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

治疗性寡核苷酸（ONs）是合成的核糖核酸（RNA）样序列，构成了一类主要的新型人类疾病治疗药物。核糖核酸识别并结合细胞中具有互补序列的同源RNA靶标，从而引发一系列细胞反应，从RNA降解到RNA靶标成熟的改变。ON在遗传性疾病的治疗中具有很高的价值，其中突变可能改变蛋白质编码rna的稳定性和成熟，这可以通过ON治疗来纠正。到目前为止，监管机构已经批准了15种on，还有更多的正在临床试验中进行评估。ON药物开发的一个主要翻译瓶颈是实现有效的体内给药到相关组织和细胞类型。全身注射后，如皮下或静脉注射，一氧化氮在血液中迅速分布，但不易穿过血脑屏障（BBB）等生物屏障。实现血脑屏障外输送对于治疗严重神经肌肉疾病（如脊髓性肌萎缩症（SMA）或肌萎缩侧索硬化症（ALS））具有重要意义，这些疾病会导致脑和脊髓的神经元变性。神经元的脑递送可能通过鞘内注射介导，即直接进入脊髓。然而，这种注射方式对于重复注射来说风险更大，也不实用。改善离子递送的一个重要策略是化学桥接（偶联）到一个分子，促进吸收到正确的组织和细胞类型。多年来，多个共轭基团得到了发展。细胞穿透肽、小分子配体和抗体促进细胞内对ON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，它将肝脏的肝细胞输送量增加了10倍。首款galnacon偶联物givosiran于2019年被批准用于治疗急性肝性卟啉症。首个抗体-寡核苷酸结合物也于2021年进入临床试验，用于增加I型肌强直性营养不良（DM1）患者肌肉组织的输送。抗体领域的最新发展是单域抗体，称为“vhs”或纳米体。纳米抗体的分子量比传统抗体小约10倍，但保留了完整的生物活性。它们在化学上更容易生产和修饰。然而，人们对它们将寡核苷酸有效载荷输送到组织中的潜力知之甚少。该提案旨在推进纳米体寡核苷酸作为脑递送载体和SMA的治疗方法。该提案分为四个目标：1-发展寡核苷酸-纳米体偶联化学。我们将评估靶向转铁蛋白受体1 （TfR1）的纳米体，并通过可切割或不可切割的连接物偶联到几种ON化学物质。将测定生物活性和摄取。可切割连接物已被用于大型偶联物，以释放靶细胞内的治疗有效载荷，但这可能对纳米体不太重要。2-开发新型纳米体，用于超越生物屏障的递送。除TfR1外，还有许多受体可用于血脑屏障外的递送。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-纳米体偶联物的药代动力学特性，与大抗体寡核苷酸偶联物和未偶联的寡核苷酸相比较。4-采用尖端串联质谱技术的先进寡核苷酸成像，用于细胞和组织内治疗性寡核苷酸的精确检测和可视化。

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.