Brain Nucleic Acid Delivery and Genome Editing via Focused Ultrasound-Mediated Blood-Brain Barrier Opening and Long-Circulating Nanoparticles

Abstract

We introduce a two-pronged strategy comprising focused ultrasound (FUS)-mediated blood-brain barrier (BBB) opening and long-circulating biodegradable nanoparticles (NPs) for systemic delivery of nucleic acids to the brain. Biodegradable poly(β-amino ester) polymer-based NPs were engineered to stably package various types of nucleic acid payloads and enable prolonged systemic circulation while retaining excellent serum stability. FUS was applied to a predetermined coordinate within the brain to transiently open the BBB, thereby allowing the systemically administered long-circulating NPs to traverse the BBB and accumulate in the FUS-treated brain region, where plasmid DNA or mRNA payloads produced reporter proteins in astrocytes and neurons. In contrast, poorly circulating and/or serum-unstable NPs, including the lipid NP analogous to a platform used in clinic, were unable to provide efficient nucleic acid delivery to the brain regardless of the BBB-opening FUS. The marriage of FUS-mediated BBB opening and the long-circulating NPs engineered to copackage mRNA encoding CRISPR-associated protein 9 and single-guide RNA resulted in genome editing in astrocytes and neurons precisely in the FUS-treated brain region. The combined delivery strategy provides a versatile means to achieve efficient and site-specific therapeutic nucleic acid delivery to and genome editing in the brain via a systemic route.

Keywords: biodegradable polymer; blood–brain barrier; brain gene therapy; long-circulating nanoparticle; systemic nucleic acid delivery.

Figure 1.. PEG-PBAE NPs, but not un-PEGylated PBAE NPs, carrying pDNA or mRNA retain colloidal stability in physiological conditions relevant to the systemic delivery of NPs to the brain.

(A) Representative agarose gel electrophoresis migration assay showing robust packaging of pDNA (left) or mRNA (right) in PEG-PBAE NPs. Numbers indicate the polymer-to-nucleic acid weight ratios. MK: molecular weight marker. (B) Representative transmission electron micrographs of PEG-PBAE NPs carrying pDNA (top) or mRNA (bottom). Scale bar = 200 nm. (C) Hydrodynamic diameters and ζ-potentials of PEG-PBAE NPs carrying pDNA or mRNA. (D) Colloidal stability of PBAE and PEG-PBAE NPs carrying pDNA or mRNA incubated in aCSF at 37 °C for up to 8 hours. (E) Median MSD values of PBAE and PEG-PBAE NPs carrying pDNA or mRNA in PBS or whole mouse serum. MSD is a square of distance traveled by an individual particulate matter within a predetermined time interval (i.e., time scale; τ = 1 s) and thus is directly proportional to the particle diffusion rate. n.s.: no significance, ****p < 0.0001 (one-way or two-way ANOVA).

Figure 2.. PEG-PBAE NPs, but not clinically used LNPs, carrying pDNA or mRNA, exhibit long circulation in bloodstream following systemic administration.

(A) Representative whole-body fluorescence images of live animals intravenously treated with PBAE NPs, PEG-PBAE NPs, or LNPs carrying Cy5-labeled pDNA or mRNA over time (N = 3 animals per group). (B) Representative fluorescence images of major organs harvested at 4-hour post-administration of NPs from the animals in Figure 2A. (C) Percentage of circulating nucleic acids determined by quantifying the Cy5 fluorescence intensity of the serum collected at 4-hour post-administration of NPs from the animals intravenously treated with PBAE NPs, PEG-PBAE NPs, or LNPs carrying Cy5-labeled pDNA or mRNA. n.s.: no significance, ****p < 0.0001 (one-way ANOVA).

Figure 3.. PEG-PBAE NPs carrying pDNA accumulate in FUS-treated brain region far greater than other major organs following systemic administration and retain the ability to mediate widespread transgene expression in the brain following serum incubation and subsequent intracranial administration.

(A) Representative fluorescence images and (B) Cy5 fluorescence intensity of brains and other major organs harvested at 4-hour post-administration of NPs from the animals intravenously treated with saline or PBAE or PEG-PBAE NPs carrying Cy5-labeled pDNA and subsequently received a FUS treatment on the right striatum (N = 3 animals per group). (C) Representative fluorescence images showing reporter ZsGreen1 expression in the brain sections at the infusion site within healthy mouse brain striatum 48 hours after the intracranial administration of freshly prepared or serum-incubated PEG-PBAE NPs carrying ZsGreen1-expressing pDNA at a pDNA dose of 0.1 mg/kg (N = 5 animals per group). Blue: nucleus; Green: ZsGreen1. Image-based quantification for (D) area and (E) intensity of ZsGreen1 expression. (F) Luciferase activity of homogenized brain tissues treated with freshly prepared or serum-incubated PEG-PBAE NPs carrying luciferase-expressing pDNA at a pDNA dose of 0.1 mg/kg via intracranial administration. n.s.: no significance, * p < 0.05, ***p < 0.001, ****p < 0.0001 (one-way ANOVA).

