2024 AIChE Annual Meeting

(175an) Enhanced Delivery of Disease-Targeted Nanobody-siRNA Conjugates

Authors

Kimmel, B., Northwestern University
Introduction: Over recent years short-interfering RNA (siRNA) research has seen growth. The delivery of naked siRNA has challenges as it is easily degradable in the bloodstream and has low cellular uptake; therefore, requiring a carrier for improving efficiency of site-specific delivery while minimizing off-target effects and to protect the siRNA from degradation before reaching the site of interest. Delivery systems including nanocarriers, peptides, antibodies, and proteins have been developed as a result, however smaller sizes are preferable for cellular uptake. siRNA delivery in vivo presents further challenges regarding its short half-life and cellular internalization – siRNA may be encapsulated by endosomes and must escape for successful delivery into the cell’s cytoplasm. Furthermore, RNA delivery into the central nervous system (CNS) and brain is hindered due to key challenges including vasculature, permeability, and penetrating the blood-brain barrier (BBB), where the tight junction of endothelial cells severely limits therapeutic entry. A nanobody is a single domain antibody fragment with a size around 15 kDa, presenting itself as a good carrier based on its small size and the ability to selectively bind protein receptors and therapeutic targets. Nanobody-siRNA conjugates have recently been shown to successfully achieve site-specific delivery of oligo payloads to knockdown AHSA1 (a house keeping gene for epidermal growth factor receptor - EGFR), vascular endothelial growth factor (VEGF), CD47, and PDL1 in cancer therapy. Nanobody-siRNA conjugates are an effective approach for targeting and regulating receptor proteins including targets in the brain due to receptor-mediated transcytosis – a noninvasive entry into the BBB and CNS.

Methods: Nanobodies can be engineered to readily cross the BBB for site specific delivery to the brain for applications in brain cancer and autoimmune diseases. In this work, we focus on the generation of a modular nanobody-siRNA conjugate platform for targeting the BBB for autoimmune diseases. T cells play a key role in the progression of multiple sclerosis by releasing antigens and cytokines that inflict inflammation and damage in the CNS or through activation of B cells, ultimately resulting in demyelination and neurodegeneration. We will investigate delivery of siRNA targeting CD4+ T cells conjugated with anti-transferrin nanobody for in vitro qPCR experiments, validating our nanobody structure with size exclusion chromatography and SDS page. The combination of parameters (siRNA gene type, nanobody, endosomolytic peptide, and linker spacing) will be optimized by using cellular uptake, stability, and impact of linker length on the delivery of siRNA.

Results: Our preliminary experiments demonstrate knockdown of luciferase in vitro to evaluate the feasibility of our structure and determine the cellular uptake with and without an endosomolytic peptide using flow cytometry and microscopy. We will also investigate the modulation of nanobody type to target different cells through transcytosis within the BBB and CNS. The top combinations of our platform will be used to evaluate delivery of a therapeutic siRNA. The impacts of gene knockdown using qPCR, western blots, and viability in vitro will be examined for future optimization.

Conclusions: Future studies will explore the nanobody-siRNA conjugate platform in vivo using flow cytometry to monitor cellular uptake and off-target effects. The impact of therapeutic siRNA will be monitored in vivo to understand the efficacy of the platform and the epitope spreading. This work will allow for more site-specific targeting across the BBB for delivery of siRNA to enable targeted knockdown of the gene expression of interest, and possibly generating novel modular nanobodies.