Ultrasound-Assisted CRISPRi-Exosome for Epigenetic Modification of α‑Synuclein Gene in a Mouse Model of Parkinson’s Disease

Currently, there is a lack of effective treatment for Parkinson’s disease (PD). In PD patients, aberrant methylation of SNCA (α-synuclein gene) has been reported and may be a potential therapeutic target. In this study, we established an epigenetic regulation platform based on an exosomal CRISPR intervention system. With the assist of focused ultrasound (FUS) opening the blood-brain barrier, engineered exosomes carrying RVG (rabies viral glycoprotein) targeting peptide, sgRNA (single guide RNA), and dCas9-DNMT3A (named RVG-CRISPRi-Exo) were efficiently delivered into the brain lesions and induced specific methylation of SNCA. In vivo, FUS combined with RVG-CRISPRi-Exo significantly improved motor performance, balance coordination, and neurosensitivity in PD mice, greatly down-regulated the elevation of α-synuclein (α-syn) caused by modeling, rescued cell apoptosis, and alleviated the progression of PD in mice. [18F]-FP-DTBZ imaging suggested that the synaptic function of the nigrostriatal pathway could be restored, which was conducive to the control of motor behavior in PD mice. Pyrosequencing results showed that RVG-CRISPRi-Exo could methylate CpG at specific sites of SNCA, and this fine-tuned editing achieved good therapeutic effects in PD model mice. In vitro, RVG-CRISPRi-Exo down-regulated SNCA transcripts and α-syn expression and relieved neuronal cell damage. Collectively, our findings provide a proof-of-principle for the development of targeted brain nanodelivery based on engineered exosomes and provide insights into epigenetic regulation of brain diseases

Profile of chromosomal alterations in all HCC samples (n = 159).

<p><b>(a)</b>, heatmap of CNAs across all chromosomes. <b>(b)</b>, the frequencies of CNAs across all chromosomes. Copy number gain and loss events of chromosomal segments were determined using log2-transformed ratio thresholds of 0.141 and −0.136 derived from the k-means clustering analysis. The color brightness is directly proportional to the frequency of CNA. Color red represents copy number gain and color blue represents copy number loss. CNA: copy number alteration.</p

Profiles of etiology-related chromosomal aberrations in HCCs.

<p><b>(a).</b> the comparison of chromosomal aberration profiles between HBV-related HCCs and HCV-related HCCs. <b>(b)</b>, the comparison of chromosomal aberration profiles between virus-related HCCs and non-virus-related HCCs. Copy number gains and losses with a significant difference in frequencies were highlighted in red and blue, respectively.</p

Kaplan-Meier plots of overall survival in patients without PVTT (A) and in patients with PVTT (B).

<p>Kaplan-Meier plots of overall survival in patients without PVTT (A) and in patients with PVTT (B).</p

Correlations between significant chromosomal aberrations either located on the same (a) or different (b) chromosomes in all HCCs.

<p>Chromosomal segments with significant gain were highlighted in red and those with significant loss in blue. Spearman's rank correlation coefficient was calculated to assess the correlations between different chromosomal copy number gains and losses at the significant level of p<0.001.</p

Stratified analysis of association between single SNPs and overall survival in HCC Patients.

a<p>In each stratified analysis, HRs were adjusted for age, gender, HBsAg, serum AFP, tumor size, BCLC stage and number of TACE except for the stratifier.</p

Distributions of clinical characteristics and multivariant Cox regression analysis of prognostic factors in HCC patients<b>.</b>

<p>Abbreviations: CI, confidence interval; HR, hazard ratio; Ref., reference.</p>a<p>Adjusted for age, gender, HBsAg, serum AFP, tumor size, BCLC stage and number of TACE where appropriate.</p

Association of SNPs with clinical outcome of HCC patients.

a<p>Multivariate analyses were adjusted for age, gender, HBsAg, serum AFP, tumor size, BCLC stage and number of TACE.</p

The functional KEGG pathways enriched with genes located on the chromosomal segments with significant CNAs in all HCCs.

<p><b>Note:</b> The pathways that were significantly affected by the identfied CNAs were determined by Fisher' exact test.</p><p>KEGG, Kyoto Encyclopedia of Genes and Genomes; CNA, copy number alteration.</p

Information of 4 collected public datasets (n = 159).

*<p>Information of virus infection for individual samples is unavailable. HBV, hepatitis B virus; HCV, hepatitis C virus; HBx, hepatitis B virus X protein.</p