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Gene-Based Therapy Strategies for Human Usher Syndrome

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Gene-based therapy strategies ResearchResearch MolecularMolecular && cellcell biologybiology of ciliated sensory cells for human Usher syndrome projectsprojects of ciliated sensory cells • Identification of novel Ciliated sensory cells in vertebrates molecules of the cytoskeleton Uwe Wolfrum, in ciliated sensory cells. Department of Cell & Matrix Biology, • Centrins in sensory cells. Institute of Zoology, Johannes Gutenberg University of Mainz • Intracellular translocations in sensory cells Germany olfactory [email protected] cells • Protein networks related to the Usher syndrome • Pathomechanisms in senso- Boston, MA June 19, 2009, ~ 30 minutes neuronal degenerations. photoreceptor hair cells photoreceptor & cells Evaluation of therapeutic Uwe Wolfrum, Cell & Matrix Biol., Institute of Zoology, Johannes Gutenberg-University of Mainz, Germany strategies Human Usher syndrome (USH) Human Usher syndrome (USH), genes • USH is the most common form of combined Type Gene locus Gene Protein Function Mouse model hereditary deaf-blindness. 1A = 1B • Prevalence: 5-10/100,000 1B 11q13.5 MYO7A myosin VIIa molecular motor Shaker-1 (sh1) 1C 11q15.1 USH1C harmonin scaffold protein Deaf circler (dfcr) • Autosomal recessive disorder 1D 10q21-q22 CDH23 cadherin 23 cell-cell adhesion Waltzer (v) • Symptoms: 1E 21q21 -- -- -- -- 1F 10q11.2-q21 PCDH15 protocadherin 15 cell-cell adhesion Ames waltzer (av) - Hearing impairment 1G 17q24-25 SANS SANS scaffold protein Jackson shaker (js) - Vestibular dysfunction 1H 15q22-23 USH1H -- -- -- - Vision loss - Retinitis pigmentosa Charles H. Usher (1913) 2A 1q41 USH2A USH2A (usherin) ECM, cell adhesion k.o. 2C 5q14.3-21.3 VLGR1b GPR98/VLGR1b GPCR, cell adhesion Mass1 (frings) • 3 clinical types (USH1-3)3 are based on severity, 2D 9q32 DFNB31 whirlin scaffold protein Whirler (wi); k.o. age of onset and progression of symptoms. 2E 10q24.3 PDZD7 PDZD7 scaffold protein 3A 3q21-25 USH3A clarin-1 cell adhesion k.o. in prep. • There are at least 13 genetic heterogeneous subtypes.subtypes 3B 20q -- -- -- -- • 10 USH causing genes are identified, so far. UW 2009 modified from Reiners et al. 2006 ExpEyeRes Diversity of Usher syndrome proteins USH protein interactome Feb. 2009 * *selected proteins USH type 1 USH type 2 Alsin MyomegalinCilia – ECM component Molecular Centrosome transport Function molecular motor Signal transduction cell adhesion protein to actin cytoskeleton Synapse organization cell adhesion protein/7-transmembrane receptor scaffold protein Endocytosis E PDZD7 (USH2E) pH regulation scaffold protein Cell polarity cell adhesion proteins D whirlin (USH2D) & cell adhesion Wnt signalling scaffold protein Cilia – USH type 3 scaffold protein Centrosome Signalling via cell adhesion protein ? Function extracellular matrix Therapy strategies for the Usher Syndrome Conclusions Ear: USH is an developmental defect – prenatal diagnoses and USH protein networks are molecular genetic treatment are necessary. integrated and linked to the cytoskeleton by the - Accessibility is very difficult. signal - Cochlea implants are efficient. processor electrode USH scaffold proteins: Cochlea harmonin, whirlin and SANS. speech In the ear, USH networks processor participate in kinocilia/stereocilia differentiation during hair cell Reiners et al. 2006 development, but also in ribbon synapse function, and in signal transduction. Eye: In photoreceptor cells, USH networks are found at the The is progression in retina implant development, but they are synapse and in the ciliary region. They may contribute to the still at early development stages. intracellular transport and to the ciliary import and delivery. Retina provides very good accessibility for molecules/agents – Defects in one protein of these networks may cause neuroprotection and molecular genetic interventions. ~ Gene based therapies dysfunction of the entire networks leading to USH. UW Gene addition - replacement Gene addition - replacement Viral gene addition - replacement: Host cells/ Vector Gene Integration Immune- Lentivirus: efficiency expression into genome response • single stranded RNA-Virus, member of Retrovirus family, Viral • maximum insert size: 7.5 kb, dividing and Lenti- • integration into genome, long term expression, non-dividing cells/ long term/ low virus yes • low immune response, infect both dividing and non-dividing cells high efficiency years dividing and Adeno-associated virus (AAV): no/ ? rAVV non-dividing cells/ long term/ • DNA virus, belongs to Parvovirus family, maximum insert size: 7 kb; under discussion no high efficiency • no/low rate integration (preferentially chromosome 19) into genome; years • long term expression, non pathogenic, no immune response; Non-viral • infect