Gene therapy for Dyskeratosis Congenita DC Davide De

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

Physical abnormalities Rare disease (prevalence: 1/1. 000) 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: DKC 1, TERC, TERT, TINF 2, NHP 2, NOP 10. The DKC 1 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 DKC 1 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 (3 rd generation) Isolation of bone marrow cells Production pseudoviral particles

Experimental Plan Evaluation of transfection efficiency Evaluation of expression level of DKC 1 (RT-PCR)

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

Future prospectives

Pitfalls and solutions ● Mutations for random insertion of vector Suicide gene to eliminate therapy (HSV tk) ● Upregulation of DKC 1 (DKC 1 endogenous + exogenous one) Apoptosis

Costs • 293 T cells 80$ www. abgent. com/products/CL 1032_293 T_Cell_Line • p. SMPUW Universal Lentiviral Expression Vector (Promoterless) Cat. No. VPK 211 Ori. Gene 415$ • p. SELECT-zeo-HSV 1 tk Invivogen (we have to contact the company) CD 34 Multi. Sorting Kit Human Cat. No. Macs 130056701 Kit PCR, restriction enzyme, ligase ecc. around 1000 € Quanti. Tect SYBR Green RT-PCR Kit (200) 850 € Endo. Free Plasmid Maxi Kit (10) 352 € • One Shot® Stbl 3™ Chemically Competent E. coli 424 € Ori. Gene Cat. No. SC 119282 185 -200$ • Southern and Western around 400 euro • Kit Elisa Quick titer lentivirus titer Kit (lentivirus-associated HIV p 24) around 400 euro RT telomerase detection Cat. No. S 7710 Millipore (we have to contact the company) Neomycin solution Cat. No. N 1142 -20 ML Sigma-Aldrich 14. 70€ growth medium , growing factor, ecc Laboratory: flasks, vials, tubes…

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 DKC 1 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. TINF 2, 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 NOP 10 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 Wu. MH et al. Bone Marrow Transplant 2001 Jun; 27(11): 1201 -9. Optimization of culture conditions to enhance transfection of human CD 34+ cells by electroporation. Maxim A. Blood. 2006 September 15; 108(6): 2095– 2105. Leukosialin (CD 43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures Poon SS, Lansdorp. PC. Current Protocol Cell Biology 2001; Chapter 18: Unit 18. 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.