Department of Cellular and
Developmental Biology
Chromosomal vectors for cystic fibrosis
gene therapy
Fiorentina Ascenzioni
The ideal gene therapy vector
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Low invasivity
Selective target
Low immunogenicity
High cloning capacity
Long term stability
Low copy number
Reduced size
Low intereference with the host genome
The chromosomal vectors
•BAC
•PAC
Mainly used
for cloning
•MAC
Minichromosome
de novo chromosomes
expression vectors for therapeutic genes
and animal transgenesis
Models to analyze the structural
features of human chromosomes
Minichromosomes: linear DNA molecules mimicking
the behaviour of a natural chromosome
•Replicate and segregate independently of
host chromosome
•1-2 copy per cell
Consist of
•Structural elements: telomeres,
centromeres and origins of
replication
•Accessory elements: selectable
markers, genes, site-specific
recombination elements
Can be engineered
•to remove sequences not relevant
to chromosome functions and to
transgene expression
•to insert your favourite transgene
Centromeres
Function
1.
2.
3.
4.
DNA
Centromere/kinetochore
assembly
Spindle microtubules capture
Sister chomatid resolution
Movement of the sister
chromatid to each spindle pole
Type I,repeated chromosome specific unit
consisting of several homogeneous
monomers
Type II, diverged chromosome units
11-mer higher order repeat
* CenpB-box
*
Diverged monomeric repeat
Centromeric chromatin
CenpA, centromere
specific histonH3 like
protein
Present in active centromere only
Proposed model for the distribution of the
constitutive CENPs
where they localize
• CenpA, nucleosomes are phased on a-I type
through interaction with CenpB
• CenpC, inner kinetochore lamina, takes part in
formation of CenpA/B/C complex
• CenpB, binds CenpB-box (17 bp in typeI alpha
and mouse minor satellite DNAs
what they do
KO mouse
• CenpA, centromere-specific H3 variant, it is
Death by 6,5 days
essential for centromeric chromatin
• CenpC, present in active but absent in inactive Death by 3,5 days
centromere
• CenpB, present both in active and inactive
Viable
centromere, absent in chr.Y
Ando et al 2002 Mol Cell Bio 22, 2229-2241
Centrochromatin
Centromeric CenpA-nucleosome, interspersed with open but not active chromatin H3 lys4-diMe
nucleosome
CenpA
H3 lys4-diMe
CenpA
H3 lys4-diMe
Cohesins
Inner Kinetochore
Outer Kinetochore
microtubule
CenpA subdomain
H3 lys4-diMe subdomain
Sullivan and Karpen, Nat Strct Mol Biol, 2004, 11, 1076-1083
Telomeres
necessary to replicate linear chromosomes but dispensable for
de novo chromosome formation. 1.5 kb of telomeric repeats are
sufficient to seed a de novo telomere
Origin of replication
it is assumed that most DNA fragments of proper
size (15-40 kb) are replication competent
How to get minichromosomes
1989, Carine et al obtained a minichromosome by irradiation of a monosomatic
CHO hybrid
Top down
1994, Brown et al obtained minichromosome from Chromosome Y by telomere
fragmentation
1995, Farr dissected human chromosome X and produced centric minichromosome
1997, Willard HF obtained de novo chromosome formation with human alphoid
DNA
1998, Ikeno et al produce de novo chromosomes from YAC clone with alpha21-I
DNA
2000, Ebersole et al assembled PAC with alpha21-I DNA competent for de novo
chromosome formation
Bottom up
Bottom up, human artificial chromosome formation is associated
with de novo centromere formation
from test tube to cells
Unlinked DNA chromosomal elements
+
+
Type I a-satellite
80-160 kb
Telomeri
1-10 kb
DNA genomico
Centromeric constructs
YAC type I a-satellite 100-1000 kb
De novo minichromosome
PAC/BAC type Ia-satellite
35-90 kb
de novo minichromosome formation is
tightly linked with alphoid DNA and
Cenp-B
alphoid
MAC formation
Type I alphoid repeat: consists of several
monomers; it contains CenpB-box
a21-I YES
a17
YES
