Transcript 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.

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