chromosomal v e ctors for cystic f i brosis gene therapy fiorentina ascenzioni
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Chromosomal vectors for cystic fibrosis gene therapy
Fiorentina Ascenzioni
Department of Cellular and Developmental Biology
The ideal gene therapy vector
Low invasivityLow invasivitySelective target Selective target Low immunogenicityLow immunogenicityHigh cloning capacityHigh cloning capacityLong term stabilityLong term stabilityLow copy numberLow copy numberReduced sizeReduced sizeLow intereference with the host genomeLow intereference with the host genome
The chromosomal vectors
•BAC•PAC
•MACMinichromosomede novo chromosomes
Mainly used for cloning
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
Can be engineered
•to remove sequences not relevant to chromosome functions and to transgene expression •to insert your favourite transgene
Consist of
•Structural elements: telomeres, centromeres and origins of replication•Accessory elements: selectable markers, genes, site-specific recombination elements
CentromeresCentromeres
1. Centromere/kinetochore assembly
2. Spindle microtubules capture3. Sister chomatid resolution4. Movement of the sister
chromatid to each spindle pole
Function DNA
Type I,repeated chromosome specific unit consisting of several homogeneous monomersType II, diverged chromosome units
11-mer higher order repeat* CenpB-box
Diverged monomeric repeat
*
Centromeric chromatin
Present in active centromere only
CenpA, centromere specific histonH3 like protein
Proposed model for the distribution of the constitutive CENPs
Ando et al 2002 Mol Cell Bio 22, 2229-2241
• CenpA, nucleosomes are phased on I typethrough 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
• CenpA, centromere-specific H3 variant, it is essential for centromeric chromatin
• CenpC, present in active but absent in inactive centromere
• CenpB, present both in active and inactive centromere, absent in chr.Y
where they localize
what they doKO mouse
Death by 6,5 days
Death by 3,5 days
Viable
Sullivan and Karpen, Nat Strct Mol Biol, 2004, 11, 1076-1083
CenpA H3 lys4-diMe CenpA H3 lys4-diMe
Centrochromatin
Cohesins
Inner Kinetochore
Outer Kinetochore
microtubule
CenpA subdomain
H3 lys4-diMe subdomain
Centromeric CenpA-nucleosome, interspersed with open but not active chromatin H3 lys4-diMe nucleosome
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
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
Top down
Bottom up
Bottom up, human artificial chromosome formation is associated with de novo centromere formation
from test tube to cells
Centromeric constructs
PAC/BAC type Isatellite 35-90 kb
YAC type I satellite 100-1000 kb
Unlinked DNA chromosomal elements
+
+DNA genomico
Telomeri1-10 kb
Type I satellite80-160 kb
De novo minichromosome
de novo minichromosome formation is tightly linked with
alphoid DNA and Cenp-B
Type I alphoid repeat: consists of several monomers; it contains CenpB-box
Type II alphoid repeat: consists of divergent monomers; does not contain Cenp-B box
Neocentromere
Non-alphoid repeats +CenpB-box
21-I YES17 YES
21-II NOY very inefficient
NO
NO
alphoid MAC formation
De novo MACs consist of amplified input DNAGrimes et al., 2002 Mol Ther 5, 798-805
Alpha 17-Iprobe
Anti-CenpA
BAC red17-I, green
