INFECTION WITH CHIMAERIC ADENOVIRUSES OF CELLS NEGATIVE FOR THE ADENOVIRUS SEROTYPE 5 COXSACKI ADENOVIRUS RECEPTOR (CAR)

Abstract: The invention relates to the field of molecular genetics and medicine. The invention discloses a method for delivering a nucleic acid of interest to a host cell by means of a gene delivery vehicle based on adenoviral material, whereby said gene delivery vehicle delivers the nucleic acid to the host cell by associating with a binding site and/or a receptor present on CAR-negative cells, said binding site and/or receptor being a binding site and/or a receptor for adenovirus subgroups D and/or F.

Description: ) t t M H o LΠ o LΠ σ LΠ HJ H < fu Ω fl> Φ H- fD Q ø H tr H μ- 0 Hi 0 (D to 0 2 f-i J fD rt P- Φ> H P. H- — rt , , ø J M fD 0 rt m fD H CO tr H TJ CO rt fD — H- H- <! 0 n 3 fu H- Hi 111 0 H rt H- J tQ ø <ϊ H- fu fD CO & o Hi Φ ø LQ Ω fD U1 fu - — 0 H- fD Λ 4 Mi ø rt H- J fD P» ø- 0 o 0 fD Mi rt rt fD IQ H- H- < Ω CD <J rt H- rt 0 fD H- 4 0 fD 0 fu 3 tr CD CO Φ -<; - ≤ o H- ^ ~. J fu ω fD 0 0 fD l P. 0 fD tr <$ rt σ> fD fU ^ H- -^ Hi 0 TJ H- Φ 0 rt Hi fD rt ø * 0 rt 0 fD Pi ^ fu Mi 0 <i o 0 - rt H- fD Mi t n α H fD ø Ω V£> CO 0 ) fD U> fD rt *» 0 ) o ' — i fu 0 * CO • 0 H- * • IQ fu tr ø- 0 fD 0, ø' PJ CO ω ) t to H M LΠ o LΠ o LΠ O LΠ tr Φ Φ 0 H- Pi φ 0 rt μ- Hi H- Φ ,_ , CO φ φ H ω tr H- tr fu CD ø J H- Φ rt fu - H U) ∞ L — • A serotype is defined on the basis of its immunological distinctiveness as determined by quantitative neutralisation with animal antisera (horse, rabbit) . If neutralisation shows a certain degree of cross-reaction between two viruses, distinctiveness of serotype is assumed if A) the hemagglutinins are unrelated, as shown by lack of cross- reaction on hemagglutination-inhibition, or B) substantial biophysical/ biochemical differences in DNA exist (Francki et al, 1991) . The nine serotypes identified last (42-51) were isolated for the first time from HIV- infected patients (Hierholzer et al 1988; Schnurr et al 1993; De Jong et al 1998) . For reasons not well understood, most of such immuno- compromised patients shed adenoviruses that were rarely or never isolated from immuno-competent individuals (Hierholzer et al 1988, 1992; Khoo et al , 1995, De Jong et al , 1998). The adenovirus serotype 5 is most widely used for gene therapy purposes. Similar to serotypes 2, 4 and 7, serotype 5 has a natural affiliation towards lung epithelia and other respiratory tissues. In contrast, it is known that, for instance, serotypes 40 and 41 have a natural affiliation towards the gastrointestinal tract . For a detailed overview of the disease association of the different adenovirus serotypes see table 1. The underlying reason for the different natural affiliations of serotypes towards specific organs can be manifold. Such reasons may include but need not be limited to the observation that serotypes differ in the route of infection or make use of different receptor molecules or internalisation pathways or that a serotype can infect many tissues/organs but it can only replicate in one organ because of the requirement of certain cellular factors for replication. As mentioned before, it is presently unknown which mechanisms are responsible for the observed differences in human disease association. ) O to to μ> μ 1 LΠ O LΠ o LΠ σ π H3 Ω 0 Φ LQ Φ H- 1 rt CO ø- > Φ i Hi Φ ø $ 0 μ- < rt μ- ø- Hi 0 rt CD tr Φ t fu fu Pi 0 Φ P. 0 0 LΠ < μ- tr H Φ 0 CO 0 φ 0 CD LQ 0 rt Hh 0 CO fu 0 Pi tr Φ LQ 0 Hi 0 0 < 0 μ- TJ Hj CQ 0 CD > - CO 0 α tr LQ ^ Hi ~ 0 0 rt TJ tr Φ π • LO LO to to H H LΠ o LΠ o LΠ O LΠ LO LO to to μ> μ> LΠ O LΠ o LΠ o Lπ binding region. The base region comprises of a penton base