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Chromosomal v e ctors for cystic f i brosis gene therapy Fiorentina Ascenzioni

Department of Cellular and Developmental Biology. Chromosomal v e ctors for cystic f i brosis gene therapy Fiorentina Ascenzioni. The ideal gene therapy vector. Low invasivity Selective target Low immunogenicity High cloning capacity Long term stability Low copy number Reduced size

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Chromosomal v e ctors for cystic f i brosis gene therapy Fiorentina Ascenzioni

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  1. Department of Cellular and Developmental Biology Chromosomal vectors for cystic fibrosis gene therapy Fiorentina Ascenzioni

  2. The ideal gene therapy vector • Low invasivity • Selective target • Low immunogenicity • High cloning capacity • Long term stability • Low copy number • Reduced size • Low intereference with the host genome

  3. Mainly used for cloning The chromosomal vectors • BAC • PAC • MAC Minichromosome de novo chromosomes expression vectors for therapeutic genes and animal transgenesis Models to analyze the structural features of human chromosomes

  4. 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 Can be engineered • to remove sequences not relevant to chromosome functions and to transgene expression • to insert your favourite transgene Structural elements: telomeres, centromeres and origins of replication Accessory elements: selectable markers, genes, site-specific recombination elements

  5. Centromeres Function 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

  6. 11-mer higher order repeat * CenpB-box Diverged monomeric repeat *

  7. Centromeric chromatin CenpA, centromere specific histonH3 like protein Present in active centromere only

  8. 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 Death by 6,5 days Death by 3,5 days Viable 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 Ando et al 2002 Mol Cell Bio 22, 2229-2241

  9. Centrochromatin CenpA H3 lys4-diMe CenpA H3 lys4-diMe Centromeric CenpA-nucleosome, interspersed with open but not active chromatin H3 lys4-diMe nucleosome Cohesins Inner Kinetochore Outer Kinetochore microtubule CenpA subdomain H3 lys4-diMe subdomain Sullivan and Karpen, Nat Strct Mol Biol, 2004, 11, 1076-1083

  10. Telomeresnecessary 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 replicationit is assumed that most DNA fragments of proper size (15-40 kb) are replication competent

  11. Bottom up How to get minichromosomes Top down 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

  12. Unlinked DNA chromosomal elements Type I a-satellite 80-160 kb + Telomeri 1-10 kb + DNA genomico De novo minichromosome YAC type I a-satellite 100-1000 kb Bottom up,human artificial chromosome formation is associated with de novo centromere formation from test tube to cells Centromeric constructs PAC/BAC type Ia-satellite 35-90 kb

  13. alphoid MAC formation 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 a21-I YES a17 YES a21-II NO aY very inefficient NO NO de novo minichromosome formation is tightly linked with alphoid DNA and Cenp-B

  14. De novo MACs consist of amplified input DNA Grimes et al., 2002 Mol Ther 5, 798-805 BAC red a17-I, green BAC/a 17I Alpha 17-I probe Anti-CenpA H3 nucleosome CenpA/CenpBAcetylated H3 Masumoto et al, 2004 Chomosome Res,12, 543-546

  15. Irradiation Cen Tel Fragmentation Constructs Cen Tel Top Down Approach Telomere Fragmentation

  16. 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

  17. 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

  18. MC1 Carine,K. Et al.(1989)Somat.Cell Mol.Genet.5:445-60 Minichromosomes generated by gamma-irradiation of human chromosome 1

  19. PFGE separation of MC1 DNase treatment MC1 0.004 U/ml MC1 0.04 U/ml S. pombe GM13139 CHO MC1 5.7 Mb wells 4.6 Mb 3.5 Mb

  20. MC1 is linear with T2AG3 telomeres Human telomeric probe

  21. GM13139 GM13139 GM13139 GM13139 GM13139 CHO CHO MC1 MC1 MC1 MC1 MC1 NdeI BglII NdeI BglII NdeI BglII NdeI BglII HindIII HindIII HindIII HindIII HindIII HindIII NdeI NdeI NdeI NdeI NdeI NdeI * * Long range restriction mapping of MC1 and human chromosome 1 (GM13139) Probes alphoid Sat2 subtel tel

  22. D1Z7 Tel Tel Sat2 MC1 Structure by Fiber FISH The two telomeres T2AG3, cy3, red D1Z7, fitc, green T2AG3, cy3, red D1Z7, fitc, green DNA,DAPI,blu T2AG3, cy3, red Sat2, fitc, green T2AG3, cy3, red Sat2, fitc, green DNA,DAPI,blu