Type II alphoid repeat: consists of divergent
monomers; does not contain Cenp-B
box
a21-II NO
aY very inefficient
Neocentromere
Non-alphoid repeats +CenpB-box
NO
NO
De novo MACs consist of amplified input DNA
Grimes et al., 2002 Mol Ther 5, 798-805
Alpha 17-I
probe
BAC red
a17-I, green
Anti-CenpA
BAC/a 17I
H3 nucleosome CenpA/CenpB Acetylated H3
Masumoto et al, 2004 Chomosome Res,12, 543-546
Top Down Approach
Telomere Fragmentation
Tel
Cen
Fragmentation
Constructs
Tel
Cen
Irradiation
Human chromosome X was reduced up to 0.85 Mb by multiple
rounds of telomere fragmenation
•Transfer into
intermediate host
(chicken DT40 cells)
• Insertion of the
Cre/loxP system
Spence et al., 2002 EMBO J 19, 5269-5280
Minichromosome features
de novo minichromosomes
•circular molecules
•5-10 Mb in size
•structure not simply related to the input DNA
•de novo chromosome formation associated with
host genome rearrangements
minichromosomes from top down
•linear with functional telomeres
•from few hundred kb to 5-10 Mb in size
•structure related to the parental chromosome
Minichromosomes generated by gamma-irradiation
of human chromosome 1
MC1
Carine,K. Et al.(1989)Somat.Cell Mol.Genet.5:445-60
PFGE separation of MC1
DNase treatment
5.7
Mb
4.6
Mb
3.5
Mb
wells
MC1 is linear with T2AG3 telomeres
Human telomeric probe
Long range restriction mapping of MC1
and human chromosome 1 (GM13139)
NdeI BglII NdeI BglII
NdeI BglII NdeI BglII
*
*
Probes
alphoid
Sat2
subtel
tel
MC1 Structure by Fiber FISH
The two telomeres
Tel
D1Z7
Sat2
Tel
T2AG3, cy3, red
Sat2, fitc, green
T2AG3, cy3, red
D1Z7, fitc, green
T2AG3, cy3, red
Sat2, fitc, green
DNA,DAPI,blu
T2AG3, cy3, red
D1Z7, fitc, green
DNA,DAPI,blu
MC1 Structure by Fiber FISH
The central region
Pericentromeric DNA
Tel
Sat2
two blocks of alphoid DNA
Sat2/D1Z5 D1Z5
D1Z7
Tel
B
D1Z7, cy3, red
Sat2, fitc, green
DNA, DAPI, blu
D1Z5, cy3, red
Sat2, fitc, green
DNA, DAPI, blu
D1Z7, cy3, red
D1Z5, fitc, green
DNA, DAPI, blu
Centromere Activity and
Centromeric Proteins
Alphoid-D1Z5
Sat2
Alphoid-D1Z5
CREST
CREST
CENP-F
MERGE
MERGE
MERGE
MC1 Structure
5.5 Mb
Pericentromeric DNA
Tel
Sat2
two blocks of alphoid DNA
Sat2/D1Z5 D1Z5
D1Z7
Active centromere
Tel
Smaller derivatives of MC1
hygro
Fragmentation construct
Sat2/D1Z5
pBluHCMVSat2/D1Z5
tel
E-GFP
pBluDGFP-Sat2/D1Z5
Transfection into CHO-MC1
Tel
Tel E-GFP
Sat2
Hyg
Sat2/D1Z5 D1Z5 D1Z7
Sat2/D1Z5 D1Z5 D1Z7
Tel
Tel
Selection
PFGE analysis
hygR clones
Construct
N. Clones
Analyzed
Reduced
PFGE
pBluHCMV-Sat2/D1Z5
39
29
none
interstitial
tel-tel fusion
pBluDGFP-Sat2/D1Z5
39
17
3
1A
3A
Probe puc
6A
7A
PFGE BluDGFP-Sat2/D1Z5 Clones FISH
pBLUHVMV-Sat2/D1Z5
1
2
3
4
5
pBLUDGFP-Sat2/D1Z5
6
7
14 15
16
17
n14
alphoid (D1Z5) probe
Sat2 probe
puc probe
Cystic Fibrosis
A model deseas for gene
therapy
•Caused by single gene mutations
•Accessible target organs
•No curative pharmacological
treatment
•The gene sequence is available
since 1989
•1/2500 affected
•Correction of 5-10% of CF- cells
restore some function in animal
models (Dorin JR et al., 1997)
CFTRp structure and channel activity
TMD1,2 hydrofobic transmembrane domains
NBF1, 2 nucleotide Binding Fold, cytoplasmic, bind ATP
R, regulatory cytoplasmid domain, controls channel opening
CFTR mutations
Classe V: reduced synthesis
alternative splicing, exon
skipping
DF 508, the most
common, affects 70% of
the CF patients
1989 CFTR gene
(Rommens et al., Science 1989)
1990 in vitro gene transfer of normal CFTR gene
(Drumm et al., Cell 1990)
1992 CFTR gene transfer in vivo cotton rats
(Rosenfeld et al., Cell 1992)
1993 First clinical trials (Zabner et al., Cell 1993)
2002 15 trials completed
2004 29 trials
Proposed gene therapy vectors for CF
Viral:
• Adenovirus, non replicating, transient expression
• Virus Adeno-associati (AAV), non replicating but
integrating vector
• Lentivirus, integrating
Synthetic
•Cationic lipid ( es. DOTAP, DOPE, DMPE etc.)