binding region and a shaft binding region. Moreover, the shaft comprises of repeated stretches of amino acids . Each of these repeated stretches may be a part . A receptor and/or binding site binding part of a fiber protein may be a single region of a fiber protein or a functional part thereof, or a combination of regions or parts thereof of at least one fiber protein, wherein said receptor and/or binding site binding part of a fiber protein, either alone or in combination with one or more other proteins of a adenovirus capsid, determines the efficiency with which a gene delivery vehicle can transduce a given cell or cell type, preferably but not necessarily in a positive way. Needless to say that said fiber and/or a capsid may comprise further modifications to adapt the fiber protein and/or the capsid to specific other needs, which a person skilled in the art will be capable of doing. A receptor and/or a binding site for adenovirus subgroups D and/or F may be any kind of molecule capable of associating with an adenovirus of subgroup D and/or F. In and/or on the surface of a cell, said receptor and/or binding site must be able to associate with said adenovirus of subgroup D and/or F provided to said cell. Said receptor and/or binding site may be part of a complex present in and/or on said cell. Said receptor and/or binding site does not need to be able to associate with an adenovirus of subgroup D and/or F all the time as long as it is capable of doing so some of the time. Said receptor and/or binding site may further also be a receptor and/or binding site for another virus and/or gene delivery vehicle, although this does not have to be so. A person skilled in the art may want to determine whether an adenovirus serotype belonging to another subgroup than D and/or F can also utilise the receptor and/or binding site for adenovirus subgroups D and/or F. In another aspect the invention provides the use of a gene delivery vehicle comprising a nucleic acid of interest and comprising adenoviral material involved in binding to a host cell, said material being from a subgroup D and/or F adenovirus, in delivering said nucleic acid of interest to a CAR-negative cell . With the knowledge of a novel pathway for the transduction of cells using adenovirus material it becomes possible to approach this novel pathway also through other means then said material derived from a subgroup D and/or F. A person skilled in the art recognises this and will be able to devise means to accomplish this for instance through the use of antibodies directed toward a crucial component of said pathway, together with a membrane fusion peptide. Such means and methods are also within the scope of the invention. In another aspect the invention provides a gene delivery vehicle being a chimaera based on at least two adenoviruses, whereby a cell recognising element of said gene delivery vehicle is based on adenoviral material from a subgroup D and/or F adenovirus, which material confers the capability of infecting CAR negative cells. Preferably, said adenoviral material is based on the fiber, penton and/or hexon proteins of a subgroup D and/or subgroup F adenovirus. To date, six different subgroups of human adenoviruses have been proposed which in total encompasses 51 distinct adenovirus serotypes. A serotype is defined on the basis of its immunological distinctiveness as determined by quantitative neutralisation with animal antisera (horse, rabbit) . If neutralisation shows a certain degree of cross- reaction between two viruses, distinctiveness of serotype is assumed if A) the hemagglutinins are unrelated, as shown by lack of cross-reaction on