BAC/ 17I
Masumoto et al, 2004 Chomosome Res,12, 543-546
H3 nucleosome CenpA/CenpB Acetylated H3
Irradiation Telomere Fragmentation
Top Down Approach
Fragmentation Constructs
CenTel
CenTel
Spence et al., 2002 EMBO J 19, 5269-5280
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
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
Minichromosome features
Minichromosomes generated by gamma-irradiation of human chromosome 1
Carine,K. Et al.(1989)Somat.Cell Mol.Genet.5:445-60
MC1
PFGE separation of MC1PFGE separation of MC1
4.6 Mb
5.7 Mb
3.5 Mb
MC1 0.04 U/m
l
wellsS. p
ombe
GM13139
MC1
CHO MC1 0.004 U
/ml
DNase treatment
MC1 is linear with T2AG3 telomeresMC1 is linear with T2AG3 telomeres
Human telomeric probe
MC1
NdeI BglII NdeI BglII
GM13139
*
*
MC1
MC1
GM13139
GM13139
NdeI BglII NdeI BglII
CHOM
C1GM
13139
HindI
II
NdeI
NdeI
NdeI
HindI
II
HindI
II
CHOM
C1GM
13139
HindI
II
NdeI
NdeI
NdeI
HindI
II
HindI
II
Long range restriction mapping of MC1 and human chromosome 1 (GM13139)
Probes alphoid Sat2 subtel tel
T2AG3, cy3, redD1Z7, fitc, green
T2AG3, cy3, redD1Z7, fitc, greenDNA,DAPI,blu
Tel Sat2 D1Z7 Tel
MC1 Structure by Fiber FISHThe two telomeres
T2AG3, cy3, redSat2, fitc, green
T2AG3, cy3, redSat2, fitc, greenDNA,DAPI,blu
D1Z7, cy3, redSat2, fitc, greenDNA, DAPI, blu
Tel Sat2 Sat2/D1Z5 D1Z5 D1Z7 Tel
Pericentromeric DNA two blocks of alphoid DNA
B
D1Z7, cy3, redD1Z5, fitc, greenDNA, DAPI, blu
D1Z5, cy3, redSat2, fitc, greenDNA, DAPI, blu
MC1 Structure by Fiber FISHThe central region
Centromere Activity and Centromeric Proteins
Alphoid-D1Z5 CREST MERGE
Sat2 CREST MERGE
Alphoid-D1Z5 CENP-F MERGE
Tel Sat2 Sat2/D1Z5 D1Z5 D1Z7 Tel
Pericentromeric DNA two blocks of alphoid DNA
MC1 Structure5.5 Mb
Active centromere
Smaller derivatives of MC1
Fragmentation construct
Transfection into CHO-MC1
Selection
PFGE analysis
Tel Sat2 Sat2/D1Z5 D1Z5 D1Z7 Tel
E-GFP
pBluHCMVSat2/D1Z5
hygro
Sat2/D1Z5
tel
Tel E-GFP Hyg Sat2/D1Z5 D1Z5 D1Z7 Tel
pBluGFP-Sat2/D1Z5
hygR clones
Construct N. Clones Analyzed Reduced
PFGE
pBluHCMV-Sat2/D1Z5 39 29 none interstitial tel-tel fusion
pBluGFP-Sat2/D1Z5 39 17 3
1A 3A
7A6A
Probe puc
pBLUGFP-Sat2/D1Z5
MC1CHO
1 2 3 4 5 6 7 14 15 16 17
pBLUHVMV-Sat2/D1Z5
Sat2 probe
PFGE FISH
n14
alphoid (D1Z5) probe
BluGFP-Sat2/D1Z5 Clones
puc probe
Cystic FibrosisA 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 domainsNBF1, 2 nucleotide Binding Fold, cytoplasmic, bind ATPR, regulatory cytoplasmid domain, controls channel opening
CFTR mutations
DF 508, the most common, affects 70% of the CF patients
Classe V: reduced synthesis
alternative splicing, exon
skipping
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?
Tel Sat2 Sat2/D1Z5 D1Z5 D1Z7 Tel
Pericentromeric DNA two blocks of alphoid DNA
MC1-CFTR5.8 Mb
Tel Sat2 D1Z5 D1Z7 Tel
Sat2/D1Z5
Pericentromeric DNA two blocks of alphoid DNA
hCFTR
IRES-geo
Southern blot analysis suggests integration of CFTR into Sat2
PEG fusion
G418 selection
30 G418 resistant clones
Integration of hCFTR locus into MC1
MC1 Sat2
CHO-MC1 transfected yeast protoplastwith Sat2 DNA
YAC-CFTR
FISH Analysis of MC1-CFTR containing clones
The probe was CFTR cDNA
P38P39
P16P37
The intersection of the P39 and human lines with y-value 1 demonstrates thepresence of half CFTR target in P39 withrespect to human T84 cells.
