Corso di Terapia genica Prof.ssa Saggio a.a. 2011/2012 LRRK2-FAMILIAR PARKINSON DISEASE: POSSIBLE APPROACH OF GENE THERAPY Cesario Claudia Giusti Valentina Milan Marika Romano Elena

PARKINSON’ S DISEASE Neurodegenerative disorder:  intracytoplasmic protein aggregates, Lewy bodies  dopaminergic neurons death Symptoms: Tremor Rigidity Bradykinesia Akinesia Postural instability Memory loss Depression Sleep disturbances

Genetic Factors (10-15%) LRRK2 gene (12q12) mutations are the most common genetic cause of familiar autosomal dominant PD  glycine to serine aminoacid substitution at codon 2019  worldwide frequency = 1.1–18.7% in familiar PD  increased kinase activity  enhanced autophosphorylation LRRK2 G2019S MUTATION

APPROACH OF GENE THERAPY CAV (Canine Adenovirus) vectors transfection in neuronal cells for the expression of: siRNA cDNA LRRK2 wt Objective: silencing of endogenous mutated gene and exogenous expression of wild-type

Choise of shRNA GFPmCherry LRRK2 WT LRRK2 G2019S CMV shRNACMV

cDNA LRRK2 esogenous construct NSEcDNA LRRK2 wtGFPRSVcDNA LRRK2 wtGFP Livello di fluorescenza RSV cDNA LRRK2 w t cDNA U1 A 3’UTR U1A wt5’ UTR LRRK2RSV cDNA LRRK2 w t cDNA U1 A 3’UTR U1A mut5’ UTR LRRK2

Preclinical research (IN VITRO) Choice of cell system:  Human neuronal cell line G2019S  Mice neuronal cell line G2019S Cre-expressing 293 cells Virions with gutless vector genome Vector construction: Gutless virus Helper virus DNA stuffer promoter cDNA LRRK2 U1A shRNA DNA stufferpromoter ITR

G2019S LRRK2 wt …the LRRK2 kinase activity,the caspase activity Measuring… and neurite loss

Preclinical research (IN VIVO) Transgenic Mice LRRK2 G2019S  (Peltékian et al., 2002) Injection control vectorInjection therapeutic vector Six mouse wtSix mouse G2019SThree mouse wtThree mouse G2019S

 behavioral testing and motor activity  Immunoassays  Northern blot  Western blot and Fingerprinting analysis  LRRK2 kinase activity  dopaminergic neuronal degeneration: tyrosine hydroxylase (TH) immunofluorescence Measuring… Control vector Therapeutic vector vector control

PITFALLS AND SOLUTIONS DHPLC : high cost  collaboration with an equipped lab FUTURE PERSPECTIVES Mus musculus Pan troglodytesHomo sapiens Discriminate wt and G2019S mutation (transcripts) RT-PCR: low sensitive for a single nt

Chemicals Plastics/Glassworks Transgenic mice individual price € CAV-2 Vector Production Kit price on order 293 cell line list price € Kit PCR about € 100 Kit Kinase Assay about € 300 Peptide Mass Fingerprinting about € 1200 Neuronal cell line about € (x2) Laboratory facilities (eg: laminar flow hood, incubators, ultracentrifuges, fluorescence microscopy, centrifuges, …) MATERIALS AND COSTS