Targeting the Transferrin Receptor to Transport Antisense Oligonucleotides Across the Mammalian Blood-Brain Barrier
Introduction
In recent years, oligonucleotide-based therapeutic technologies, such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), have been widely applied in the treatment of various neurological disorders. The basis for their application is that these technologies can selectively regulate target RNA molecules, which are often difficult to modulate through other therapeutic approaches. Particularly after the approval of Nusinersen for the treatment of spinal muscular atrophy in 2016, the potential of ASOs in central nervous system (CNS) diseases has received further attention and research. However, due to their inherent biophysical properties, such as large molecular weight, charged nature, and backbone chemistry, oligonucleotides have difficulty penetrating the blood-brain barrier (BBB), necessitating their direct delivery to the cerebrospinal fluid (CSF) through intrathecal injection to affect the CNS. This delivery method has many limitations, such as uneven distribution of the drug in deep brain regions and potential adverse events associated with intrathecal injection. Therefore, finding an efficient and safe delivery method has become an urgent problem to solve.
Research Background and Source
This research paper, titled “Targeting the Transferrin Receptor to Transport Antisense Oligonucleotides Across the Mammalian Blood-Brain Barrier,” was written by Scarlett J. Barker and her team, with the research conducted in collaboration between Denali Therapeutics Inc. and Ionis Pharmaceuticals. The paper was published in the journal “Science Translational Medicine” on August 14, 2024. This study aims to develop a delivery platform based on transferrin receptor 1 (TfR1) to transport ASOs to the mammalian brain through systemic injection, overcoming the limitations of existing technologies.
Research Methods
Experimental Design and Research Subjects
This study first engineered TfR1 binding molecules, named oligonucleotide transport vehicles (OTVs), to test their biodistribution and RNA degradation effects in TfR1 gene-edited mice (Tfr1mu/hu KI mice) and non-human primates. The authors described the research process in detail through the following steps:
1. OTV Molecule Production and Characteristics
The research team expressed and purified OTV molecules containing engineered Fc domains using CHO cell lines. These domains can specifically bind to TfR1 without affecting normal transferrin binding. Subsequently, the team chemically synthesized and stably site-specifically conjugated target ASOs to OTVs, analyzing the purity and binding ratio of the products using mass spectrometry and gel filtration chromatography.
2. Cellular Uptake and Transport Experiments
The researchers verified OTV uptake and distribution in cells through in vitro experiments, including binding experiments on HTFR1-expressing cells and determining the affinity of OTV for HTFR1 using surface plasmon resonance (SPR) technology. Additionally, the distribution of dual-labeled OTVs in neuronal cells was observed in detail using confocal microscopy, confirming that OTVs could be absorbed by cells and primarily localized in late endosomes and lysosomes.
3. Animal Experiments
In mice and non-human primates, the research team systemically injected OTVs intravenously and collected tissue samples to measure ASO concentrations in the brain and peripheral tissues, as well as the degradation effect on the target RNA (MALAT1). Particularly in mice, single-nucleus RNA sequencing technology was applied to analyze MALAT1 levels in various brain cell types in detail.
Research Results
1. OTV Construction and Binding Characteristics
Experiments showed that site-specifically conjugated OTV molecules maintained high affinity for TfR1 while not affecting ASO binding and function. This property ensures that OTVs can efficiently cross the blood-brain barrier and release ASOs.
2. Cellular Distribution and Uptake Experiments
Cell experiment results showed that OTVs could be efficiently absorbed by cells and primarily localized in endosomes and lysosomes. Compared to naked ASOs, the subcellular distribution of OTV molecules did not change significantly, indicating that OTVs can effectively carry ASOs and enhance their stability within cells.
3. In Vivo Effects
In mouse models, systemically injected OTVs significantly increased ASO concentrations in the brain and peripheral tissues, especially showing better MALAT1 RNA degradation effects in deep brain structures and muscle tissues. Single-nucleus sequencing results showed that OTVs significantly reduced MALAT1 levels in all major brain cell types, indicating that OTVs can be widely distributed and exert their effects.
In non-human primates, systemic injection of OTVs achieved a more uniform ASO biodistribution. Compared to intrathecal injection, OTVs more effectively delivered ASOs to deep brain regions, reducing high-concentration accumulation of the drug at the injection site and related side effects.
Conclusions and Significance
Novelty and Value of the Research
Through this study, the authors proposed a novel ASO delivery platform, OTV, based on TfR1. This method not only overcomes the limitations of traditional delivery methods, achieving efficient delivery of ASOs across the blood-brain barrier, but also significantly improves the distribution and efficacy of the drug in the CNS and other hard-to-reach peripheral tissues. Considering the potential applications of ASOs in gene therapy, the OTV platform has the potential to be used in the treatment of various neurodegenerative diseases and peripheral neuropathies in the future, opening up new avenues for ASO therapy.
This research not only has important scientific value but also provides new ideas and technical support for the clinical application of ASO drugs. It is expected to greatly improve the efficacy and safety of existing gene therapies, bringing new hope and treatment options to patients with related diseases.