Gene therapy for hearing loss

编码蛋白质otoferlin的基因OTOF的突变通常会导致先天性深度耳聋（DFNB 9）。耳传递素是从听觉感觉细胞到随后的神经元的突触传递所必需的。迄今为止，患有这种形式的耳聋的患者接受人工耳蜗植入，它直接用电信号刺激听觉通路，从而使人们能够理解语言。然而，耳蜗植入物几乎不传输频率信息，并且仅对有限的声音强度动态范围进行编码。由于在这种形式的耳聋中，内耳在形态上几乎是完全完整的，因此将otoferlin cDNA转移到感觉细胞中有望恢复听力。这种因果基因疗法可能使听力几乎没有任何质量限制，但尚未开发用于人类患者的日期。最近，我的小组成功地将大otoferlin cDNA导入小鼠听觉感觉细胞的重组病毒的手段。这是具有挑战性的，因为没有已知的病毒载体适合于将大cDNA转移到这种类型的细胞中。我们通过"双AAV"方法克服了这一挑战，该方法用于部分恢复耳聋otoferlin敲除小鼠的听力。在该方法中，将otoferlin的cDNA分裂成两种腺相关病毒（AAV），其在靶细胞的细胞核中重新组装以允许全长otoferlin mRNA的转录。该提案的重点是测试这些AAV的变体，这些变体将增强正确的尾对头多聚化，旨在增加蛋白质表达，从而进一步改善听力。此外，我将采用一种改进的方法注射到内耳中，并使用不同的AAV血清型来测试这是否适合于增加转导的感觉细胞的速率。接下来，突触和听觉神经纤维应在耳传递蛋白的再表达后表征。然后，我们将把这种基因治疗方法应用于两种具有人类突变的小鼠模型。此外，我的目标是使用这种方法来测试在体内的cDNA变体，例如在otoferlin和两个不同的剪接变体的磷酸化模拟突变，在噪声诱导的突触丢失的潜在作用。将用免疫荧光法将Otoferlin表达水平与野生型水平进行比较。将通过细胞膜片钳电生理学来评估重新表达的耳传递蛋白的功能，以记录突触囊泡的Ca2+触发的融合。小鼠的听力将通过听觉脑干记录和行为测试来测量。我的目标是完善DFNB 9的因果疗法，使其接近临床应用。

Mutations in the gene OTOF, encoding the protein otoferlin, typically cause congenital profound deafness (DFNB9). Otoferlin is required for synaptic transmission from auditory sensory cells to subsequent neurons. To date, patients with this form of deafness receive cochlear implants, which stimulate the auditory pathway directly with electrical signals, thereby enabling people to comprehend speech. However, cochlear implants hardly transmit frequency information and encode only a limited dynamic range of sound intensity. Since in this form of deafness the inner ear is morphologically almost entirely intact, a transfer of otoferlin cDNA into the sensory cells is expected to restore hearing. Such a causal gene therapy could potentially enable hearing with hardly any quality restrictions, yet has not been developed for human patients to date.Recently, my group succeeded to transfer the large otoferlin cDNA into auditory sensory cells of mice by means of a recombinant virus. This was challenging because no viral vector was known that is suitable to transfer large cDNAs into this type of cells. We overcame this challenge by means of a "dual-AAV" method, which served to partially restore hearing in deaf otoferlin knock-out mice. In this method, the cDNA of otoferlin was split to two adeno-associated viruses (AAVs), which re-assemble in the nuclei of target cells to allow transcription of the full length otoferlin mRNA. This proposal focuses on testing variants of these AAVs that shall enhance the correct tail-to-head multimerization, aiming to increase protein expression and thus to further improve hearing. In addition, I will employ a refined method for injection into the inner ear, and a different AAV serotype to test if this is suitable to increase the rate of transduced sensory cells. Next, the synapses and auditory nerve fibers shall be characterized after re-expression of otoferlin. We will then apply this gene therapy approach to two mouse models with human mutations. Moreover, I aim to use this method to test cDNA variants in vivo, e.g. phosphomimetic mutations in otoferlin and two different splice variants, for a potential role in noise-induced synapse loss. Otoferlin expression levels will be compared to wild-type levels with immunofluorescence. The function of re-expressed otoferlin will be assessed by cellular patch-clamp electrophysiology to record Ca2+-triggered fusion of synaptic vesicles. Hearing in mice will be measured by auditory brainstem recordings and behavioral tests.My goal is to refine the causal therapy for DFNB9 to bring it close to clinical application.