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Gene therapy for Dyskeratosis Congenita (DC) Davide De Rocco Lorenzo Errichelli Marco Fabiani Valentina Flamini A.A. 2011-2012.

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Presentation on theme: "Gene therapy for Dyskeratosis Congenita (DC) Davide De Rocco Lorenzo Errichelli Marco Fabiani Valentina Flamini A.A. 2011-2012."— Presentation transcript:

1 Gene therapy for Dyskeratosis Congenita (DC) Davide De Rocco Lorenzo Errichelli Marco Fabiani Valentina Flamini A.A. 2011-2012

2 Physical abnormalities Rare disease (prevalence: 1/1.000.000) Individuals with mutations in one of the known DC genes have variable clinical phenotypes Nature, Human genetics: Testing telomerase

3 Genes involved in DC ● Mutations in one of the six genes have been identified in approximately half of individuals who meet clinical diagnostic criteria for DC: DKC1,TERC, TERT, TINF2, NHP2, NOP10. The DKC1 gene provides instructions for making a protein called dyskerin, which is involved in manteinig the structures of telomers The essential telomerase components. Mutations in telomerase and telomere components lead to syndromes of telomere shortening.

4 Testing Telomere length according to age in patients with dyskeratosis congenita and their relatives

5 Therapy In DKC1 mutations are more frequent than in the other genes (11-36%). Bone marrow failure (BMF) is the most important aspect of this disease. Objectives: ● To obtain a functional Telomerase complex ● To estabilish normal lenght of telomeres

6 Experimental Plan Expression vector (3rd generation) Production pseudoviral particles Isolation of bone marrow cells

7 Experimental Plan Evaluation of expression level of DKC1 (RT-PCR) Evaluation of transfection efficiency

8 Experimental Plan Evaluation: telomerase enzymatic activity lenght of telomeres (Q-FISH e Southern Blot)

9 Future prospectives

10 Pitfalls and solutions ● Mutations for random insertion of vector Suicide gene to eliminate the therapy (HSV tk) ● Upregulation of DKC1 (DKC1 endogenous + exogenous one) Apoptosis

11 Costs 293T cells 80$ www.abgent.com/products/CL1032_293T_Cell_Linewww.abgent.com/products/CL1032_293T_Cell_Line pSMPUW Universal Lentiviral Expression Vector (Promoterless) Cat.No. VPK211 OriGene 415$ pSELECT-zeo-HSV1tk Invivogen (we have to contact the company) CD34 MultiSorting Kit Human Cat.No. Macs130056701 Kit PCR, restriction enzyme, ligase ecc. around 1000 € QuantiTect SYBR Green RT-PCR Kit (200) 850 € EndoFree Plasmid Maxi Kit (10) 352 € One Shot® Stbl3™ Chemically Competent E. coli 424 € OriGene Cat.No. SC119282 185-200$ Southern and Western around 400 euro Kit Elisa Quick titer lentivirus titer Kit (lentivirus-associated HIV p24) around 400 euro RT telomerase detection Cat. No. S7710 Millipore (we have to contact the company) Neomycin solution Cat.No. N1142-20ML Sigma-Aldrich 14.70€ growth medium, growing factor, ecc Laboratory: flasks, vials, tubes…

12 References Alter BP et al., Blood. 2007 Sep 1;110(5):1439-47. Very short telomere length by flow fluorescence in situ hybridization identifies patients with dyskeratosis congenita. Knight SW et al.,Am J Hum Genet. 1999 Jul;65(1):50-8. X-linked dyskeratosis congenita is predominantly caused by missense mutations in the DKC1 gene Vulliamy T et al., Nat Genet. 2004 May;36(5):447-9. Disease anticipation is associated with progressive telomere shortening in families with dyskeratosis congenita due to mutations in TERC. Marrone A et al.,Blood. 2007 Dec 15;110(13):4198-205.Telomerase reverse-transcriptase homozygous mutations in autosomal recessive dyskeratosis congenita and Hoyeraal-Hreidarsson syndrome. Vulliamy TJ et al., Blood Cells Mol Dis. 2005 May-Jun;34(3):257-63.Mutations in the reverse transcriptase component of telomerase (TERT) in patients with bone marrow failure Savage SA et al.,.Am J Hum Genet. 2008 Feb;82(2):501-9. TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita. Vulliamy TJ, et al. Biochimie. 2008 Jan;90(1):122-30.Dyskeratosis congenita: the diverse clinical presentation of mutations in the telomerase complex. Walne AJ et al.Hum Mol Genet. 2007 Jul 1;16(13):1619-29.Genetic heterogeneity in autosomal recessive dyskeratosis congenita with one subtype due to mutations in the telomerase-associated protein NOP10 Ricks DM et al..Stem Cells Dev. 2008 Jun;17(3):441-50.Optimized lentiviral transduction of mouse bone marrow-derived mesenchymal stem cells Qasim W,.et al. Mol Ther. 2007 Feb;15(2):355-60.Lentiviral vectors for T-cell suicide gene therapy: preservation of T-cell effector function after cytokine-mediated transduction. ghr.nlm.nih.gov/condition/dyskeratosis-congenita WuMH et al. Bone Marrow Transplant 2001 Jun;27(11):1201-9. Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation. Maxim A. Blood. 2006 September 15; 108(6): 2095–2105. Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures Poon SS, LansdorpPC. Current Protocol Cell Biology 2001; Chapter 18: Unit18.4. Quantitative fluorescence in situ hybridization (Q-FISH) Grabowski P. Blood 2005. 105: 4807-12. Telomere lenght as a prognostic parameter in chronic lymphocytic leukemia with special referenze to VH gene mutation status Baird DM. Exp Gerontol 2005; 40: 363-8. New developments in telomere lenght analysis Chiu CP et al. Stem Cells 1996 Mar; 14(2): 239-48. Differential expression of telomerase activity in hematopoietic progenitors from adult human bone marrow.


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