hemagglutination-inhibition, or B) substantial biophysical/ biochemical differences in DNA exist (Francki et al, 1991) . The nine serotypes identified last (42-51) were isolated for the first time from HIV-infected patients (Hierholzer et al 1988; Schnurr et al 1993;). For reasons not well understood, most of such immune-compromised patients shed adenoviruses that were rarely or never isolated from immune-competent individuals (Hierholzer et al 1988, 1992; Khoo et al , 1995, De Jong et al , 1998). The usefulness of these adenoviruses or cross-immunising adenoviruses to prepare gene delivery vehicles may be seriously hampered, since the individual to which the gene delivery vehicle is provided, will raise a neutralising response to such a vehicle before long. There is thus a need in the field of gene therapy to provide gene delivery vehicles, preferably based on adenoviruses, which do not encounter pre-existing immunity and/or which are capable of avoiding or diminishing neutralising antibody responses. Thus preferably, a gene delivery vehicle of the invention further comprises an element from adenovirus 35 or a functional equivalent thereof, responsible for at least partially avoiding an immune response against adenovirus 35. A functional equivalent/homologue of adenovirus 35 (element) for the purpose of the present invention is an adenovirus (element) which, like adenovirus 35, encounters pre-existing immunity in less than about 10% of the hosts, at least in a significant geographic region of the world, to which it is administered for the first time, or which is capable in more than about 90% of the hosts, at least in a significant geographic region of the world, to which it is administered to avoid or diminish the immune response. Typical examples of such adenoviruses are adenovirus serotypes 34, 26 and 48. In another embodiment a gene delivery vehicle according to the invention comprises an element of adenovirus 16 or a functional equivalent thereof, which element confers said virus with an enhanced capability to infect smooth muscle cells and/or synoviocytes . A functional equivalent of an element of adenovirus 16 in this respect is an element from LO LO to to H- 1 Lπ O LΠ o LΠ o LΠ LO LO t to μ> μ> LΠ O LΠ o LΠ o LΠ tr Ω 3 0 0 φ 3 3 fu 0 TJ ø H- 1 Hi rt 0 μ- Q 01 rt 0 Φ tr 0 ) CO Φ rt to Hj CD to Φ φ PJ Ω 0 μ- 0 rt P. μ- J 0 μ- < ø φ J > : Hj rt H- J μ- G fυ O 0 TJ ø TJ ^ μ- ø φ 0 LQ PJ 0 Pi φ CD μ- φ ø Ω ►P Q 0 0 0 φ rt rt 0 0 μ- Ω ø φ rt ø CD tr 0 Φ ø μ- to ø LQ φ CQ Φ 0 Φ fu φ ►P Ω Hi ø tr ) Φ rt ø ø μ- Ω 0 0 Φ Ω 0 Ξ φ 0 ø' μ- Hti Φ Ω Hi fυ Φ fυ μ- Ω ø μ- LO LO to to H LΠ O LΠ o LΠ o LΠ <! rt CQ φ to fu rt Hj CO to VD J TJ 3 P. s; TJ fu 3 rt rt rt fu μ- to 3 ø * 3 3 ) 3 CO Hi Φ Hj fu Hh fu Pi tr Φ fu ) CΛ Hj Φ P> Φ Φ fu Ω 0 0 Φ O $. 0 0 fu Φ O Φ φ Φ Hj P) φ 0 Ω fu μ- Hh μ- Pi φ TJ μ- μ- O Φ Hi 3 Hj Hi μ- O Pi <J μ- rt 3 rt CO rt φ ^ Λ 0 rt 0 μ- μ- H- 1 Pi Pi to Hh Hh 3 μ- Pi rt Pi φ rt to Φ Pi tr 0 ø- μ- ø' 0 Ω 0 to Ω rt 3 μ- to Φ 0 PJ < μ- μ- 01 Ω tr μ- PJ 0 0 Hi 0 O O 0 tr Φ rt Hj fU Hj μ- H{ μ- Φ Ω 3 ø Ω Hj Hj fu ø PJ 0 Φ ø μ- ø Hj rt ø Pi > O Pi Φ Pi O Φ 0 μ- Ω φ Φ Φ G rt J fu 0 > fυ 3 μ- rt LQ P ) H- J LQ Pi Ω Φ 0 LQ Pi • rt Ω O rt TJ ø TJ 0 tr rt 3 Ω O tr Φ PJ μ- Hj Φ LQ fu Pi Ω Hi TJ O tr Φ 0 μ- Ω H- 1 fu 0 μ- 3 — ' Pi ø <! rt ^ φ 03 φ rt Z φ 0 0 Hj 0 TJ tr ≥! μ- ) ø μ- * μ- Pi 0 fu φ ^ Φ 0 • 0 O Φ μ- ) rt φ Hi φ CQ Hi Φ O Φ LQ rt LQ Ω Ω co ø μ- G μ- Ω LQ 0 φ LQ Ω H- 1 Φ μ- Hh Φ Ω 0 CO tr tr fu O fu rt μ- Ω Hi to ø Φ fu Φ <! φ Hj Φ P. o Hj Ω LQ Φ Hj 03 fu μ- Φ Φ PJ Hh rt Hi rt PJ 0 μ- fu u ø rt Φ 0 fu rt μ- fu LQ 3 φ Hi Φ 0 0 Ω HJ H Pi 0 μ- μ- Hi Hi to ø PJ fu μ- fu P ø' Hi rt ø' ø Ω 0 μ- fu 0 Hj Ω 3 CQ O Φ P ) Φ 