both dividing and non-dividing cells; dividing and short term • depend on a helper virus to replicate (Dependovirus). DNA only non-dividing cells no no Non-viral gene addition – replacement by nanoparticle: very low efficiency • unlimited size for delivery, very poor integration, dividing and PEG nano- up to 3 months stays episomal no non-dividing cells/ • no immunological problems; expression termination ? particle analyzed • low efficiency in transfection of both dividing and non-dividing cells high efficiency • alternative “carriers”: liposomes, DNA-protein conjugates … Gene addition via rAAVs in retinitis Gene addition projects for USH genes pigmentosa/LCA2 patients USH1B (myosinVIIa) - Consortium – Welp/Brown families The New England Journal of Medicine, 2008 David Williams, San LA, U.S.A. - Lentivirus successful transfer Brief report into mouse RPE Safety and Efficacy of Gene Transfer for Leber’s Congenital Amaurosis Alberto Aurichio, Univ. of Naples, Italy - recombinant Albert M. Maguire ….. Jean Bennett, Philadelphia, U.S.A. adeno-associated virus (modified rAVV5) … USH1C (harmonin) – Consortium planned Suchert family The New England Journal of Medicine, 2008 Uwe Wolfrum, Mainz,* Germany; - recombinant adeno-associated virus Brief report (rAVV5); PEG nanoparticle Effect of Gene Therapy on Visual Function in Leber’s Congenital Amaurosis USH2A -Consortium ? James W. B. Bainbridge ….. Robin R. Ali, London, England W. Kimberling, Omaha, Peter Francis, Portland, U.S.A.* … USH3A (clarin-1) - Consortium – Alexander family – “Hope for Vision” Human Gene Therapy, 2008 John Flannery, Berkeley, U.S.A.*; Eeva-Marja Sankila, Helsinki, Finland; Phase I Trial of Leber Congenital Amaurosis due to RPE65 Mutations by Ocular Kris Palczewski, Cleveland; David A. Saperstein, Seattle, U.S.A. Subretinal Injection of Adeno-Associated Virus Gene Vector: Short-Term Results /QLT Inc., Vancouver, Canada … William W. Hauswirth……. Samuel G. Jacobson, Florida and Pennsylvania, U. S. A. - recombinant adeno-associated virus (rAVV5) *Cooperation with W.W. Hauswirth, Gainesville, U.S.A. Recombinant adeno-associated virus (rAAV) Application of biomolecules in the eye Adeno-associated virus (AAV) infected cell AAV genome AAV CAP REP helper virus ITR REP CAP ITR + REP: viral replications-proteins, transcription, species specific integration and packaging replication CAP: capsid proteins X recombinant AAV (rAAV) infected cell rAAV rAAV genome Harmonin a1 helper virus Harmonin a1 + X ITR USH1C ITR X Application of biomoleculs: a. vitreal ITR USH1C ITR replication b. subretinal Strategies for treatment of USH1C Harmonin (USH1C)-basedUSH1C protein network in the retina – Wolfrum Team Mainz USH2 proteins USH1 cadherins • gene addition using recombinant adeno-associated virus (rAAV), VLGR1b Pcdh15 USH2A Cdh23 Collaboration: W.W.Hauswirth, Gainesville, USA Extra NBC3 • gene addition using polyethylen glycol (PEG) nanoparticles, Cyto Collaboration: M. Naash, Oklahoma City, USA Harmonin PDZ1 PDZ2 CC1 CC2 PST PDZ3 • gene repair by homologous recombination with zinc finger nucleases (USH1C) SANS (ZFN) delivered by nanoparticles, SANS SANS Collaboration: (D. Carroll, Utah, USA) SANS USH1 proteins Myo7A filamin A Myo7A • translational readthrough with modified aminoglycosides β-cateinin Collaboration: T. Bassov, Haifa, Israel Myo7A T. Ben-Yosef, Haifa, Israel F-actin UW 09 Gene addition Non-viral gene addition by DNA PEG nanoparticles via recombinant adeno-associated viruses (rAAV5) Cys-polylysin polyethylene glycole CK30 PEG maleimide – photoreceptor cell specific DNA CK30PEG10k ITR USH1C ITR rAAV5 Opsin:: GFP harmonin a1 delivered by subretinal injected Interaction of PEG-maleimide with CK30 to form polycation CK PEG into Ush1c -/- mouse retina 30 that compacts DNA vector to form Trifluoracetate (TFA) Acetate charged-neutral nanoparticles. Lysine counter ion determines shape of the particles Dr. K. Copernicus Therapeutics, Cleveland, Ohio, USA. GFP/ Nagel-W DAPI-fluorescence image 100nm 100nm of mature mouse retina MN 08, modified . Ellipsoids < 18nm Rods < 8nm Nora Overlack Mol. characteristics of DNA PEG nanoparticles • PEG (Polyethylenglycol), CK30 (30 lysins - cystein) + plasmid DNA promoter Rod MOP-221bp SV 40 Poly A Cell specificity Rod & Cone IRBP-1.3kb e.g. harmonin a1 (USH1C) cDNA 0.9 kb All cells CBA-280bp • ~ 8 nm; neutral charge; no toxicity; no immunoreactions; no cellular degradation; high in vivo transfections efficiency. Alternative molecular genetic • bind to cell surface exposed Nucleolin – internalization and transport into the nucleus (~ 15 min) & stays episomal. therapy strategies nucleus Rhodamin DNA PEGs anti-nucleolin nuclear for USH ? pore episomal post translational modification DNA PEGs nucleolin Modifiziert: Chen et al. (2008) Mol Ther 16: 333 p.R31X point mutation in USH1C
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