•Cationic polymer (PEI, polylysine, dendrimers )
Barriers
Extracellulars
intracellulars
Does MC1 represent a good vector for CF gene therapy?
Pericentromeric DNA
Tel
Sat2
two blocks of alphoid DNA
Sat2/D1Z5 D1Z5
D1Z7
Tel
MC1-CFTR
5.8 Mb
Pericentromeric DNA
Tel
Sat2
hCFTR
two blocks of alphoid DNA
D1Z5
D1Z7
Tel
Sat2/D1Z5
IRES-Bgeo
Southern blot analysis suggests integration of CFTR into Sat2
Integration of hCFTR locus into MC1
YAC-CFTR
MC1 Sat2
CHO-MC1 transfected
with Sat2 DNA
yeast protoplast
PEG fusion

G418 selection

30 G418 resistant clones
FISH Analysis of MC1-CFTR containing clones
P16
P39
P37
P38
The probe was CFTR cDNA
P39 clone contains one copy of the CFTR gene
As demonstrated by competitive and limiting dilution PCR reactions
pg competitor 625
4,8
P39
T84
P39 regression
T84 regression
10
500 bp
ratio T/S
competitor
1
CFTR target
0,1
1
PCR products obtained by competitive methods
on P39 clone
10
100
1000
pg standard
The intersection of the P39 and human
lines with y-value 1 demonstrates the
presence of half CFTR target in P39 with
respect to human T84 cells.
CFTR activity in MC1-CFTR clones
Northern analysis of the indicated RNA
CFTR
2000
1000
Actin
0
T84, human epithelial cells
CHO, hamster ovary cells
MC1, CHO cells with MC1
L and P, CHO-MC1 cells containing CFTR
T84
16
37
38
39
CFTR activity in MC1-CFTR clones
SDS-PAGE of cell lysates immunoprecipitated
with an antibody to the human CFTR and phosphorylated
HT29, human epithelial cells
CHO, hamster ovary cells
MC1, CHO cells with MC1
P, CHO-MC1 cells containing CFTR
CFTR immunolocalization
Anti-CFTR
MATG1031
MC1
P16
P38
P 37
P 39
Plasma membrane CFTR
FACS analysis of the P clones labelled with the monoclonal
antibody MATG 1031 directed to the human CFTRp
4
B
A
200
basal
150
positive cells
(% of basal)
% positive cells
3
2
1
0
forskolin
100
50
0
37
38
39
37
38
39
A: cytofluorimetric analysis of viable cells B: same as in A but with untreated
incubated with MATG1031 and with FITC- (basal) and
clone
treated (forskolin) cells
conjugated secondary antibody
Functional analysis of CFTR protein
36Cl- efflux from cells stimulated with CTP.cAMP
P39
P38
P37
Functional analysis of CFTR protein
A
B
P38 
MC1
P39
MC1
8 exp
10 exp
C
P38
+glib O
8 exp
D
P39
+glib 
10 exp
Analysis of the therapeutic effects of the minichromosome
require its transfer into appropriate models
Epithelial CF
MC1-CFTR
corrected
The ideal host of MC1-CFTR should
•recapitulate CF defects
•enable the expression of a functionale CFTR
•acquire the minichromosome by…..
