结果免疫前各组听阈差异无显著性(F=0.07,P>0.05),免疫后对照组和C组听阈和内耳形态学未见明显改变,B组(9/30只)和D组(7/28只)部分动物出现听力损失.组间听阈差异有显著性(F=9.12,P<0.01).实验组动物血清IgG均显著性升高(F=7.46,P<0.01),B、C和D组与对照组相比差异有显著性.31000蛋白完全分布于耳蜗神经纤维,而42000～45000和60000蛋白分布广泛,螺旋神经节、Corti器、血管纹和螺旋韧带均有分布.结论粗制内耳抗原中31000和60000亚组份均能独立诱发自身免疫性内耳病,31000蛋白具有很高的组织特异性,可能作为一种标志性蛋白用于自身免疫性内耳病的临床诊断.     

化学性诱变和内耳基因突变

随着人类基因组序列的完成,学者们系统地确定基因功能及其在人类疾病中的作用已成为生命科学研究的中心。小鼠突变对于展现哺乳动物基因功能和模拟人类疾病具有关键性的作用,对分析和探讨疾病的发病机制具有特别重要意义。小鼠全基因组的突变率很低,常规的基因敲除(knock-out)和转基因(transgenesis)突变规模小,且研究的是已知基因的功能。近年,采用化学诱导突变的表型筛选(phenotype screens)已成为日益关注的目标,乙烷亚硝基脲(N-ethyl-N-nitrosourea,ENU)诱变的大规模表型筛选能获得大量新突变,确定了一些新基因,同时也发现了与听觉及平衡功能有关的突变和基因。本文对近年采用ENU诱变表型筛选的方法和在确定新的耳聋基因中的作用进行概述。     

小鼠内耳COCH基因的表达

目的观察小鼠内耳COCH基因的表达。方法体外转录合成地高辛标记的反义RNA探针,内耳样本冰冻切片,进行原位杂交反应。结果发现除已报道的耳蜗螺旋韧带、壶腹嵴间质等内耳组织外,COCH基因在内淋巴囊周围间质成纤维细胞亦表达阳性,其mRNA水平(原位杂交染色平均积分光密度0.68±0.25)与耳蜗螺旋韧带(0.77±0.19)相似,强于壶腹嵴间质(0.47±0.21)。结论COCH基因在小鼠内淋巴囊周围间质细胞高表达,为COCH基因内耳功能的后续研究及其与梅尼埃病关系的探究提供了线索。     

Drug delivery to the inner ear using gene therapy

The last 10 years have seen the development of numerous strategies for the delivery of genes to the inner ear. Besides being a useful research tool,gene therapy has significant promise as a potential clinical treatment. The human inner ear is easily accessible through either the round window or the stapes footplate. It is now possible to choose a variety of vectors to target a variety of different tissues. Modification of promoters yields different expression patterns as well as differences in degree of expression. Several animal studies have also demonstrated that expression of exogenous genes in the cochlea does not result in loss of hearing function. A variety of potential clinical applications are already evident from these early studies. Protective strategies such as prevention of neuronal degeneration and protection of auditory hair cells from oxidative stress are potential examples where gene therapy may be useful. As the understanding of gene therapy improves, investigators will be able to move toward targeted single-gene replacement to treat disorders such as connexin mutations and applying gene therapy to sensory cell replacement.     

听觉损伤后毛细胞再生与聋病基因治疗策略

听觉损伤可引起耳蜗毛细胞和听觉神经元的不可逆性损伤,从而导致永久性的感音神经性耳聋。虽然内耳所独具的结构是基因治疗非常独特和重要的靶器官,但能否实现损伤后毛细胞的再生是其前提条件。国内外研究者们做了大量的探索并取得重要突破,从非哺乳动物到哺乳动物毛细胞再生,从前庭毛细胞到耳蜗毛细胞再生,从未成熟期到成年期毛细胞再生,从离体培养毛细胞再生到在体毛细胞再生,整整经历了近半个世纪。但是毛细胞的再生并不等同于听力完全恢复。针对内耳基因治疗时间窗问题,我们根据听觉损伤后不同的病理状态,提出听觉损伤后毛细胞再生和基因治疗的基本策略：（1）毛细胞纤毛损伤阶段,是基因治疗的最好时机,通过完全修复或纤毛再生达到功能的完全或部分恢复;（2）内耳毛细胞虽有损伤但没有坏死,支持细胞和神经纤维基本正常,所以有恢复形态和功能的机会,这个阶段导入Math1基因应该有效,是基因治疗的最关键时机;（3）毛细胞严重损伤但支持细胞尚存,是毛细胞再生的抢救阶段,而且还可以争取在Corti器细胞构架没有塌陷之前进行干细胞导入,所以这个阶段内细胞移植可能有效地实现听力恢复;（4）Corti器完全失去构架,仅仅残留上皮层或瘢痕化,基因导入完全无效,即使干细胞导入也会面临困难,如何重塑Corti器构架是巨大挑战。为了实现耳聋基因治疗临床应用的可能性。我们还探索了最有效简便的外源基因内耳导入方式以及高效安全可靠的基因载体比如纳米载体的研发。毛细胞再生研究已经取得突破性进展,但还面临诸多挑战。通过不懈的努力,聋病基因治疗的最终临床应用一定会实现。     

