Production and optimization of cell-specific and brain-targeted recombinant adeno-associated virus tools for target validation in Parkinson’s disease models
Abyari, Soroush (2022-05-12)
Production and optimization of cell-specific and brain-targeted recombinant adeno-associated virus tools for target validation in Parkinson’s disease models
(12.05.2022)
Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.
avoin
Julkaisun pysyvä osoite on:
https://urn.fi/URN:NBN:fi-fe2022053039368
https://urn.fi/URN:NBN:fi-fe2022053039368
Tiivistelmä
Parkinson’s disease (PD) is a heterogeneous disorder associated with the progressive loss of dopaminergic neurons in specific brain regions. The underlying molecular events in PD result from the complicated interplay of genetics and the environment. Currently, there is no disease-modifying therapy for PD and developing novel therapeutics requires a deeper understanding of the early neuropathological mechanisms of the disease. The first aim of this project was to generate recombinant adeno-associated virus (rAAV) vectors to deliver transgenes in a cell-type-specific manner. The transgenes transfer phosphomutants of three target proteins implicated in PD pathogenesis for further studies. The second aim of this project was to optimize the produced rAAVs to achieve the most efficient integration into target dopaminergic cells.
The rAAVs were generated using a special capsid, enabling them to systematically infect specific cells. Following production, rAAVs transduction efficiency was optimized by cell transduction assays and visualized by immunofluorescence staining.
In total, 11 high titer rAAVs were generated with titer ranging from 0.2 - 5.6 x 1013 vg/ml. All rAAVs were functionally working with different transduction efficiency. The best labeling titer and infection incubation time were optimized. The results show that our rAAVs were reliably transducing target cells and can be used for in vitro and in vivo experiments. The follow-up experiments will assess the effects of phosphomutants delivered by rAAVs on cellular mechanisms of PD to validate new target proteins as potentially involved in the early stages of the disease and pave the way for further studies.
The rAAVs were generated using a special capsid, enabling them to systematically infect specific cells. Following production, rAAVs transduction efficiency was optimized by cell transduction assays and visualized by immunofluorescence staining.
In total, 11 high titer rAAVs were generated with titer ranging from 0.2 - 5.6 x 1013 vg/ml. All rAAVs were functionally working with different transduction efficiency. The best labeling titer and infection incubation time were optimized. The results show that our rAAVs were reliably transducing target cells and can be used for in vitro and in vivo experiments. The follow-up experiments will assess the effects of phosphomutants delivered by rAAVs on cellular mechanisms of PD to validate new target proteins as potentially involved in the early stages of the disease and pave the way for further studies.