0 0 rt 0 LQ Ω Pi • Φ Φ fu μ- Φ LQ μ- Φ ø Ω φ Φ O fu Φ 0 Pi rt Ω ø Ω ø tr ø Φ rt ø Ω μ- Pi Ω O Hj fu 0 Hj α fu μ- Hj tr rt tr rt 0 rt Φ rt tr ø Φ Φ Ω Φ 2 Hi TJ TJ ø rt rt fu * rt μ- tr fu 0 <! μ- Ω μ- CD Φ Pi H{ Φ H- 1 0 μ- TJ rt Hj O O 3 " rt Φ μ- " ø' ø Φ ø fu μ- 0 0 3 CQ Φ 3 μ- H- 1 Hj J to μ- 0 Φ 0 Hh LQ O Φ Hi 3 μ- ø Φ ø Ω H! ø M φ Hh μ- ω 0 < Φ 0 ø LQ Hi Hh Φ Hj Φ O ø r fu < O fu 0 0 CD TJ rt fu 0 ø μ- TJ H-> fυ Φ fu TJ LQ Φ φ Pi rt φ Φ TJ LQ 0 rt ø ø 0 ø ø CD φ tr Hi < Hj Hi φ rt μ- X TJ φ rt Hj 0 μ- ø- Ω O Φ φ Pi μ- Φ Hi CO 03 Φ 0 S- rt o CQ φ φ μ- Ω μ- to TJ Hj 0 Hi CD ø- Hj 0 H Φ 0 Hh fu Ω fu μ- O Φ Φ • 3 μ- Φ 0 0 ø P. 0 ø < Hi Φ LQ Hh Φ Φ Φ Φ Hh Hj . rt ø ø 3 ø rt Pi 0 O" PJ rt to CQ 0 rt Hi (- ■ Φ P ) Φ Ml 0 0 Λ Hi Pi φ • φ Φ O 0 < rt -3 φ ^ ø' ø μ- CO Φ O Φ Φ Hj 0 0 Hj to H O Φ fu ø TJ Ω φ tr O Hj Hh 0 0 Φ 0 μ- rt CD rt TJ to Hi 3 Φ rt Φ Hj Φ CO 0 Hi CD Pi rt Hj rt Φ Hj Φ 0 0 rt 0 Hh rt to ø ø 0 tr M μ- fu rt Φ 0 tr 0 J tr Φ 0 ø- H-" ^ 0 Ω tr Hj μ- Ω fu Φ rO ø tr ø- Pi Ω Hi tr rt μ- fu O PJ Ω 0 O Pi Φ rt fu Ω Hi Ω Φ rt Hj TJ φ to Hj Hj • LQ Φ Φ 0 0 0 Pi < 0 O O Hh ø ø- TJ Hi O 0 CQ ø- Φ Φ Hi fυ Φ Φ O Hj μ- to ø Φ Φ Hi H H 0 Φ O Φ μ- μ- 0 σ M ^ ø Pi rt 3 < Ω LQ Φ TJ Φ 0 tr Hj ø • LQ μ- t ø to Φ r 3 0 TJ μ- rt rt Φ μ- P. rt 0 • Φ tr μ- CO rt Pi 0 Hi μ> 0 ø- 3 Ω O 0 fu ø Hi Hi tr to 0 O ø 0 μ- 3 TJ CD μ- Hi ø ø 0 . Ω 1 CO Hh PJ H φ o H- 1 Pi 0 03 Ω μ- 0 Φ * Φ <! LQ Ω ø O 2! rt ø Φ 0 3 Hi £ rt Φ Hi fu . 0 tr ø Hi Φ μ- Pi TJ rt CO CO Hi Φ Φ 0 H μ- μ- Hj < 3 fu φ Φ H φ μ- Ω 0 rt μ- Hh Φ φ μ- Φ μ- fυ Hi ω O 0 rt Ω fu φ 0 tr Pi H- J Ω 0 LQ J Φ fu 3 03 Ω μ- Hj Ω O < 0 rt fu φ rt ø LQ ø * fu rt ø tr ø Pi PJ μ- Hi 0 03 fu Ω LQ rt 0 O φ < μ- H- 1 (P tr CO O μ- rt Φ rt Φ 0 "< Ω μ- TJ fυ ^^ 0 Ξ Φ Hi H- J fu μ- PJ PJ 0 J 0 Ω φ 0 ø Φ 0 ø μ- μ- Ω LQ Φ ø PJ M ø Φ Hi 0 0 μ- Ω rt 0 O rt ø 0 Φ O CD 0 0 0 fu 0 rt CQ LQ tr TJ n Φ 0 Hj LQ Pi μ- μ- φ 0 ^ O rt φ ø Hj < to 0 Hi ø 0 0 tr Hj φ Pi Hj Hj rt 0 rt Pi O Φ TJ Hj μ- 0 Hi Ω Φ Φ Hj P ) CQ to Hi rt rt φ Φ o ø μ- fu tr 0 Pi ≤ ø 3 J O O Hh Hi Φ H- J Hj Φ μ- 0 fu TJ φ 0 tr 0 Hi o Ω ø tr PJ Φ 03 μ- O 3 O 0 H{ 0 £ ø μ- to μ- Ω Pi Hh Pi Hi Hj 0 Φ LQ 0 φ G LQ Hj Hh o 3 Pi Hj rt Φ tr ø Ω u fu O Φ Φ μ- Φ LQ Φ Hh Hj Pi Pi Hh Hi Hi 0 fu μ- Φ TJ 0 μ- fu tr TJ 3 Ω CO 0 CQ rt Ω tr Hi rt Hj φ Φ rt 0 fu Φ Φ ø Φ ^ Hi tr fu Hj Hi i φ Hj μ- TJ tj" O fu 0 0 0 Φ tr Hj Φ TJ 0 CD 0 01 Ω Hi Ω Ω μ- o P. α Φ O Φ ø Φ μ- μ- 0 μ- < 3 Hj TJ φ Φ Pi 0 LQ fu Ω TJ 0 Φ 0 μ- Ω rt Φ Φ μ- < 03 Ω μ- ø ø rt Pi μ- fu Φ § • H μ- Hi TJ CQ TJ Φ μ- rt φ P> 0 1 ø ø <J tr μ- rt 0 - 0 rt 1 0 Φ ø fu tr Hi 0 ø μ- μ- ^ fu rt LQ PJ μ- Hi tr Pi μ- 0 fu . Φ μ- Hi rt μ- Ω rt μ- φ Ω rt μ- O Hi H- J ^ rt ø H- μ- Ω 03 μ- 0 <J < tr Φ Φ O CO to μ- 0 to ^ Ω fu μ- μ- co Ω t 1 rt Φ Ω ø ø 0 0 O Hh fu μ- 0 03 Pi Hi 0 O φ O 0 φ Φ rt PJ fu 0 0 Φ K Φ fu 01 fυ 0 fu α 0 rt Hj rt Hh Λ ø ø ø tr CQ μ- μ- μ- μ- rt Pi ø μ- fu rt Ω Ω φ φ 03 01 μ- 0 " fu CO 0 ø to G Φ 0 ø rt μ- μ- ø μ- μ- μ- μ- fu fυ < 03 fυ 0 to Φ Φ < Ω rt ø LQ ^ ø 3 Cj 1 to CD TJ 0 φ Pi Pi rt TJ μ- Φ O μ- rt υ 0 < μ- fu tr LQ Φ Φ • μ- fυ ø μ- μ- ø- Hi Hh μ- Ω Φ Hj 0 Φ 0 ø Hi to Hi Ω ø Φ μ- Φ ø 0 CQ Hh μ- rt rt 0 LQ O 03 rt CQ Φ ø PJ - Hh Hi 03 In a preferred embodiment, one of said nucleic acid molecules is relatively small and the other is relatively large. This configuration is advantageous because it allows easy manipulation of said relatively small nucleic acid molecule allowing for example the generation of a large number of small nucleic acid molecules comprising different nucleic acid of interest for instance for the generation of an adenovirus vector library. Said configuration is also desired because it allows the production of a large batch of quality tested large nucleic