[笔试] [903] 考试题目及中文翻译

主题	好友	212 积分

项目经理

发消息

电梯直达

1楼

发表于 2008-11-10 13:02 |显示全部楼层 |倒序浏览

考完了，不想回头看。不过还是忍不住去查了这专利。准备了这长时间，觉得不甘心呀。

(WO/2003/104467) MEANS AND METHODS FOR THE PRODUCTION OF ADENOVIRUS VECTORS

FIELD OF THE INVENTION The invention relates to nucleic acid delivery vehicles and use thereof, more in particular the invention relates to recombinant adenoviral vectors and the use thereof.

DETAILED DESCRIPTION The present invention discloses methods and means that solve certain difficulties related to diminished complementation of non-group C adenoviral vectors in Ad5 packaging/complementing cells. Although in the Ad5 complementing cell lines functional Ad5 E1B-55K expression is present, it was found that only very low titers of adenoviral vectors could be produced when the adenoviral backbone was of a non-group C adenoviral origin; this finding implies a [a class="hl hl-2" name="a-2-36" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-37"]serotype[/a])-specificity in the interaction of E1B-55K with another (viral) protein. It is disclosed here that this [a class="hl hl-2" name="a-2-37" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-38"]serotype[/a])-dependency can be circumvented by providing E4-orf6 protein compatible with the E1B-55K protein provided by the complementing cell line. As discussed herein, E1B-55K and E4- orf6 form a complex that is involved in inhibiting transport of cellular mRNA's from the nucleus to the cytoplasm, while the complex is also involved in stimulation of transport of viral mRNA's from the nucleus to the cytoplasm. It has been observed by the present inventors that proper complementation of viral vectors in packaging cells requires the presence of [a class="hl hl-5" name="a-5-5" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-5-6"]E1B[/a])-[a class="hl hl-4" name="a-4-2" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-4-3"]55K[/a]) and E4-orf6 gene products that are compatible.

Packaging cells are also referred to as complementing cells, if the cells comprise certain sequences encoding proteins that complement functions not provided by the vector that should be packaged. Compatible'as used herein therefore means that a complex between the available E1B-55K gene product is able to form a functional complex with the available E4-orf6 gene product in a sense that this protein complex supports viral replication, propagation and/or packaging to a level that is comparable to the wild-type situation or that is comparable to the situation found when a recombinant adenovirus [a class="hl hl-2" name="a-2-38" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-39"]serotype[/a]) 5 vector is produced on a Ad5 complementing cell line such as [a class="hl hl-3" name="a-3-5" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-3-6"]293[/a]) or PER. C6. Vector replication in packaging cells is efficient if, during production period in which the virus is formed, the cell comprises at least E1B-55K protein and E4-orf6 protein that are compatible. Preferably, the E1B-55K and E4-orf6 sequences are from adenoviruses within the same adenovirus subgroup (such as A, B, C, D, E or F). More preferably, the E1B-55K and E4-orf6 sequences are from the same [a class="hl hl-2" name="a-2-39" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-40"]serotype[/a]). Since established cell lines-are available in the art that are capable of supporting the growth of adenoviruses of subgroup C, such as [a class="hl hl-2" name="a-2-40" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-41"]serotype[/a]) 5, it is even more preferred that the E1B-55K and E4-orf6 genes are derived from adenovirus [a class="hl hl-2" name="a-2-41" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-42"]serotype[/a]) 5. As will be understood by the skilled person compatibility may be determined in complementation tests or assays as such in the realm of those skilled in the art of adenoviral vector production. The person skilled in the art will also understand that the present invention can also be used for the production of any adenovirus [a class="hl hl-2" name="a-2-42" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-43"]serotype[/a]) on any complementing cell line as long as the [a class="hl hl-5" name="a-5-6" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-5-7"]E1B[/a])-[a class="hl hl-4" name="a-4-3" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-4-4"]55K[/a]) and E4-orf6 proteins are compatible.

It has further been observed that the E4-orf6 gene product, matching'with the [a class="hl hl-5" name="a-5-7" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-5-8"]E1B[/a]) in the complementing cell line, can be provided by the adenoviral vector, by replacing the E4-orf6 in the adenoviral vector of choice with an E4- orf6 encoding sequence that is compatible with the [a class="hl hl-5" name="a-5-8" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-5-9"]E1B[/a]) gene present within the packaging cell line. This modification was surprisingly found not to have a severe effect on the stability, replication, packaging, assembly and production of the vector.