三种内耳抗原与自身免疫性内耳病的相关性研究

目的 探讨3种纯化的内耳抗原与自身免疫性内耳病的关系,并确定其在耳蜗的表达。方法 以粗制内耳抗原的3种亚组分（31000、42000－45000和60000蛋白）作为抗原,分别免疫动物（B、C和D组）,观察听阈、血清IgG水平和内耳形态学的改变,并应用免疫组织化学方法确定其在耳蜗的表达。A组为对照组,以不含抗原的聚丙烯酰凝胶匀浆液代替内耳抗原。结果 免疫前各组听阈差异无显著性（F＝0．07,P＞0．05）,免疫后对照组和C组听阈和内耳形态学未见明显改变,B组（9／30只）和D组（7／28只）部分动物出现听力损失。组间听阈差异有显著性（F＝9．12,P＜0．01）。实验组动物血清IgG均显著性升高（F＝7．46,P＜0．01）,B、C和D组与对照组相比差异有显著性。31000蛋白完全分布于耳蜗神经纤维,而42000－45000和60000蛋白分布广泛,螺旋神经节、Corti器、血管纹和螺旋韧带均有分布。结论 粗制内耳抗原中31000和60000亚组份均能独立诱发自身免疫性内耳病,31000蛋白具有很高的组织特异性,可能作为一种标志性蛋白用于自身免疫性内耳病的临床诊断。     

Transgene expression in neonatal mouse inner ear explants mediated by first and advanced generation adenovirus vectors

The mouse serves as a valuable model for treatment leading to the prevention and therapy of inner ear disease. Transgenic correction of genetic inner ear disease in mice may help develop treatment for human genetic inner ear disease. In mutations involving hair cells (HCs) or supporting cells (SCs), it is necessary to insert the wild-type transgenes directly into these cells. We used inner ear explants to characterize the transgenic expression using adenovirus-mediated reporter genes (bacterial lacZ). The variable parameters were the age of the explants (P1-P5), the type of vector (first and advanced generation adenovirus) and the genotype of the mouse (wild-type versus shaker-2 mutant). Transduction of cochlear HCs was detected at P1 and in some of the P3 cochleae. Low efficiency transduction of SCs was observed in P1 explants, but the efficiency increased with age and reached high levels at P5. The pattern of transduction was similar regardless of the genotype and the type of vector used. The data demonstrate that differentiating HCs and SCs in mouse explants can be transduced by adenovirus vectors, suggesting that cultures of mouse ears are a valuable model for developing inner ear gene therapy protocols.     

Autonomic nerve and inner ear

Summary The vascular change could be produced in the inner ear and in the visceral organ by the electrical stimulation of autonomic nerve. But the effects of autonomic dysfunction seemed to be non-specific as described already in the earlier literature.The physiologic changes in the inner ear due to the dysfunction was that in the oxygen tension. The stimulation of sympathetic nerve produced the increase in the oxygen tension, while the stimulation of parasympathetic nerve produced the decrease in it, with an increase in the beginning of stimulation. Since the cochlear microphonics remained unchanged during the stimulation of autonomic nerve, I think that the autonomic dysfunction does not influence severely upon the cochlea. Now I am inclined to think that the effect of autonomic dysfunction would dominantly influence upon the visceral organs, but not upon the inner ear.

---

Zusammenfassung Durch elektrische Reizung cerebraler Zentren des autonomn Nervensystems konnten Durchblutungsstörungen im Innenohr and in visceralen Organen hervorgerufen werden.Entsprechend früheren Literaturangaben scheinen these Effekte nicht spezifisch zu sein.Die physiologische Folge einer solchen Reizung war eine Veränderung der Sauerstoffspannung im Innenohr. Die Stimulation sympathischer Zentren bewirkte dabei einen Anstieg, während die Stimulation der parasympathischen Zentren nach einem initialen Anstieg einen endgültigen Abfall der Sauerstoffspannung zur Folge hatte.Weil aber die Mikrophonpotentiale unverändert blieben, wird angenommen, daß die vegetativen Dysfunktionen keine stärkere Beeinflussung der Cochlea bewirken. Die Wirkungen dieser Dysfunktionen auf die visceralen Organe überwiegen.