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Terapia genica 18/4/2012 Mucopolysaccharidosis and adenovectors (CAV) Anna Borroso Gianmarco Rinaldi Petra Tomassini Silvia Santopolo.

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Presentation on theme: "Terapia genica 18/4/2012 Mucopolysaccharidosis and adenovectors (CAV) Anna Borroso Gianmarco Rinaldi Petra Tomassini Silvia Santopolo."— Presentation transcript:

1 Terapia genica 18/4/2012 Mucopolysaccharidosis and adenovectors (CAV) Anna Borroso Gianmarco Rinaldi Petra Tomassini Silvia Santopolo

2 Abstract The mucopolysaccharidoses (MPSs) are lysosomal storage disorders caused by the accumulation of glycosaminoglycans in the lysosomes that leads to a cascade of multisystemic disease manifestations. The disease is caused by mutations in one of the enzymes that catalyze GAGs degradation. The most severe forms of MPSs involve progressive degeneration of the Central Nervous System with consequent cognitive impairment. Current therapies are targeted to the replacement of the defective enzyme, but the impossibility of therapeutic molecules or vectors to cross the Blood Brain Barrier limits the treatment to the non- neural symptoms. The aim of new emerging strategies is the reduction of the substrate production, rather than promote its degradation. According with this strategy, we propose to inhibit the gene expression of the enzymes involved in the first steps of GAG biosynthesis using shRNAs expressed by an helper-dependent canine adenoviral vector. Our aim is to effectively treat cognitive impairment, which is still now uncured.

3 - Lysosomal storage disorder - Deficiency of one of the enzyme involved in degradation of GAGs. - Seven distinct clinical types(I-II-III-IV- VI-VII-IX) - Autosomal recessive disorder (the exception is MPS II that is X-linked) - Affect multiple organ systems and lead to organ failure - Cognitive impairment - Reduced life expectancy Mucopolysaccharidosis

4 Site of the enzyme deficiencies

5 - autosomal recessive - 1: frequence - 3 type: - Hurler Syndrome - Hurler-Scheie Syndrome - Scheie Syndrome - deficient activity of α -L-iduronidase (IDUA) (chromosome 4) - more than 100 different mutations MPS I

6 StrategyMechanismLimits Degradation of stored GAGs via restoration of enzyme activity ERT, Enzyme replacement Therapy CNS access across the Blood Brain Barrier; Repeated administration. HSCT, Hematopoietic Stem Cell Treatment CNS access across the BBB Gene therapy, Numerous eterogeneus different mutantions across the entire gene: it needs to be entirely substitueted Immunogenicity of IDUA; Diffusion of the vector into the brain; Inibition of new GAGs synthesis Inibition of new GAGs synthesis (substrate reduction therapy) (substrate reduction therapy) Inhibitors of enzymes involved in GAGs biosynthesis (Genistein) Side Effects (e.g. decreased male fertility) Gene Theray with siRNA: Inhibition of the expression of enzymes involved in GAG biosynthesis Delivery Existing Therapies

7 Objectives Therapeutic strategy to recover cognitive impairment Reduction of GAG Reduction of GAG accumulation throught accumulation throught inhibition of their syntesis inhibition of their syntesis use of siRNAs use of siRNAs use of Canine Adenoviral Vector use of Canine Adenoviral Vector - low immunogenicity - efficently trasduce neurons

8 3 rd generation 3 rd generation CAV-2 multi-shRNA-GFP How to design the shRNAs

9 CAV-2 multi-shRNA-GFP - Test cell vitality - Test vector presence: light microscopy observation of GFP fluorescence - siRNAs expression: Northern Blot - mRNAs target levels: Real time PCR - Target proteins levels: Western Blot - Reduction of GAG biosynthesis: measurement of incorporation of 35 S from a radioactive sodium sulfate into PG - Level of GAG: Blyscan Assay - siRNAs off-target effects: Microarray Experimental plan primary cultures of neurons from wt mice

10 CAV-2 multi-shRNA-GFP primary cultures of neurons from MPS I mice - Test cell vitality - Test vector presence: light microscopy observation of GFP fluorescence - siRNAs expression: Northern Blot - mRNAs target levels: Real time PCR - Target proteins levels: Western Blot - Reduction of GAG biosynthesis: measurement of incorporation of 35 S from a radioactive sodium sulfate into PG - Level of GAG: Blyscan Assay - siRNAs off-target effects: Microarray - Reduction of the number of lysosomal inclusions: light microscopy Experimental plan

11 MPS I newborn mice CAV-2 multi-shRNA-GFP - Test vector diffusion: GFP fluorescence observation in brain sections - Target mRNAs levels: Real Time PCR - Target proteins levels: Western Blot - Reduction of GAG storage: light microscopy observation of number and aspect of lysosomal inclusions - Absence of desease synthomps: cognitive test - Eventual IFN siRNAs response: IFN target genes levels of expression

12 Materials and costs Necessity of an early treatment in newborn patients Future human trial Necessity of a prenatal diagnosis Necessity of a long-therm expression of the shRNA (e.g.: hybrid vector able to integrate in the host genome) Pitfalls and solutions MPS I mice (IDUA -/- ): provided by Dr. E. F. Neufeld, USA Blyscan Glycosaminoglycan Kit: 120 Assays 478 € Kit Microarray: 500€ Anticorpi vari: 500€ Strumentazioni e varie: €


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