(M-486) Feasibility of The Noninvasive Gene Delivery/Editing to Large Brain Areas

Neurological disorders, such as neurodevelopmental disorders (NDDs), often have a genetic basis, making gene therapy an appealing treatment approach. Recombinant adeno-associated viruses (rAAVs) are a promising vector for central nervous system (CNS) gene therapy but, delivering  these vectors to the CNS presents significant challenges due to the presence of the blood-brain barrier (BBB) and the risks associated with invasive delivery methods. focused ultrasound-induced BBB opening (FUS-BBBO) brings a new sight for the delivery of gene therapy vectors. However, current FUS-BBBO techniques are limited to local or regional targeting, which may not be sufficient for brain-wide pathologies. To address this limitation, we propose a strategy for multi-site FUS-BBBO targeting of large brain areas over fewer sessions for gene delivery and editing. We suggest combining FUS-BBBO with clustered regularly interspaced short palindromic repeats (CRISPR) systems for RNA-guided genome editing of DNA sequences. CRISPR allows for precise and efficient gene editing in vivo by using single-guided RNAs (sgRNAs) to direct a DNA nuclease, typically CRISPR-associated 9 (Cas9), to modify or delete target genomic sequences. Overall, our proposed strategy represents a promising direction for CNS gene therapy, with significant implications for the treatment of a wide range of neurological disorders.

We aimed to determine the maximum safe volume of the brain that can be targeted using focused ultrasound-induced BBB opening (FUS-BBBO). To achieve that, we performed FUS on mice by targeting the brain incrementally from 11 to 105 brain sites (Fig. 1a-d).  We chose 0.27 MPa of pressure, a frequency of 1.53 MHz, and a 10 ms pulse length repeated once per second for 30 burst per site and to assess safety and BBBO efficacy respectively we evaluated Evan Blue dye (EBD) delivery by histological hemorrhagic and EBD extravasation analyses on neuro-anatomic regions (striatum, thalamus, midbrain, & hippocampus). Following parameter optimization, a complementary insonation procedure was carried out to intravenously deliver AAV-mediated green fluorescent protein (GFP), and 3 weeks post-treatment, fluorescence imaging and analysis of GFP expression were performed.

To evaluate potential applications in treating genetic disorders that affect widespread brain regions, we delivered a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system via AAV to the brains (22 sites) of transgenic mice (Ai9). Ai9 mice have a loxP-flanked STOP cassette that prevents transcription of a red fluorescent protein (tdTomato), and by delivering the CAS9, it excises the stop cassette, and tdTomato can express in the targeted area. Three weeks post-treatment, fluorescence imaging and analysis of tdTomato expression were performed to evaluate the efficacy of the treatment.

 Histology analysis revealed pressure of 0.27 MPa with 1.53 MHz frequency, 1% duty cycle and 1 Hz pulse repetition frequency for 30 pulses is safe and effective, as brightfield microscopy showed no brain hemorrhage and positive EBD signal (Fig. 1e). Using this pressure, we delivered AAVs (1E10 viral particles/gram, intravenous) carrying GFP under constitutive CAG promoter via FUS-BBBO throughout and We observed successful gene delivery at 100% of the targeted sites that could be recovered by tissue histology (Fig. 1e). For 11-site targeting, the average transduction efficiency was 32.92±2.766%; for 22 sites, it was 44.99±2.422%, and for 105 sites targeting, it was 60.82±3.191% which was safe with no significant mouse weight loss, death, or neuronal loss. We observed that increasing the number of target sites significantly increased the transduction efficiency at each site, suggesting that widespread gene delivery with FUS-BBBO is an advantageous method for gene delivery.

Histological analysis of tdTomato expression in Ai9 mice showed successful genome editing in the brain after noninvasive delivery of CRISPR and gRNA (Fig. 1f). We observed 24.1±2.1% transduction efficiency at the targeted sites, compared to significantly lower efficiency of 2.8±1% at the contralateral control sites (p< 0.0001, two-tailed paired t-test, numbers represent means with 95% CI, n=110 sites per group in 5 mice) We did not observe significant differences in gene editing in different targeted regions with the tdTomato fluorescence present in 9.8±2.8% of hippocampus and 6.2±3.5% of midbrain and it was 5.2±2.2% of the striatum cells ( p< 0.0001, one-way ANOVA, with Tukey’s post-hoc test). Our results show that FUS-BBBO can be used for noninvasive genome editing in the brain.

The use of FUS-BBBO for gene delivery and genome editing in the brain could have important implications for the treatment of neurological disorders that affect large areas of the brain. This is because FUS-BBBO allows for the precise and noninvasive delivery of therapeutic agents to targeted areas of the brain, potentially reducing the need for more invasive surgical procedures.