Cytogenetic analysis and functional analysis of candidate CF cells
CFBE
CFT1
N chr.7
polarized epithelia
CFBE
7
+
CFT1
3/4
+/-
CFPAC
3
+
IB3
2/3
-
FRT
nd
+
Microcells fusion
Cells
IB3
DF508/W1282X
N
clones
15
Donor P37
Recipient IB3
Positive to
neoPCR
Positive to
DF PCR
Positive to
corresponding
WT PCR
IB3/8
IB3/11
IB3/8
IB3/11+/-*
IB3/8
IB3/11
* Mixed clone, in fact repetition of DF-PCR after 2-3 passages was negative
To control CFTR activity we produced stable transfected clone with pCMV-CFTR
cl2, cl4, cl5
PCR analysis of IB3 clones
neo PCR to confirm the presence of
the marker
CFTR-DF508 and
the corresponding
wt to identify the recipient
CFTR-DF508
neo
1
neg
1
2
4
5
8
11
13 14
4
5
11
IB3
P37
neg 15 IB3 P37
PCR neo sui cloni IB3/P37
CFTR-Wt
Expected results of the controls: P37 IB3
Neo PCR
pos neg
CFTR-DF508
CFTR-Wt
8
neg
pos
pos
pos
1
4
5
8
11
IB3
P37
Rotterdam 05
WP1: Evaluation of the therapeutic effects of CFTR-MC1 in CF cultured cells
FISH analysis of the IB3 clones
Probes
Clones
Chrom1
Pericentromeric
Sat2
Chrom1
Centromeric
pAL1/D1Z5
CHO
Human
IB3/11
pos
pos
Pos
+/-
IB3/8
pos
pos
Neg
Pos
Rotterdam 05
WP1: Evaluation of the therapeutic effects of CFTR-MC1 in CF cultured cells
FISH analysis of IB3-11
points to human chromosome 1
points to MC1-CFTR
IB3
centromeric probe
IB3-11
IB3-8
Centromeric probe
IB3
IB3-11
IB3-11
IB3-11
IB3-11
CHO probe
Pericentromeric probe
FISH analysis of IB3-8
Pericentromeric DNA
Tel
Sat2
two blocks of alphoid DNA
D1Z5
D1Z7
Tel
Sat2/D1Z5
IB3-8
Centromeric probe
(pAL1)
Pericentromeric probe
(Sat2)
Points to MC1-CFTR
IB3-8
Rotterdam 05
WP1: Evaluation of the therapeutic effects of CFTR-MC1 in CF cultured cells
FISH analysis of IB3-8
points to human chromosome 1
points to MC1-CFTR
Pericentromeric sat2 probe
Conclusion
cl8 rescued from IB3/P37 microcell-fusion experiment
is IB3/MC1-CFTR
FISH probes
Clone
cen and
pericen
CHO
Human
identity
IB3/8
Pos
Neg
Pos
IB3/MC1-CFTRP37
IB3/11
Pos
Pos
+/-
CHO/MC1-CFTRP37
To be analyzed
the protein
the functional activity
Progetto FFC #11/2004
Valutazione della patogenicità di ceppi
ambientali e clinici di Burkholderia
cepacia complex da soli ed in presenza
di Pseudomonas aeruginosa
A. Bevivino, F. Ascenzioni, A. Bragonzi
Durata 1 anno. Finanziamento €30.000
Distribuzione delle specie
Ambiente naturale
IIIB
B. cepacia gnv I
B. multivorans
IIIC
Ambiente clinico:
espettorato pazienti CF
B. cenocepacia
B. stabilis
B. vietnamiensis
IIIA
B. dolosa
B. ambifaria
IIIB
IIID
B. anthina
B. pyrrocinia
Esistono differenze nel grado
di patogenicità tra ceppi
ambientali e clinici del B.
cepacia complex?
Cosa accade in presenza di P.
aeruginosa?
Scopo del progetto
 Valutazione della patogenicità di
isolati
ambientali e clinici appartenenti alle diverse
linee filogenetiche di B. cenocepacia, mediante
(i) analisi della capacità di adesione e invasione
dell’epitelio cellulare respiratorio CF e non-CF
(ii) analisi della capacità di colonizzazione degli
epiteli respiratori murini in un modello di
infezione cronica
 Valutazione dell’influenza di P. aeruginosa, il
principale patogeno per i pazienti CF, sulla
capacità di invasione ed infezione dei ceppi
presi in esame
Disegno sperimentale
1. Allestimento di un pannello di ceppi
ambientali e clinici di B. cenocepacia
2. Screening dei ceppi mediante saggi di
infezione in vitro, utilizzando colture di
cellule epiteliali CF e non CF
3. Screening dei ceppi mediante saggi di
infezione in vivo, utilizzando il modello
murino di infezione cronica polmonare
4. Analisi della capacità di invasione ed
infezione in vitro ed in vivo dei ceppi in
presenza di P. aeruginosa
Università di Roma “La
Sapienza”
Fiorentina Ascenzioni
Institute for Experimental
Treatment of Cystic Fibrosis,
Milano Massimo Conese
Daniela Carpani Sante di Gioia
Salvatore Carrabino
Cristina Auriche Elisabetta Testa
Lucia Rocchi
Piera Fradani Laura Fico
Livia Civitelli, Emanuele Fanella, Laboratorio di Genetica Molecolare,
Enea di Domenico,
Gaslini Olga Zegarra-Moran
Nicoletta Pedemonte
Emanuela Caci
Dr.ssa A. Bevivino
L. Pirone, dottoranda
Dr. S. Tabacchioni, Dr. C. Dalmastri, Dr.
L. Chiarini