Objective/Rationale:
Overexpression of LRRK2 in mammalian cells, either transient or as stable cell lines, has proven cumbersome and quite inefficient. This can be partly attributed to the large size and complex structure of the protein.
Project Description:
Lentiviral vectors are efficient tools for gene transfer into mammalian cells, including in cell lines that are difficult to transfect, in primary neurons and as well as in vivo. However, LRRK2 cDNA together with a heterologous CMV promoter, and all cis-acting viral elements present in the vector construct and required for normal vector production constitute ~11.4 kb, which is larger than the practical packaging limits based on the size of the viral genome of the HIV-1 (9.2 kb) from which LV are derived. Despite the size limitations, at the lab for Neurobiology and Gene Therapy and the Leuven Viral Vector Core we have been able to develop LV vectors encoding LRRK2 with a functional titer of 106 transducing units per ml (TU/ml), using an optimized production process.
We will produce large scale batches of LRRK2 LV vectors, which will allow more efficient overexpression of LRRK2 (wild type and muntant) in difficult-to-transfect cell types and facilitate the generation of stable LRRK2 overexpressing cell lines.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
The availability for the research community of LRRK2 encoding lentiviral vectors will facilitate the overexpression of LRRK2 in difficult-to-transfect cell types and facilitate the generation of stable LRRK2 overexpressing cell lines. This will contribute to modeling LRRK2 function in cells and to develop and validate novel therapeutics.
Anticipated Outcome:
We will produce large batches of different forms of LRRK2 encoding lentiviral vectors, which will be available for the LRRK2 research community.