Self-delivering RNAi to promote axon regeneration

描述(申请人提供)：神经损伤后，中枢神经系统(CNS)中的神经元不会再生其受损的轴突。随着正常神经元分化而发展的内在信号在阻止轴突再生方面发挥着重要作用。几个本征 通过对基因敲除小鼠的研究，已经确定了再生的负调控因素。我们将设计和测试新的自我递送RNA干扰(SdRNAi)化合物，作为评估再生的内在障碍作为治疗目标的一种方法。我们将使用新的sdRNAi技术，它不需要任何额外的递送配方，以降低炎症和非特异性效应的风险，这些都是RNAi注射到中枢神经系统特别令人担忧的。SdRNA已经在体内被证明是有效的，不需要特殊的递送包装或脂质体。对于临床开发，确定是否需要在细胞体附近注射针对内在调节的sdrna，或者是否可以注射sdrna将是很重要的。 在受伤的轴突部位注射。在后一种情况下，sdRNA可能作用于神经末梢，也可能被逆行运输到细胞体。作为传递技术的重点，我们将研究sdRNAs在体外分别应用于细胞体和轴突，然后在体内通过注射到运动皮质或损伤脊髓中。这些实验将帮助我们设计合适的给药策略来治疗脊髓损伤。我们将针对通过基因敲除技术验证的三个目标创建sdRNA。我们将设计、合成和测试针对PTEN、TSC1和KLF9的sdRNAs，所有这些靶点都被证明在沉默时可以促进轴突再生。每个候选者都将在体外的原代神经元中进行评估，以抑制表达，并促进轴突生长。对星形胶质细胞增殖的影响将通过原代胶质细胞培养来研究。为了评估传递，我们将测试sdRNA对在隔室中培养的原代培养的轴突生长的影响，这种隔室允许向轴突或细胞体隔室专门添加药物。铅sdRNA的有效性将在大鼠脊髓损伤的挫伤模型中进行体内测试。我们将评估是否需要局部或局部应用sdRNA来诱导体内基因沉默和刺激轴突再生。受伤大鼠的功能恢复也将被评估为临床前验证。

DESCRIPTION (provided by applicant): After neurotrauma, neurons in the central nervous system (CNS) do not regenerate their injured axons. Intrinsic signals that progress with normal neuronal differentiation play a significant role in preventing axon regeneration. Several intrinsic negative regulators of regeneration have been identified through studies of knock-out mice. We will design and test novel self-deliverable RNA interference (sdRNAi) compounds as an approach to evaluate intrinsic barriers to regeneration as therapeutic targets. We will use novel sdRNAi technology, which does not require any additional delivery formulation, to reduce the risk of inflammation and non-specific effects, which are of special concern with RNAi injected into the CNS. sdRNA has been proven effective in vivo without the need for special delivery packaging or liposomes. For clinical development, it will be important to determine if sdRNA against intrinsic regulators needs to be injected in the vicinity of the cell body, or if it can be injected at the site of the injured axons. In the latter case, sdRNA might act at the nerve termina or might be retrogradely transported to the cell body. As a focus on delivery techniques, we will investigate sdRNAs applied to cell bodies and axons separately in vitro, and then by injection into the motor cortex or injured spinal cord in vivo. These experiments will help us design appropriate delivery strategies to treat spinal cord injury. We will create sdRNAs against three targets validated through knockout techniques. We will design, synthesize, and test sdRNAs to PTEN, TSC1 and KLF9, all targets shown to promote axon regeneration when silenced. Each candidate will be evaluated in in vitro in primary neurons for knockdown of expression, and for promotion of neurite outgrowth. The effect on astrocyte proliferation will be studied with primary glial cultures. To evaluate delivery, we will test the effect of sdRNA on axon growth in primary cultures plated in compartmental chambers that allow addition of agents specifically to the axonal compartment or to the cell body compartment. The effectiveness of the lead sdRNA will be tested in vivo in a rat contusion model of spinal cord injury. We will evaluate if local or distl application of sdRNA is required to induce gene silencing in vivo and stimulate regeneration of axons. Functional recovery of injured rats will also be assessed to for preclinical validation.