Rittiner, J. | bioRxiv Science

bioRxiv2025

SNCA-targeted epigenome therapy for Parkinsons disease alleviates pathological and behavioral perturbations in a mouse model

Alpha-synuclein (SNCA) overexpression is implicated in Parkinsons disease (PD) pathogenesis, making SNCA downregulation a promising therapeutic strategy. We developed a SNCA-targeted epigenome therapy using an all-in-one lentiviral vector (LV) carrying deactivated CRISPR/(d)Cas9, gRNA targeted at SNCA-intron1, and either the catalytic domain of DNA-methyltransferase3A (DNMT3A), or a synthetic repressor molecule of Kruppel-associated box (KRAB)/ methyl CpG binding protein 2 transcription repression domain (MeCp2-TRD). Therapeutic efficacy was evaluated in a new PD mouse model, generated with an adeno-associated viral vector carrying an engineered minigene comprised of the human (h)A53T-SNCA expressed via the human native regulatory region. Both therapeutic vectors reduced expression of -synuclein in the substantia nigra (SN), with LV/dSaCas9-KRAB-MeCP2(TRD) demonstrating greater repression. LV/dSaCas9-KRAB-MeCP2(TRD) also significantly reduced pathological -synuclein aggregation and phosphorylation (Ser129), and preserved tyrosine hydroxylase expression in the SN and the striatum. Behavioral analysis following LV/dSaCas9-KRAB-MeCP2(TRD) injection, showed significant improvement in motor deficits characteristic of our PD-mouse model. Safety assessments found normal blood counts, serum chemistry, and weights. Collectively, we provide in vivo proof-of-concept for our SNCA-targeted epigenome therapy in a PD-mouse model. Our results support the systems therapeutic potential for PD and related synucleinopathies and establish the foundation for further preclinical studies toward investigational new drug enablement.

neuroscience↗

bioRxiv2023

All-in-one AAV-delivered epigenome-editing platform: proof-of-concept and therapeutic implications for neurodegenerative disorders

Safely and efficiently controlling gene expression is a long-standing goal of biomedical research, and the recently discovered bacterial CRISPR/Cas system can be harnessed to create powerful tools for epigenetic editing. Current state-of-the-art systems consist of a deactivated-Cas9 nuclease (dCas9) fused to one of several epigenetic effector motifs/domains, along with a guide RNA (gRNA) which defines the genomic target. Such systems have been used to safely and effectively silence or activate a specific gene target under a variety of circumstances. Adeno-associated vectors (AAVs) are the therapeutic platform of choice for the delivery of genetic cargo; however, their small packaging capacity is not suitable for delivery of large constructs, which includes most CRISPR/dCas9-effector systems. To circumvent this, many AAV-based CRISPR/Cas tools are delivered in two pieces, from two separate viral cassettes. However, this approach requires higher viral payloads and usually is less efficient. Here we develop a compact dCas9-based repressor system packaged within a single, optimized AAV vector. The system uses a smaller dCas9 variant derived from Staphylococcus aureus (Sa). A novel repressor was engineered by fusing the small transcription repression domain (TRD) from MeCP2 with the KRAB repression domain. The final dSaCas9-KRAB-MeCP2(TRD) construct can be efficiently packaged, along with its associated gRNA, into AAV particles. Using reporter assays, we demonstrate that the platform is capable of robustly and sustainably repressing the expression of multiple genes-of-interest, both in vitro and in vivo. Moreover, we successfully reduced the expression of ApoE, the stronger genetic risk factor for late onset Alzheimers disease (LOAD). This new platform will broaden the CRISPR/dCas9 toolset available for transcriptional manipulation of gene expression in research and therapeutic settings.

bioengineering↗