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Drug Delivery

Targeted or Localized Drug Delivery Systems I

Focused ultrasound and long-circulating nanoparticle-mediated nucleic acid delivery and genome editing in the brain

Thursday, October 24, 2024
9:15 AM – 9:30 AM EST
Location: Room 317

Introduction: Nanoparticle (NP)-based nucleic acid delivery to the brain via the systemic route requires the ability of NPs to avoid liver accumulation, stably circulate in the bloodstream, traverse the blood-brain barrier (BBB), and deliver the payloads throughout the disease area within the brain. A BBB penetrable adeno-associated virus serotype 9-based gene therapy was approved by the FDA for treating spinal muscular atrophy but was also shown to accumulate in other organs, including the liver. Here, we introduce a combination of BBB-opening with focused ultrasound (FUS)/microbubbles (MBs) and long-circulating poly(ethylene glycol)-coated poly(β-amino ester) (PEG-PBAE) NPs that enables efficient and safe systemic nucleic acid delivery to the brain.

Materials and

Methods: PEG-PBAE NPs were prepared by packaging various nucleic acids, including plasmid DNA (pDNA), mRNA, or a blend of mRNA and single-guide RNA (sgRNA), with a mixture of PBAE and PEG-PBAE polymers. PEG-PBAE NPs were analyzed by gel electrophoresis, transmission electron microscopy, dynamic light scattering, and multiple particle tracking analysis. Animals received intravenous PEG-PBAE NPs and MBs and FUS treatment. Reporter protein expression was assessed by live-animal imaging, tissue homogenate-based luciferase assay, and confocal microscopy.

Results, Conclusions, and Discussions: PEG-PBAE NPs were engineered and confirmed to package pDNA or mRNA (Figures 1A and 1B). PEG-PBAE NPs exhibited excellent colloidal stability in relevant physiological environments, including artificial cerebrospinal fluid and mouse whole serum (Figures 1C and 1D). Systemically administered PEG-PBAE NPs exhibited stable systemic circulation for at least up to 4 hours (Figures 1E, 1F, and 1G). Accordingly, the combination of systemic administered PEG-PBAE NPs and FUS/MB-mediated BBB-opening resulted in efficient NP accumulation preferentially at the FUS-applied site in the brain with negligible off-target accumulation (Figures 1H and 1I). Freshly prepared and serum-incubated PEG-PBAE NPs showed comparable widespread transgene expression in the brain (Figures 1J and 1K). Systemic PEG-PBAE NPs and FUS/MB treatment achieved reporter protein production by pDNA or mRNA payloads in astrocytes and neurons at the FUS-applied site (Figures 1L, 1M, 1N, and 1O). Likewise, systemic PEG-PBAE NPs carrying mRNA encoding CRISPR-associated protein 9 and sgRNA targeting loxP-flanked STOP cassette in the Ai9 mice chromosome resulted in efficient tdTomato expression, indicating successful gene editing, in astrocytes and neurons at the FUS-applied site regardless of gender (Figures 1P, 1Q, 1R, and 1S). Systemic PEG-PBAE NPs and FUS/MB treatment provide a highly versatile means to enable efficient and site-specific therapeutic nucleic acid delivery and gene editing in the brain.

Acknowledgements (Optional):