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Published byVivian Elliott Modified over 9 years ago
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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
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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
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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
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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
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Choise of shRNA GFPmCherry LRRK2 WT LRRK2 G2019S CMV shRNACMV
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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
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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
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G2019S LRRK2 wt …the LRRK2 kinase activity,the caspase activity Measuring… and neurite loss
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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
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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
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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
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Chemicals Plastics/Glassworks Transgenic mice individual price € 258.34 CAV-2 Vector Production Kit price on order 293 cell line list price € 306.84 Kit PCR about € 100 Kit Kinase Assay about € 300 Peptide Mass Fingerprinting about € 1200 Neuronal cell line about € 685.24 (x2) Laboratory facilities (eg: laminar flow hood, incubators, ultracentrifuges, fluorescence microscopy, centrifuges, …) MATERIALS AND COSTS
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