Engineered Retrogradely Transported, Barcoded AAVs Elucidate Molecular Pathways Involved in the Pathogenesis in Parkinson’s Disease (PD)

MARTINO AVALLONE; JOAQUIN PARDO; SARA PALO; ULRICH PFISTERER; JANA RAJOVA; MALIN ÅKERBLOM; MARCUS DAVIDSSON; TOMAS BJÖRKLUND

Los Angeles

Congreso; ASCGT 2023; 2023

Here we describe a novel strategy based on AAV engineering and transgenic animals. The MNM008 AAV capsid, capable of retrograde transport in DA neurons, is injected in the striatum of TH-Cre ratsand DAT-Cre mice. The vector carries uniquely barcoded aSyn or nonpathogenic tagBFP.Injection into the striatum with Cre-recombinase restriction of the expression to DA neurons of the SN, we avoid possible injection-related inflammation and cell loss. The nuclei from the micro dissected SN area are enriched for DA neurons using FANS. This is achieved thanks to the viral vector prep containing a Cre-inducible histone-linked GFP (H2B-GFP), which is only expressed in the population of interest. Additionally, we used a recently adopted marker (Nurr1) in FANS to study the dopaminergic neurons in WT animals. We tested two emerging and one mature single-cell sequencing approaches. The first one is a single-cell combinatorial fluidic indexing protocol (Scifi) which recently showed to increase the 10X Genomics cells load capacity to 100 folds. The second one is using Parse Bioscience technology, which appends barcodes to each transcript by progressing cells through split-pool combinatorial barcoding steps. This allows to sequence up to 1 M using any standard biology lab equipment. The third approach is using the gold standard 10X Genomics that has been widely adopted in single-cell studies. The molecular barcoding will allow us to accurately measure the aSyn expression level in each DA neuron arranging them into disease stages in what we call pseudo-time.Moreover, this approach will identify causative cascades of changes at the transcriptional level.