Automated Production of RALA/Nucleic Acid Nanoparticles from Bench to Patient Dose

RNAi疗法有可能改变医疗保健干预措施，这一点在过去2年中批准了2种用于威胁生命的疾病的产品。RNAi疗法被设计为瞬时减少缺陷基因以用于治疗目的。这是一个快速增长的市场，临床试验中有109种基于RNAi的疗法（2018年7月）\[Wu X.和Turnball A. P. 2018\]。然而，仍然存在围绕RNAi疗法的问题，包括到达适当的组织，然后确保细胞内递送到目的地位点。最近的研究还表明，患有糖尿病、高血压或吸烟者等潜在健康状况的人肺上皮中ACE-2受体的数量增加[Leung J.M，2020]。研究揭示，COVID-19利用ACE-2受体进入细胞，以产生更多的病毒颗粒，从而感染更多ACE-2受体阳性细胞\[Kuba K. 2005年]。ACE-2受体对许多生物功能起作用，但如果表达可以在短时间内降低，它可以降低病毒的感染性，并有助于平衡健康恢复。RNAi可用于瞬时降低该ACE-2受体的表达，但仅当存在适当的递送系统时。pHion（贝尔法斯特SME）已经开发了一种用于RNAi递送的解决方案，该解决方案是安全的，不会进一步加剧免疫系统，优先将治疗剂递送至肺并且是成本有效的，最终使得RNAi疗法能够被广泛采用。创新的中心是使用一种名为RALA的肽，该肽被设计用于将RNAi浓缩成纳米颗粒（NP），这些纳米颗粒具有穿过细胞膜所必需的特性，从而逃脱内体，将货物高效地递送到细胞质中。在设计用于降低ACE-2表达的RNAi和RALA肽NP之间形成的NP不需要冷链储存，并且可以储存数月而不损失功能性。然而，我们还没有一种方法来支持这些NP的大规模生产。事实上，对于核酸工业来说，最大的障碍之一将是制造新的治疗方法。因此，与COVID-19所带来的挑战的特定主题明确一致，该项目旨在加速和优化RALA/RNAi治疗药物对患者剂量的扩大，以便“为未来做好准备”。拟议的9个月项目旨在开发使用微流体自动化生产功能性纳米颗粒的最佳条件，这些纳米颗粒可以很容易地转移到临床剂量。我们还将开发最佳的冻干工艺，以确保高度稳定的功能性产品。最后，随着监管框架的到位，并证明我们可以将我们的过程转移到外部，以扩大到临床剂量，我们将能够很好地将这种疗法应用于临床，并将RALA定位为肺部RNAi疗法的首选递送系统。

RNAi therapeutics have the potential to transform healthcare interventions as evidenced by the approval of 2 products in the last 2 years for life threatening diseases. RNAi therapy is designed to transiently reduce a defective gene for therapeutic purposes. It is a rapidly growing market with 109 RNAi based therapeutics in clinical trials (July 2018) \[Wu X. and Turnball A.P. 2018\]. However, there are still issues that surround the RNAi therapeutics which include getting to the appropriate tissue and then ensuring intracellular delivery to the destination site. Recent studies have also indicated that those with underlying health conditions such as diabetes, high-blood pressure or smokers have an increased number of ACE-2 receptors in the lung epithelium \[Leung J.M, 2020\]. Studies have revealed that COVID-19 uses the ACE-2 receptor to enter cells in order to produce more viral particles that can infect more ACE-2 receptor positive cells \[Kuba K. 2005\]. The ACE-2 receptor plays a role for many biological functions but if expression could be lowered for a short period of time it could reduce the infectivity of the virus and help tip the balance towards healthy recovery. RNAi could be used to transiently reduce expression of this ACE-2 receptor but only if there is an appropriate delivery system. pHion (Belfast SME) have developed a solution for RNAi delivery that is safe, does not further exacerbate the immune system, preferentially delivers the therapeutic to the lung and is cost-effective, ultimately enabling widespread adoption of the RNAi therapy. The innovation centres around the use of a peptide termed RALA that is designed to condense RNAi into nanoparticles (NPs) that have the properties necessary to cross cell membranes, escape endosomes delivering the cargo to the cytoplasm with high efficiency. The NPs formed between the RNAi which is designed to reduce ACE-2 expression and the RALA peptide NPs do not require cold chain storage and can be stored for many months without losing functionality. However, we do not as yet have a methodology in place to support the large-scale production of these NPs. Indeed, for the nucleic acid industry, one of the greatest hurdles will be the manufacture of novel therapeutics. Therefore with clear alignment to the specific theme of challenges as a result of COVID-19, this project is designed to accelerate and optimise the scale-up of the RALA/RNAi therapeutic to patient doses in order to be 'future ready'. The proposed 9 month project is designed to develop the optimal conditions for the automated production of functional NPs using microfluidics that can be readily transferred to clinical doses. We will also develop the optimal lyophilisation process to ensure a highly stable functional product. Finally, with regulatory framework in place and proof that we can transfer our process externally to scale up to clinical doses, we will be well positioned to take this therapy to the clinic and to position RALA as the go-to delivery system for RNAi therapeutics to the lung.