acid molecule. The amplification of large nucleic acid molecules for instance in bacteria is difficult in terms of obtaining sufficient amounts of said large nucleic acid. The amplification of large nucleic acid molecules for instance in bacteria is also difficult to control because a small modification of said large nucleic acid is not easily detected. Moreover, for reasons not quite understood some large vectors are more stable in bacteria or yeasts than others. Said configuration however, allows the generation of a standard batch of a large nucleic acid molecule which can be thoroughly tested, for instance through generating a control adenovirus of which the efficiency and the reliability of production is known, and determining said parameters of a new batch of large nucleic acid molecule. Once validated said batch may be used for the generation of a large number of different adenovirus vectors through combining said large molecule with a large number of different small nucleic acid molecules. Said system therefore also allows for the selection and/or manipulation of vectors comprising a large nucleic acid molecule of the invention to allow a suitable yield of intact large nucleic acid. In another embodiment said cell comprising nucleic acid encoding El-region proteins further comprises a nucleic acid encoding an adenovirus E2-region and/or an adenovirus E4- region protein. Preferably, said cell further comprising nucleic acid encoding an adenovirus E2-region and/or an adenovirus E4-region protein is a derivative of PER.C6. In another aspect the invention provides a receptor and/or a binding site for adenoviruses type D and/or F, present on or associated with CAR negative cells. Preferably said receptor and/or a binding site is present on K562 cells, amniotic fluid derived cells and/or primary fibroblast cells. In yet another aspect, the invention provides the use of receptor and/or a binding site for adenoviruses type D and/or F, present in and/or on a cell, for the delivery nucleic acid to said cell. In yet another embodiment the invention provides the use of a gene delivery vehicle according to anyone of claims 1-14, in a pharmaceutical. In another aspect the invention provides a capsid protein derived from a subgroup D and/or a subgroup F adenovirus or a functional part, derivative and/or analogue thereof. Preferably, said protein is a fiber protein. The invention further provides a nucleic acid encoding a capsid protein of the invention. Preferably, said nucleic acid comprises a fiber sequence from a subgroup D and/or a subgroup F as depicted in figure 7. Detailed description. It has been demonstrated in mice that upon in vivo systemic delivery of recombinant adenovirus serotype 5 for gene therapy purposes approximately 99% of the virus is trapped in the liver (Herz et al, 1993) . Therefore, alteration of the adenovirus serotype 5 host cell range to be able to target other organs in vivo is a major interest of the invention. The initial step for successful infection is binding of adenovirus to its target cell, a process generally thought to be mediated through fiber protein. The fiber protein has a trimeric structure (Stouten et al, 1992) with different lengths depending on the virus serotype (Signas et al 1985; Kidd et all 1993) . Different serotypes have polypeptides with structurally similar N and C termini, but different middle stem regions. N-terminally, the first 30 amino acids are involved in anchoring of the fiber to the penton base (Chroboczek et al, 1995) , especially the conserved FNPVYP region in the tail (Arnberg et al 1997) . The C-terminus, or knob, is generally thought to be responsible for