Figure 4.. PEG-PBAE NPs carrying pDNA mediate reporter protein production in astrocytes and neurons precisely in the FUS-treated brain region following systemic administration.

(A) Bioluminescence images of brains harvested at 48-hour post-administration of NPs from animals intravenously treated with PEG-PBAE NPs carrying luciferase-expressing pDNA at a pDNA dose of 1.25 or 2.5 mg/kg and subsequently received a FUS treatment on right striata (N = 5 animals per group). (B) Quantification of luciferase activity in the homogenized hemispheres from the brains in Figure 4A. (C) Representative confocal micrograph of brain harvested at 48-hour post-administration of NPs from animals intravenously treated with PEG-PBAE NPs carrying ZsGreen1-expressing pDNA at a pDNA dose of 2.5 mg/kg pDNA and subsequently received a FUS treatment on right striata (N = 5 animals per group). Blue: nucleus; Green: ZsGreen1. Representative confocal micrographs showing reporter ZsGreen1 production in untreated or FUS-treated areas of (D) GFAP- and (E) NeuN-stained brains from the animals identically treated as in Figure 4C. Blue: nucleus; Green: ZsGreen1; Red: astrocyte (GFAP) or neuron (NeuN). (F) Representative H&E-stained histological images of untreated or FUS-treated areas of the brains harvested from the animals identically treated as in Figure 4C. n.s.: no significance, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA).

Figure 5.. PEG-PBAE NPs, but not clinically used LNPs, carrying mRNA mediate reporter protein production in astrocytes and neurons precisely in the FUS-treated brain region following systemic administration.

(A) Bioluminescence images of brains harvested at 24-hour post-administration of NPs from animals intravenously treated with PEG-PBAE NPs carrying luciferase-expressing mRNA at an mRNA dose of 0.5 mg/kg and subsequently received a FUS treatment on right striata (N = 5 animals per group). (B) Quantification of luciferase activity in the homogenized hemispheres from the brains in Figure 5A. (C) Representative confocal micrograph of brain harvested at 24-hour post-administration of NPs from animals intravenously treated with PEG-PBAE NPs carrying mCherry-expressing mRNA at an mRNA dose of 0.5 mg/kg and subsequently received a FUS treatment on right striata (N = 5 animals per group). Blue: nucleus; Green: mCherry. Representative confocal micrographs showing reporter mCherry production in untreated or FUS-treated areas of (D) GFAP- and (E) NeuN-stained brains from the animals identically treated as in Figure 5C. Blue: nucleus; Green: mCherry; Red: astrocyte (GFAP) or neuron (NeuN). (F) Representative H&E-stained histological images of untreated or FUS-treated areas of the brains harvested from the animals identically treated as in Figure 5C. n.s.: no significance, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA).

Figure 6.. PEG-PBAE NPs carrying Cas9-expressing mRNA and sgRNA targeting the STOP cassette (sgRNA 298) mediate genome editing in astrocytes and neurons precisely in the FUS-treated regions of Ai9 mouse brains following systemic administration.

(A) Agarose gel and (B) TBE gel electrophoresis migration assay showing robust co-packaging of Cas9-expressing mRNA and sgRNA 298 in PEG-PBAE NPs. Numbers indicate the polymer-to-nucleic acid weight ratios. MK: molecular weight marker. (C) Representative confocal micrographs of the brains harvested at 5-day post-administration of NPs from female (upper) and male (bottom) Ai9 mice intravenously treated with PEG-PBAE NPs carrying Cas9-expressing mRNA and sgRNA 298 at an mRNA and an sgRNA dose each of 0.5 mg/kg and subsequently received a FUS treatment on right striata (N = 6 animals per group). Blue: nucleus; Green: tdTomato. Representative confocal micrographs showing reporter tdTomato production in untreated or FUS-treated areas of (D) GFAP- and (E) NeuN-stained brains from the animals identically treated as in Figure 6C. Blue: nucleus; Green: tdTomato; Red: astrocyte (GFAP) or neuron (NeuN).