  23. Pericentromeric DNA two blocks of alphoid DNA Tel Sat2 Sat2/D1Z5 D1Z5 D1Z7 Tel B D1Z7, cy3, red Sat2, fitc, green DNA, DAPI, blu MC1 Structure by Fiber FISH The central region D1Z7, cy3, red D1Z5, fitc, green DNA, DAPI, blu D1Z5, cy3, red Sat2, fitc, green DNA, DAPI, blu

  24. Alphoid-D1Z5 CREST MERGE Sat2 CREST MERGE Alphoid-D1Z5 CENP-F MERGE Centromere Activity and Centromeric Proteins

  25. Pericentromeric DNA two blocks of alphoid DNA Tel Sat2 Sat2/D1Z5 D1Z5 D1Z7 Tel MC1 Structure 5.5 Mb Active centromere

  26. hygro pBluHCMVSat2/D1Z5 Sat2/D1Z5 E-GFP tel pBluDGFP-Sat2/D1Z5 Tel Sat2 Sat2/D1Z5 D1Z5 D1Z7 Tel Tel E-GFP Hyg Sat2/D1Z5 D1Z5 D1Z7 Tel Smaller derivatives of MC1 Fragmentation construct Transfection into CHO-MC1 Selection PFGE analysis

  27. 1A 3A 7A 6A Probe puc

  28. pBLUHVMV-Sat2/D1Z5 pBLUDGFP-Sat2/D1Z5 CHO MC1 1 2 3 4 5 6 7 14 15 16 17 alphoid (D1Z5) probe Sat2 probe BluDGFP-Sat2/D1Z5 Clones PFGE FISH n14 puc probe

  29. 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)

  30. 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

  31. Classe V: reduced synthesis alternative splicing, exon skipping CFTR mutations DF 508, the most common, affects 70% of the CF patients

  32. 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

  33. 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 )

  34. Barriers Extracellulars intracellulars

  35. Does MC1 represent a good vector for CF gene therapy? Pericentromeric DNA two blocks of alphoid DNA Tel Sat2 Sat2/D1Z5 D1Z5 D1Z7 Tel

  36. Pericentromeric DNA two blocks of alphoid DNA Tel Sat2 D1Z5 D1Z7 Tel Sat2/D1Z5 hCFTR MC1-CFTR 5.8 Mb IRES-Bgeo Southern blot analysis suggests integration of CFTR into Sat2

  37. YAC-CFTR MC1 Sat2 Integration of hCFTR locus into MC1 CHO-MC1 transfected yeast protoplast with Sat2 DNA PEG fusion  G418 selection  30 G418 resistant clones

  38. FISH Analysis of MC1-CFTR containing clones P16 P37 P38 P39 The probe was CFTR cDNA

  39. 625 4,8 pg competitor competitor 500 bp CFTR target P39 clone contains one copy of the CFTR gene As demonstrated by competitive and limiting dilution PCR reactions PCR products obtained by competitive methods on P39 clone 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.

  40. CFTR activity in MC1-CFTR clones Northern analysis of the indicated RNA T84, human epithelial cells CHO, hamster ovary cells MC1, CHO cells with MC1 L and P, CHO-MC1 cells containing CFTR MC1 CHO P16 P37 P34 T84 P36 L4 P38 P39 CFTR Actin

  41. 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

  42. CFTR immunolocalization P16 P38 P 37 P 39 MC1 Anti-CFTR MATG1031

  43. Plasma membrane CFTR A: cytofluorimetric analysis of viable cells incubated with MATG1031 and with FITC- conjugated secondary antibody B: same as in A but with untreated (basal) and treated (forskolin) cells FACS analysis of the P clones labelled with the monoclonal antibody MATG 1031 directed to the human CFTRp

  44. Functional analysis of CFTR protein 36Cl- efflux from cells stimulated with CTP.cAMP P39 P38 P37

  45. A B D C Functional analysis of CFTR protein P38  MC1 P39 MC1 8 exp 10 exp P38 +glib O P39 +glib  10 exp 8 exp

  46. MINICROMOSOMI Epithelial CF MC1-CFTR corrected Analysis of the therapeutic effects of the minichromosome require its transfer into appropriate models

  47. 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

  48. Microcells fusion Donor P37 Recipient IB3 * 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

  49. neo neg 1 2 4 5 8 11 13 14 neg 15 IB3 P37 CFTR-F508 neo PCR to confirm the presence of the marker CFTR-F508 and the corresponding wt to identify the recipient 1 4 5 8 11 IB3 P37 PCR neo sui cloni IB3/P37 CFTR-Wt 1 4 5 8 11 IB3 P37 Expected results of the controls: P37 IB3 Neo PCR pos neg CFTR-F508 neg pos CFTR-Wt pos pos PCR analysis of IB3 clones

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