initial interaction with the cellular adenovirus receptor. After this initial binding secondary binding between the capsid penton base and cell-surface integrins is proposed to lead to internalisation of viral particles in coated pits and endocytosis (Morgan et al, 1969; Svensson et al, 1984; Varga et al, 1992; Greber et al, 1993; Wickham et al , 1994). Integrins are αβ-heterodimers of which at least 14 α- subunits and 8 β-subunits have been identified (Hynes et al , 1992) . The array of integrins expressed in cells is complex and will vary between cell types and cellular environment. Although the knob contains some conserved regions, between serotypes, knob proteins show a high degree of variability, indicating that different adenovirus receptors might exist. For instance, it has been demonstrated that adenoviruses of subgroup C (Ad2 , Ad5) and adenoviruses of subgroup B (Ad3) bind to different receptors (Defner et al , 1990) . By using baculovirus produced soluble CAR as well as adenovirus serotype 5 knob protein, Roelvink et all concluded via interference studies that all adenovirus serotypes, except serotypes of subgroup B, enter cells via CAR (Roelvink et al, 1998) . The latter, which is now generally accepted in the field, if valid should thus limit the complexity of using different serotypes for gene therapy purposes. Besides the involvement in cell binding, the fiber protein also contains the type specific γ-antigen, which together with the ε-antigen of the hexon determines the serotype specificity. The γ-antigen is localised on the fiber and it is known that it consists of 17 amino acids (Eiz et al, 1997) . The anti-fiber antibodies of the host are therefore directed to the trimeric structure of the knob. To obtain re-directed infection of recombinant adenovirus serotype 5, several approaches have been or still are under investigation. Wickham et al has altered the RGD (Arg, Gly, Asp) motif in the penton base which is believed to be responsible for the α v β 3 and α v βs integrin binding to the penton base. They have replaced this RGD motif by another peptide motif which is specific for the CΛ 4 βι receptor. In this way targeting the adenovirus to a specific target cell could be accomplished (Wickham et al, 1995, 1996) . Krasnykh et al has made use of the HI loop available in the knob. This loop is, based on X-ray crystallographies, located on the outside of the knob trimeric structure and therefore is thought not to contribute to the intramolecular interactions in the knob (Krasnykh et al, 1998) . However, complete CAR independent infection was not observed. It is an object of the present invention to provide a method and means by which an adenovirus can infect cells negative for the CAR protein. Therefore, the generation of chimaeric adenoviruses based on adenovirus serotype 5 with a modified fiber gene is described. For this purpose, two or three plasmids, which together contain the complete adenovirus serotype 5 genome, were constructed. From a plasmid the DNA encoding the adenovirus serotype 5 fiber protein was essentially removed and replaced by linker DNA sequences which facilitate easy cloning. This plasmid subsequently served as template for the insertion of DNA encoding for fiber protein derived from different adenovirus serotypes (human or animal) . The DNAs derived from the different serotypes were obtained using the polymerase chain reaction technique in combination with (degenerate) oligonucleotides. At the former El location in the genome of adenovirus serotype 5, any gene of interest can be cloned. A single transfection procedure of the two or three plasmids together resulted in the formation