MICA: Single-domain antibody oligonucleotides conjugates for brain delivery of oligonucleotide therapeutics

治疗性寡核苷酸(ONS)是人工合成的类核糖核酸(RNA)序列，构成了人类疾病治疗的一大类新疗法。ONS识别并结合细胞中互补序列的同源RNA靶标，从而触发一系列细胞反应，从RNA降解到RNA靶标的改变成熟。ONS在遗传病的治疗方面具有很高的兴趣，在遗传病中，突变可能会改变编码蛋白质的RNA的稳定性和成熟度，这可以通过ON疗法纠正。到目前为止，已有15个ONS获得了监管部门的批准，还有更多的ONS正在进行临床试验。药物开发的一个主要翻译瓶颈是实现有效的体内递送到相关组织和细胞类型。全身注射后，如皮下或静脉注射，ONS在血液中迅速分布，但不容易越过血脑屏障(BBB)等生物屏障。实现超越血脑屏障的交付对于治疗严重的神经肌肉疾病至关重要，如脊髓性肌萎缩症(SMA)或肌萎缩侧索硬化症(ALS)，这些疾病会导致大脑和脊髓中的神经元退化。ONS的脑内传递可能通过鞘内注射，即直接进入脊髓。然而，这种注射模式对于重复注射来说风险更大，也不切实际。改善ONS释放的一个重要策略是化学桥联(接合)到促进摄取到合适的组织和细胞类型的分子。多年来，已开发出多种共轭结构。细胞穿透肽、小分子配体和抗体促进细胞内对药物的摄取。一个非常成功的例子是GalNAc(N-乙酰半乳糖胺)配体，它使肝脏向肝细胞的转运增加了10倍。第一个GalNAc-on结合物吉沃西兰于2019年被批准用于治疗急性肝性卟啉。第一个抗体-寡核苷酸结合物也于2021年进入临床试验，用于在强直性肌营养不良I型(DM1)的背景下增加对肌肉组织的递送。抗体领域的最新进展是单域抗体，即所谓的“VHH”或纳米体。纳米抗体的相对分子质量比传统抗体小约10倍，但仍具有完全的生物活性。它们更容易生产和化学修饰。然而，人们对它们将寡核苷酸有效载荷运送到组织中的潜力知之甚少。这项提案旨在推动纳米体寡核苷酸作为脑输送载体和治疗SMA的药物。该提议分为四个目标：1-发展寡核苷酸-纳米体结合化学。我们将评估以转铁蛋白受体1(TfR1)为靶标的纳米体，并通过可切割或不可切割的连接子连接到几个化学上的纳米体。将对生物活性和摄取进行检测。可切割连接物已被用于大的连接物，以在靶细胞内释放治疗有效载荷，但这可能对纳米体不那么关键。2-开发新的纳米体，用于超越生物屏障的输送。除了TfR1以外的许多受体可以被利用来传递到血脑屏障以外的地方。我们将为四种受体开发纳米体，并评估它们将药物输送到大脑的潜力。3-建立寡核苷酸-纳米体结合物的药代动力学特性，与大抗体寡核苷酸结合物和未结合的寡核苷酸进行比较。4-通过尖端串联质谱仪进行先进的寡核苷酸成像，以精确检测和可视化细胞和组织中的治疗性寡核苷酸。

Therapeutic oligonucleotides (ONs) are synthetic ribonucleic acid (RNA)-like sequences which constitute a major class of novel therapeutics for human disease. ONs recognize and bind a cognate RNA target of complementary sequence in the cell and thereby trigger a range of cellular responses, from RNA degradation to altered maturation of the RNA target. ONs are of high interest for the treatment of genetic diseases, where mutations may alter stability and maturation of protein coding RNAs which can be corrected by ON therapy. 15 ONs have been approved by regulatory authorities so far, and many more are being evaluated in clinical trials.A major translational bottleneck in ON drug development is achieving effective in vivo delivery to relevant tissues and cell types. Upon systemic injection, e.g. subcutaneous or intravenous, ONs distribute rapidly through the bloodstream but do not readily cross biological barriers like the blood-brain barrier (BBB). Achieving delivery beyond the BBB is of prime relevance for the treatment of severe neuromuscular disorders such as spinal muscular atrophy (SMA) or amyotrophic lateral sclerosis (ALS), which cause neuronal degeneration in brain and spinal cord. Brain delivery of ONs might be mediated through intrathecal injection, i.e. directly into the spinal cord. This mode of injection is however riskier and unpractical for repeated injections. A prominent strategy to improve the delivery ONs is chemical bridging (conjugation) to a molecule facilitating uptake into the right tissue and cell type. Over the years, multiple conjugate moieties have been developed. Cell-penetrating peptides, small-molecule ligands, and antibodies facilitate the intracellular uptake of ON drugs. A very succesful example is the GalNac (N-Acetylgalactosamine) ligand, which increases delivery to hepatocytes of the liver by a factor 10. The first GalNac-ON conjugate, givosiran, was approved for the treatment of acute hepatic porphyria in 2019. A first antibody-oligonucleotide conjugate also entered clinical trials in 2021 for increased delivery to muscle tissue in the context of myotonic dystrophy type I (DM1). A recent development from the antibody field are single-domain antibodies, as known as "VHHs" or nanobodies. Nanobodies are about 10 times smaller in molecular weight than conventional antibodies but retain full biological activity. They are easier to produce and modify chemically. However, little is known about their potential for delivery of oligonucleotide payloads into tissues. This proposal aims to advance nanobody-oligonucleotides as delivery vehicles for brain delivery and as therapeutics for SMA. The proposal is broken down into four aims:1- Develop oligonucleotide-nanobody conjugation chemistries. We will evaluate nanobodies targeting the transferrin receptor 1 (TfR1) and conjugated to several ON chemistries through cleavable or non-cleavable linkers. Biological activity and uptake will be assayed. Cleavable linkers have been used for large conjugates to release therapeutic payloads within target cells, but this may be less critical for nanobodies.2- Develop novel nanobodies for delivery beyond biological barriers. Many receptors other than TfR1 could be harnessed for delivery beyond the BBB. We will develop nanobodies for four receptors and evaluate their potential for drug delivery into the brain.3- Establish pharmacokinetics properties of oligonucleotide-nanobody conjugates, as compared to large antibody oligonucleotide conjugates and unconjugated oligonucleotides.4- Advance oligonucleotide imaging by cutting-edge tandem mass spectrometry, for precise detection and visualization of therapeutic oligonucleotides within cells and tissues.