2024 AIChE Annual Meeting
(175an) Enhanced Delivery of Disease-Targeted Nanobody-siRNA Conjugates
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.