of a recombinant chimaeric adenovirus. Although successful introduction of changes in the adenovirus serotype 5 fiber and penton-base have been reported, the complex structure of knob and the limited knowledge of the precise amino acids interacting with CAR render such targeting approaches laborious and difficult. To overcome the limitations described above we used preexisting adenovirus fibers to maximise the chance of obtaining recombinant adenovirus which can normally assemble in the nucleus of a producer cell and which can be produced on pre-existing packaging cells. By generating a chimaeric adenovirus serotype 5 based fiber library containing fiber proteins of all other human adenovirus serotypes, we have developed a technology which enables rapid screening for a recombinant adenoviral vector with preferred infection characteristics . In one aspect this invention describes chimaeric adenoviruses and methods to generate these viruses that have an tropism different from that of adenovirus serotype 5. This chimaeric adenovirus serotype 5 is able to infect cell types which do not express the CAR protein much more efficiently both in vi tro and in vivo than the adenovirus serotype 5. Such cells include but are not limited to endothelial cells, smooth muscle cells, dendritic cells, neuronal cells, glial cells, synovical cells, primary fibroblasts, cells from the amniotic fluid, hemopoietic stem cells, and monocytic/ macrophage cells etc. In another aspect the invention describes the construction and use of plasmids consisting of distinct parts of adenovirus serotype 5 in which the gene encoding for fiber protein has been replaced with DNA derived from alternative human or animal serotypes. This set of constructs, in total encompassing the complete adenovirus genome, allows for the construction of unique chimaeric adenoviruses customised for transduction of particular cell types or organ (s) . In all aspects of the invention the chimaeric adenoviruses may, or may not, contain deletions in the El region and insertions of heterologous genes linked either or not to a promoter. Furthermore, chimaeric adenoviruses may, or may not, contain deletions in the E3 region and insertions of heterologous genes linked to a promoter. Furthermore, chimaeric adenoviruses may, or may not, contain deletions in the E2 and/ or E4 region and insertions of heterologous genes linked to a promoter. In the latter case E2 and/ or E4 complementing cell lines are required to generated recombinant adenoviruses . Example 1 : Generation of adenovirus serotype 5 genomic plasmid clones <• The complete genome of adenovirus serotype 5 has been cloned into various plasmids or cosmids to allow easy modification of parts of the adenovirus serotype 5 genome, while still retaining the capability to produce recombinant virus. For this purpose the following plasmids were generated: 1. pBr/Ad.Bam-rlTR (ECACC deposit P97082122) In order to facilitate blunt end cloning of the ITR sequences, wild-type human adenovirus type 5 (Ad5) DNA was treated with Klenow enzyme in the presence of excess dNTPs . After inactivation of the Klenow enzyme and purification by phenol/chloroform extraction followed by ethanol precipitation, the DNA was digested with BamHI . This DNA preparation was used without further purification in a ligation reaction with pBr322 derived vector DNA prepared as follows: pBr322 DNA was digested with EcoRV and BamHI , dephosphorylated by treatment with TSAP enzyme (Life Technologies) and purified on LMP agarose gel (SeaPlaque GTG) . After transformation into competent E. coli DH5a (Life Techn.) and analysis of ampicilline resistant colonies, one clone was selected that showed a digestion pattern as expected for an insert extending from the BamHI site in Ad5 to the right ITR. Sequence analysis of the cloning border at the right ITR revealed that the most 3' G residue of the ITR was missing, the remainder of the ITR was found to be correct . Said missing G residue is complemented by the other ITR during replication. 