Presentation on theme: "Gene therapy for Dyskeratosis Congenita (DC) Davide De Rocco Lorenzo Errichelli Marco Fabiani Valentina Flamini A.A. 2011-2012."— Presentation transcript:
Gene therapy for Dyskeratosis Congenita (DC) Davide De Rocco Lorenzo Errichelli Marco Fabiani Valentina Flamini A.A
Physical abnormalities Rare disease (prevalence: 1/ ) Individuals with mutations in one of the known DC genes have variable clinical phenotypes Nature, Human genetics: Testing telomerase
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.
Testing Telomere length according to age in patients with dyskeratosis congenita and their relatives
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
Experimental Plan Expression vector (3rd generation) Production pseudoviral particles Isolation of bone marrow cells
Experimental Plan Evaluation of expression level of DKC1 (RT-PCR) Evaluation of transfection efficiency
Experimental Plan Evaluation: telomerase enzymatic activity lenght of telomeres (Q-FISH e Southern Blot)
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
Costs 293T cells 80$ 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. Macs 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. SC $ 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. N ML Sigma-Aldrich 14.70€ growth medium, growing factor, ecc Laboratory: flasks, vials, tubes…
References Alter BP et al., Blood Sep 1;110(5): Very short telomere length by flow fluorescence in situ hybridization identifies patients with dyskeratosis congenita. Knight SW et al.,Am J Hum Genet 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 May;36(5): Disease anticipation is associated with progressive telomere shortening in families with dyskeratosis congenita due to mutations in TERC. Marrone A et al.,Blood Dec 15;110(13): Telomerase reverse-transcriptase homozygous mutations in autosomal recessive dyskeratosis congenita and Hoyeraal-Hreidarsson syndrome. Vulliamy TJ et al., Blood Cells Mol Dis May-Jun;34(3): Mutations in the reverse transcriptase component of telomerase (TERT) in patients with bone marrow failure Savage SA et al.,.Am J Hum Genet Feb;82(2): TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita. Vulliamy TJ, et al. Biochimie Jan;90(1): Dyskeratosis congenita: the diverse clinical presentation of mutations in the telomerase complex. Walne AJ et al.Hum Mol Genet Jul 1;16(13): 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 Jun;17(3): Optimized lentiviral transduction of mouse bone marrow-derived mesenchymal stem cells Qasim W,.et al. Mol Ther Feb;15(2): 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): Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation. Maxim A. Blood 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 : Telomere lenght as a prognostic parameter in chronic lymphocytic leukemia with special referenze to VH gene mutation status Baird DM. Exp Gerontol 2005; 40: New developments in telomere lenght analysis Chiu CP et al. Stem Cells 1996 Mar; 14(2): Differential expression of telomerase activity in hematopoietic progenitors from adult human bone marrow.