It is a specific aspect of the invention that one is now able to efficiently produce adenovirus serotypes different than those from subgroup C on cell lines normally applied for the production of adenovirus [a class="hl hl-2" name="a-2-43" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-44"]serotype[/a]) 5 or other [a class="hl hl-2" name="a-2-44" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-45"]serotype[/a]) from subgroup C, such as [a class="hl hl-2" name="a-2-45" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-46"]serotype[/a]) 1,2 and 6. The present invention provides methods for the production of non-group C adenoviruses without the necessity of separately providing the complementing (packaging) cell with E4-orf6 because the E4-orf6 sequence, that is compatible with the complementing E1B-55K sequence, is incorporated in the adenoviral backbone.

The present invention provides a recombinant adenovirus vector comprising structural and non-structural elements of an adenovirus of a first [a class="hl hl-2" name="a-2-46" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-47"]serotype[/a]), wherein said vector further comprises a sequence encoding a functional E4-orf6 protein, or a functional part, derivative and/or analogue thereof, wherein said sequence is selected from the group consisting of: a) an E4-orf6 encoding sequence derived from an adenovirus of a second [a class="hl hl-2" name="a-2-47" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-48"]serotype[/a]) different from said first [a class="hl hl-2" name="a-2-48" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-49"]serotype[/a]); b) an E4-orf6 encoding sequence derived from an adenovirus of said first [a class="hl hl-2" name="a-2-49" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-50"]serotype[/a]) comprising a deletion, mutation, addition and/or substitution in one or more codons; and c) an E4-orf6 encoding sequence comprising a fusion between a part of an E4-orf6 encoding sequence derived from a second [a class="hl hl-2" name="a-2-50" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-51"]serotype[/a]) different from said first [a class="hl hl-2" name="a-2-51" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-52"]serotype[/a]) and a part of an E4-orf6 encoding sequence derived from a third [a class="hl hl-2" name="a-2-52" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-53"]serotype[/a]), wherein said third [a class="hl hl-2" name="a-2-53" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-54"]serotype[/a]) may be identical to-, or different from said first [a class="hl hl-2" name="a-2-54" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-55"]serotype[/a]). In one embodiment, the present invention provides a recombinant adenovirus vector according to the invention, wherein said first [a class="hl hl-2" name="a-2-55" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-56"]serotype[/a]) and said second [a class="hl hl-2" name="a-2-56" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-57"]serotype[/a]) are from different adenovirus subgroups.

In a preferred embodiment, a recombinant adenovirus vector according to the invention is provided, wherein said first [a class="hl hl-2" name="a-2-57" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-58"]serotype[/a]) is from a subgroup other than subgroup C and wherein said E4-orf6 encoding sequence is derived from an adenovirus [a class="hl hl-2" name="a-2-58" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-59"]serotype[/a]) of subgroup C. More preferred is a recombinant adenovirus according to the invention, wherein said first [a class="hl hl-2" name="a-2-59" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-60"]serotype[/a]) is from subgroup B and said second [a class="hl hl-2" name="a-2-60" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-61"]serotype[/a]) is from subgroup C. Even more preferably, said E4-orf6 encoding sequence is derived from adenovirus [a class="hl hl-2" name="a-2-61" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-62"]serotype[/a]) 5.

It has been recognized by those skilled in the art that different levels of neutralizing antibodies circulate in humans that are directed against different serotypes. It has been found that certain adenoviral serotypes encountered high titers of such neutralizing antibodies and that many individuals in different populations carried neutralizing antibodies against such serotypes. It was also found that certain serotypes were only neutralized in a minority of the samples (WO 00/70071). Apparently, certain serotypes from subgroup B were only neutralized in a small group of samples.

Therefore, in a preferred embodiment of the present invention, recombinant adenovirus vectors according to the invention are provided, wherein said first [a class="hl hl-2" name="a-2-62" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-63"]serotype[/a]) is selected from the group consisting of adenovirus serotypes 11,14, 16, 21, 34,35 and 50. Highly preferred are recombinant adenoviral vectors, wherein said first [a class="hl hl-2" name="a-2-63" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-64"]serotype[/a]) is [a class="hl hl-2" name="a-2-64" href="http://www_wipo_int/pctdb/en/fetch_jsp?SEARCH_IA=EP2003050125&DBSELECT=PCT&C=10&TOTAL=11&IDB=0&TYPE_FIELD=256&SERVER_TYPE=19-10&ELEMENT_SET=B&START=1&SORT=41249385-KEY&QUERY=%28FP%2Fadenovirus%29+AND+%28NPCC%2Fcn%29+AND+%28DE%2FE1B+AND+DE%2F55k+AND+DE%2Fserotype+AND+DE%2F293%29+&RESULT=9&DISP=25&FORM=SEP-0%2FHITNUM%2CB-ENG%2CDP%2CMC%2CAN%2CPA%2CABSUM-ENG&IDOC=269116&IA=EP2003050125&LANG=ENG&DISPLAY=DESC#a-2-65"]serotype[/a]) 11 or 35, while these encountered neutralizing antibodies only in a very small percentage of the tested samples.

