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

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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.