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