Intranasal Delivery of Telomerase Reverse Transcriptase mRNA for Therapy ofTraumatic Brain Injury

摘要 据估计，美国每年发生约300万例创伤性脑损伤（TBI）病例。 TBI可导致精神和运动功能的严重损害或死亡。没有有效 改善中重度TBI后认知能力的治疗。许多新的药理学方法， TBI治疗不够有效。基因治疗可以提供一个未满足的解决方案，以防止几个 神经退行性疾病，包括TBI。mRNA治疗剂/疫苗的最新进展已经吸引了 由于他们有能力解决未满足的临床需求而受到极大关注。例如， COVID-19 mRNA疫苗有助于控制全球流行病。有效的mRNA疗法需要 脂质纳米颗粒（LNP）载体，以保护mRNA免受降解。端粒，重复非编码DNA 序列，在组织修复和衰老中具有关键作用。血液导致大脑中端粒缩短 流动损伤和细胞死亡相关的炎症，并影响组织再生能力。的催化亚基 端粒酶是一种在细胞分裂过程中负责维持端粒长度（TL）的酶， 逆转录酶（TERT）。TL功能障碍与神经炎性和神经退行性疾病有关。 并建议作为TBI结果的标志。此外，TERT被证明是重要的， 神经元存活和认知，保护免受氧化应激和阻断神经元凋亡。而TERT 是神经系统疾病的潜在靶点，没有研究评估RNA治疗以解决TBI中的TL， 报道至今。在这里，我们提出了一个转型的治疗方法，创伤性脑损伤治疗，其中包括鼻内 (IN)TERTmRNA- LNP递送至脑。LNP在通往靶TBI组织的途中保护mRNA。立即 我们工作的重点是由头部撞击引起的大脑正常功能受损。我们的数据 表明LNP的IN递送绕过BBB，增强药物向大脑的转运。我们的团队已经 表明TERTmRNA可以在体外和体内增强TL，改善其他退行性疾病的预后， 条件我们还发现TBI患者的端粒缩短，TERT水平降低， 小鼠模型 我们的方法可能会从根本上改变TBI以及其他脑部疾病的治疗方法。在这 在探索性项目中，我们的中心假设是，TERTmRNA的IN给药将使暂时的 在TBI后恢复认知功能的受影响脑组织中的TERT表达。我们的具体目标是： (1)体外mRNA-LNP的设计、表征和生物相容性：将研究四种TERT-mRNA-LNP系统。 设计、表征并在体外用各种脑细胞进行评估;（2）评估生物分布， 在TBI小鼠模型中IN施用TERTmRNA-LNP的治疗效率（控制性皮质脑损伤） 影响，CCI）。报告基因mRNA和荧光标记的LNP在脑内的生物分布 TBI将 被执行。将基于TERT水平、TL、行为测试和免疫学标志物来测试效率。 这一探索性项目可能是开发急需的TBI疗法的第一步。

ABSTRACT It is estimated that annually ~3 million Traumatic brain injury (TBI) cases occur in the U.S. Moderate to severe TBI can cause significant impairments in mental and motor functions or death. There are no effective therapies to improve cognitive abilities after moderate-severe TBI. Many novel pharmacological approaches for TBI therapy are not effective enough. Gene therapy can provide an unmet solution in protecting against several neurodegenerative disorders, including TBI. Recent advances in mRNA therapeutics/ vaccines have drawn significant attention due to their ability to tackle unmet clinical needs. For instance, the prompt development of COVID-19 mRNA vaccines aided in controlling the pandemics worldwide. Efficient mRNA therapies require a Lipid Nanoparticles (LNP) carrier to protect mRNA from degradation. Telomeres, repetitive non-coding DNA sequences, have a pivotal role in tissue repair and aging. Telomere shortening in the brain results from blood flow impairment and cell-death related inflammation and affects tissue regeneration ability. A catalytic subunit of telomerase, an enzyme responsible for maintaining telomere length (TL) during cell division, is telomerase reverse transcriptase (TERT). TL dysfunction has been implicated in neuroinflammatory and neurodegenerative processes and proposed as a marker for TBI outcomes. Additionally, TERT was shown to be important in neuronal survival and cognition, protecting from oxidative stress and blocking neuronal apoptosis. While TERT is a potential target in neurological disorders, no studies evaluating RNA therapy to address TL in TBI were reported yet. Here we propose a transformational therapeutic approach for TBI therapy which includes intranasal (IN) TERT mRNA- LNP delivery to the brain. LNP protect mRNA en route to the target TBI tissue. The immediate focus of our work is an impairment in the brain's normal function caused by an impact to the head. Our data show that IN delivery of LNP bypasses BBB, enhancing drug transport to the brain. Our teams have demonstrated that TERT mRNA can enhance TL in vitro and in vivo improving prognosis in other degenerative conditions. We have also shown that there is a shortening of telomeres and reduction in TERT levels in our TBI mouse model. Our approach may radically change therapy for TBI, as well as other brain disorders. In this exploratory project, our central hypothesis is that IN administration of TERT mRNA will enable temporary expression of TERT in the affected brain tissue restoring cognitive functions after TBI. Our Specific aims are: (1) Design, characterization, and biocompatibility of mRNA-LNP in vitro: four TERT-mRNA-LNP systems will be designed, characterized, and assessed in vitro with various brain cells; (2) Evaluate the biodistribution and therapeutic efficiency of IN administration of TERT mRNA-LNPs in a mouse model of TBI (Controlled cortical impact, CCI). Biodistribution of reporter mRNA and fluorescently labeled LNP in the brains of mice with TBI will be performed. Efficiency will be tested based on TERT levels, TL, behavioral tests, and immunological markers. This exploratory project can be the first step in the development of a much-needed therapy for TBI.