0,1
1
10
1 10 100 1000
pg standard
ratio
T/S
P39 T84 P39 regression T84 regression
PCR products obtained by competitive methods on P39 clone
625 4,8
competitor
CFTR target
pg competitor
500 bp
P39 clone contains one copy of the CFTR geneAs demonstrated by competitive and limiting dilution PCR reactions
CFTR activity in MC1-CFTR clones
MC
1
T84
CH
O
L4 P16
P34
P36 P37
P38
P39
CFTR
Actin
Northern analysis of the indicated RNA
T84, human epithelial cellsCHO, hamster ovary cellsMC1, CHO cells with MC1L and P, CHO-MC1 cells containing CFTR
0
1000
2000T8416373839
SDS-PAGE of cell lysates immunoprecipitated with an antibody to the human CFTR and phosphorylated
HT29, human epithelial cellsCHO, hamster ovary cellsMC1, CHO cells with MC1P, CHO-MC1 cells containing CFTR
CFTR activity in MC1-CFTR clones
P16 P38
P 37 P 39
MC1Anti-CFTRMATG1031
CFTR immunolocalization
FACS analysis of the P clones labelled with the monoclonalantibody MATG 1031 directed to the human CFTRp
0
50
100
150
200
po
siti
ve c
ells
(
% o
f b
asa
l)
37 38 39
clone
B
forskolin
basal
0
1
2
3
4
% p
osi
tive
ce
lls
37 38 39
A
A: cytofluorimetric analysis of viable cellsincubated with MATG1031 and with FITC-conjugated secondary antibody
B: same as in A but with untreated(basal) and treated (forskolin) cells
Plasma membrane CFTR
Functional analysis of CFTR protein
36Cl- efflux from cells stimulated with CTP.cAMP
P39 P38 P37
A B
C D
P38 MC1 P39
MC1
P39+glib
P38+glib O
8 exp 10 exp
10 exp8 exp
Functional analysis of CFTR protein
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
N chr.7 polarized epithelia CFBE 7 +
CFT1 3/4 +/-
CFPAC 3 +
IB3 2/3 -
FRT nd +
CFBE
CFT1
Cells N
clones
Positive to
neoPCR
Positive to
F PCR
Positive to
corresponding WT PCR
IB3
F508/W1282X
15 IB3/8
IB3/11
IB3/8
IB3/11+/-*
IB3/8
IB3/11
* Mixed clone, in fact repetition of F-PCR after 2-3 passages was negative
To control CFTR activity we produced stable transfected clone with pCMV-CFTRcl2, cl4, cl5
Microcells fusion Donor P37 Recipient IB3
PCR analysis of IB3 clones
neo
PCR neo sui cloni IB3/P37
neg 1 2 4 5 8 11 13 14 neg 15 IB3 P37
neo PCR to confirm the presence of the marker
Expected results of the controls: P37 IB3Neo PCR pos negCFTR-F508 neg posCFTR-Wt pos pos
1 4 5 8 11 IB3 P37
CFTR-F508
1 4 5 8 11 IB3 P37
CFTR-Wt
CFTR-F508 and the corresponding wt to identify the recipient
Clones Chrom1 Pericentromeric
Sat2
Chrom1 Centromeric
pAL1/D1Z5
CHO Human
IB3/11 pos pos Pos +/-
IB3/8 pos pos Neg Pos
FISH analysis of the IB3 clones
Rotterdam 05 WP1: Evaluation of the therapeutic effects of CFTR-MC1 in CF cultured cells
Probes
IB3
IB3-8
IB3-11 IB3-11
FISH analysis of IB3-11 points to human chromosome 1 points to MC1-CFTR
CHO probe
Rotterdam 05 WP1: Evaluation of the therapeutic effects of CFTR-MC1 in CF cultured cells
centromeric probe
IB3Centromeric probe
IB3-11
Pericentromeric probe
IB3-11IB3-11
Tel Sat2 D1Z5 D1Z7 Tel
Sat2/D1Z5
Pericentromeric DNA two blocks of alphoid DNA
Pericentromeric probe (Sat2)
IB3-8
IB3-8
Centromeric probe (pAL1)
FISH analysis of IB3-8
Points to MC1-CFTR
Rotterdam 05 WP1: Evaluation of the therapeutic effects of CFTR-MC1 in CF cultured cellsFISH analysis of IB3-8 Pericentromeric sat2 probe
points to human chromosome 1 points to MC1-CFTR
Clone cen and pericen
CHO Human identity
IB3/8 Pos Neg Pos IB3/MC1-CFTRP37
IB3/11 Pos Pos +/- CHO/MC1-CFTRP37
Conclusioncl8 rescued from IB3/P37 microcell-fusion experiment
is IB3/MC1-CFTR
FISH probes
To be analyzed the proteinthe functional activity
Progetto FFC Progetto FFC
#11/2004#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. BragonziDurata 1 anno. Finanziamento €30.000
B. cepacia gnv I
B. multivorans
B. cenocepacia
B. stabilis
B. vietnamiensis
B. dolosa
B. ambifaria
B. anthina
B. pyrrocinia
Distribuzione delle specie
Ambiente naturale
Ambiente clinico:espettorato pazienti CF
IIIB
IIIB
IIIC
IIIA
IIID
Esistono differenze nel Esistono differenze nel grado di patogenicità tra grado di patogenicità tra ceppi ambientali e clinici ceppi ambientali e clinici del del B. cepacia B. cepacia complex? complex?
Cosa accade in presenza di Cosa accade in presenza di P. aeruginosaP. 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 AscenzioniCristina Auriche Elisabetta Testa Lucia RocchiPiera Fradani Laura FicoLivia Civitelli, Emanuele Fanella, Enea di Domenico,
Institute for Experimental Treatment of Cystic Fibrosis, Milano Massimo ConeseDaniela Carpani Sante di GioiaSalvatore Carrabino
Laboratorio di Genetica Molecolare, Gaslini Olga Zegarra-MoranNicoletta Pedemonte Emanuela Caci
Dr.ssa A. Bevivino L. Pirone, dottoranda Dr. S. Tabacchioni, Dr. C. Dalmastri, Dr. L. Chiarini
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