The vectors of the present invention can be used in different settings such as gene therapy, functional genomics, tumour vaccination and/or anti-viral vaccination. For this, it is necessary that the adenoviral vector functions as a gene delivery vehicle, wherein a non-native gene is incorporated into the adenoviral genome. The adenoviral particle can subsequently be targeted specifically to target cells of interest ; the adenovirus binds to that specific cell either through capsid-receptor binding or through other means, and delivers the transgene. Targeting of adenoviruses can be performed in many different ways. Persons skilled in the art of adenoviral vector targeting will be aware of all the different possibilities that are applied to deliver the adenoviral vectors to the cells of interest. Such possibilities include but are not limited to capsid alterations (fiber, hexon and/or penton modifications, such as deletions, swaps between fibers of different serotypes, and additions of peptides and/or other binding moieties), wherein chimeric fibers are produced that recognize a receptor present on the cell of interest, or wherein the binding of the penton-base is utilized. Other possibilities are linking targeting moieties to the capsid proteins, wherein for instance binding peptides, known and strong binding proteins, or antibodies or parts thereof are linked to the capsid proteins to achieve specific targeting. Such vectors can all be produced using the methods and means provided by the present invention. Therefore, the present invention also discloses recombinant adenovirus vectors according to the invention, further comprising a sequence encoding a non-adenoviral protein, polypeptide or peptide.

Such sequences can be present on different locations within the adenoviral backbone, but preferably it is located in the E1 region, which is lacking in the recombinant adenoviral vectors of the invention. The E1 region is complemented by (the complementation) elements present in the complementing cells. The direction of the promoter, transgene and other regulatory sequences can be directed towards the left-, as well as to the right inverted terminal repeat.

The present invention can also be used for the production of viral vectors based on adenovirus and/or on other viruses such as the Adeno-Associated Virus (AAV), wherein the combination, such as an Ad-AAV chimeric virus, can integrate into the host cell genome. Several methods are known in the art for generating integrating adenoviruses.

Generally, the invention is also useful for the production of adenovirus forms that (specifically, or non-specifically) can integrate.

As mentioned, several non-adenoviral transgenes can be cloned into the recombinant adenoviral vectors of the present invention. These do not only include regulatory nucleic acid sequences such as enhancers, promoters (e. g. strong non- adenoviral promoters such as the cytomegalovirus promoter, the SV40 promoter and the RSV promoter) and polyadenylation signals, but also heterologous genes for therapeutic purposes. Therefore, in one aspect of the invention, recombinant adenovirus vectors according to the invention are provided, wherein said non-adenoviral protein, polypeptide or peptide is selected from the group consisting of: a cell- death inducing polypeptide, an antigenic determinant of a pathogenic organism, a tumor specific antigen, a viral protein, a hormone and a cytokine. Examples of pathogenic organisms are, but are not limited to bacteria, viruses and fungi. Non-limiting examples of non-adenoviral factors, proteins, polypeptides and petides are transcription factors, intracellular signalling proteins, phosphatases, kinases, apoptosis inhibiting factors, receptor antagonists, soluble forms of membrane-bound receptors, RNA inhibitors, anti-sense RNA's, decoy factors, ribozymes, and more specifically, thymidine kinase, erythropoietin, novel-erythropoiesis stimulating protein (NESP), IL3, ceNOS, gamma-interferon and gap100. Non-adenoviral viral proteins can be cloned into the recombinant adenoviral vectors provided by the methods and means of the present invention for vaccination purposes. Such viral proteins include, but are not limited to, gag, pol, env, nef, etc. for HIV vaccines, E6 and E7 proteins for Human Papilloma Virus vaccines, circumsporozoite proteins from Plasmodium protozoa for malaria vaccines, rotavirus components for rotavirus vaccines, ebola proteins for ebola vaccines, the F and G gene products from Respiratory syncytial virus (RSV) for RSV vaccines, HA and NA for influenza vaccines, etc.