HPGS and Lentivector
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Transcript HPGS and Lentivector
Corso di Terapia Genica anno
2011-2012
Hutchinson Gilford Progeria Syndrome
and Lentivector
Group members:
Alessandro Angerilli
Angela Di Bello
Elena Grossi
Marta Marzullo
Hutchinson-Gilford disease: Clinical Features
Premature development of “segmental” features recalling aging
Severe growth retardation
Skeletal alteration
Amyotrophy, lipodystrophy and skin atrophy
Alopecia
Extremely severe atherosclerosis
The mean age at diagnosis is 2.9 years, while
death occurs at a mean age of 13.5 years
Molecular characterization
In 2003 the disease's genetic basis was identified: HGPS is caused by point mutations that
increase utilization of an alternative splice donor site in exon 11 of LMNA (the gene encoding
lamin C and prelamin A)
Chromosome 1
(1p22)
NORMAL SEQUENCE
C1824T
GGTGGGC
GGTGGGT
Prelamin A vs Progerin processing
HA- lamin A
anti-HA
HA- progerin
anti-HA
Gene therapy approaches
Morpholino
oligonucleotides
wt
GFP
mut GFP
FTI
ex9 ex10 ex11
ex12
GGC>GGT
ex9 ex10 ex11
ex12
Clinical Trial in
progress (2009-2012)
Goals:
Avoid the production of progerin caused by aberrant
splicing
Recovery of Lamin A isoform
Rescue of the wild type phenotype
New gene therapy approach based on TALENs
(Transcriptional Activator Like Effectors Nucleases)
focused on the classical mutation occurring in HGPS:
C1824T
Naturally occurring TALE:
Recognition code:
[one di-residue – one nt]
Engineered TALEN:
Application of TALEN's technology to HPGS disease:
1) Customization of TALENs to bind
sequences flanking the mutated region
1) TALENs-mediated deletion of a specific
three-nucleotide codon containing the
mutation
CAREFUL TO
THE
FRAMESHIFT!
AACACCTGGGGCTGCGGGAACCACCCAGCTCTCATCAACAACACCT
TTGTGGACCCCGACGCCCTTGGTGGGTCGAGAGTAGTTGTTGTGGA
exon 11 - C1824T
The deleted
region is part of
C-term tail = not
essential to
protein function!
(SWISS PROT)
3rd Generation Lentivectors:
Lentiviral expression vector
Ψ
Lentiviral Packaging plasmids
HTNV
In vitro experiments:
hESC and iPSC
transduction
transduced cells
Neo
Only cells with transgene Tet-On
Only GFP-positive
- 1st A control-group trunsduced with an empty lentiviral vector
Checkpoints:
-2nd Test the presence of TALENs by Western Blot technique.
-3rd Test the removal of the trinucleotide sequence by real time PCR
-4th Verify if differentiated cells express the Lamin A isoform by Western Blot
In vivo experiments:
Choose an efficient animal model
mice
(atherosclerosis mice)
LmnAG608G transgenic
Vector injection 8-month mice:
Local carotid arteries injection through “Remedy catheter” and diffused injection through tail vain
4 groups of 5 mice for each mode of injection:
• 5 individuals LmnAG608G trunsduced with TALEN-HNTV LVs ( 2·107 TU/mouse)
• 5 individuals WT trunsduced with TALEN-HNTV LVs ( 2·107 TU/mouse)
• 5 individuals LmnAG608G trunsduced with Adenoviral vectors (1–1.5 109 pfu)
• 5 individuals WT trunsduced with Adenoviral vectors (1–1.5 109 pfu)
An other control? The same treatment in APOE mice
In vivo experiments:
Five weeks after injection
• GFP expression
- MOLECULAR
• Western blot
ANALYSIS
• ELISA
- MORPHOLOGICAL
ANALYSIS
To test vector expression
and tropism
To test and quantify
progerin expression level
• Histological analysis of aortic section and heart
• Observation of nuclear architecture
- PATOLOGICAL ANALYSIS
α-actin
RFP-VECTOR
merge
I.
AdV
Zhong Qian et al. 2006
AORTIC SECTION
Material and costs:
•Cloning kit
490€ (each clone)
•Lentiviral vector 416€
•TALEN 5000€
•iPS AND hESC 3000 €
•C57BL/6J mice(WT) 50 €
•Transgenic mouse 200€
•Molecular analysis (antibodies, reagent prc,
western blot...) 3000€
•ELISA kits 4000€
•Immuno-histochemical analysis
•General patological analysis
200-300€
200€
References:
“A-type lamins and Hutchinson-Gilford progeria syndrome: pathogenesis and
therapy”. Gonzalez et al., Frontiers in Bioscience S3, 1133-1146, June 1, 2011
“Molecular bases of progeroid syndromes”. Navarro et al., Human Molecular
Genetics, 2006, Vol. 15, Review Issue No. 2 R151–R161
“HGPS and related premature aging disorders: From genomic identification to
the first therapeutic approaches”, Pereira et al., Mechanisms of Ageing and
Development 129 (2008) 449–459
“Reversal of the cellular phenotype in the premature aging disease
Hutchinson-Gilford Progeria Syndrome”, Scaffidi and Misteli, Nat Med. 2005
April; 11(4): 440–445.
“Efficient design and assembly of custom TALEN and other TAL effector-based
constructs for DNA targeting”; Cermak et al. Nucleic Acids Research, 2011,
Vol. 39, No. 12
References:
“A Human iPSC Model of Hutchinson Gilford Progeria Reveals Vascular
Smooth Muscle and Mesenchymal Stem Cell Defects”; Zhang et al.; Cell Stem
Cell. 2011 Jan 7;8(1):31-45. Epub 2010 Dec 23.
“Generic engineering of human pluripotent cells using TALE nucleases”;
Hockemeyer D. et al.; Nat Biotechnol. 2011 Jul 7;29(8):731-4.
“Targeting vascular injury using Hantavirus-pseudotyped lentiviral
vectors”.Qian Z et al.;Mol Ther. 2006 Apr;13(4):694-704. Epub 2006 Jan 23
“Progressive vascular smooth muscle cell defects in a mouse model of
Hutchinson-Gilford progeria syndrome”; Varga et al.; Proc Natl Acad Sci U S
A. 2006 Feb 28;103(9):3250-5.
“Lamin A/C, laminopathies and premature ageing”; Liu and Zhou; Histol
Histopathol (2008) 23: 747-763