Transcript Gene therapy for Dyskeratosis Congenita (DC)

Gene therapy for
Dyskeratosis Congenita (DC)
Davide De Rocco
Lorenzo Errichelli
Marco Fabiani
Valentina Flamini
A.A. 2011-2012
Physical abnormalities
Rare disease (prevalence: 1/1.000.000)
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: 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)
Isolation of bone marrow cells
Production pseudoviral particles
Experimental Plan
Evaluation of transfection efficiency
Evaluation of expression level of DKC1 (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
the therapy (HSV tk)
●
Upregulation of DKC1 (DKC1 endogenous + exogenous one)
Apoptosis
Costs
•
293T cells 80$ www.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…
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.