2. pBr/Ad.Sal-rlTR (ECACC deposit P97082119) pBr/Ad.Bam-rlTR was digested with BamHI and Sail. The vector fragment including the adenovirus insert was isolated in LMP agarose (SeaPlaque GTG) and ligated to a 4.8 kb Sall-BamHI fragment obtained from wt Ad5 DNA and purified with the Geneclean II kit (Bio 101, Inc.) . One clone was chosen and the integrity of the Ad5 sequences was determined by restriction enzyme analysis. Clone pBr/Ad. Sal-rlTR contains adeno type 5 sequences from the Sail site at bp 16746 up to and including the rITR (missing the most 3 ' G residue) . 3. pBr/Ad.Cla-Bam (ECACC deposit P97082117) wt Adeno type 5 DNA was digested with Clal and BamHI, and the 20.6 kb fragment was isolated from gel by electro-elution. pBr322 was digested with the same enzymes and purified from agarose gel by Geneclean. Both fragments were ligated and transformed into competent DH5a. The resulting clone pBr/Ad.Cla-Bam was analysed by restriction enzyme digestion and shown to contain an insert with adenovirus sequences from bp 919 to 21566. 4. pBr/Ad.Aflll-Bam (ECACC deposit P97082114) Clone pBr/Ad.Cla-Bam was linearised with EcoRI (in pBr322) and partially digested with Aflll. After heat inactivation of Aflll for 20' at 65°C the fragment ends were filled in with Klenow enzyme. The DNA was then ligated to a blunt double stranded oligo linker containing a Pad site (5'- AATTGTCTTAATTAACCGCTTAA-3 ' ) . This linker was made by annealing the following two oligonucleotides: 5 1 - AATTGTCTTAATTAACCGC-3 ' and 5 ' -AATTGCGGTTAATTAAGAC-3 ' , followed by blunting with Klenow enzyme. After precipitation of the ligated DNA to change buffer, the ligations were digested with an excess Pad enzyme to remove concatameres of the oligo. The 22016 bp partial fragment containing Ad5 sequences from bp 3534 up to 21566 and the vector sequences, was isolated in LMP agarose (SeaPlaque GTG) , religated and transformed into competent DH5a. One clone that was found to contain the Pad site and that had retained the large adeno fragment was selected and sequenced at the 5' end to verify correct insertion of the Pad linker in the (lost) Aflll site. 5. pBr/Ad.Bam-rITRpac#2 (ECACC deposit P97082120) and pBr/Ad.Bam-rITR#8 (ECACC deposit P97082121) To allow insertion of a Pad site near the ITR of Ad5 in clone pBr/Ad.Bam-rlTR about 190 nucleotides were removed between the Clal site in the pBr322 backbone and the start of the ITR sequences. This was done as follows: pBr/Ad.Bam-rlTR was digested with Clal and treated with nuclease Bal31 for varying lengths of time (2 ' , 5', 10' and 15'). The extent of nucleotide removal was followed by separate reactions on pBr322 DNA (also digested at the Clal site) , using identical buffers and conditions. Bal31 enzyme was inactivated by incubation at 75°C for 10 minutes, the DNA was precipitated and resuspended in a smaller volume of TE buffer. To ensure blunt ends, DNAs were further treated with T4 DNA polymerase in the presence of excess dNTPs. After digestion of the (control) pBr322 DNA with Sail, satisfactory degradation (~150 bp) was observed in the samples treated for 10' or 15'. The 10' or 15' treated pBr/Ad.Bam-rlTR samples were then ligated to the above described blunted Pad linkers (See pBr/Ad.AflII-Bam) . Ligations were purified by precipitation, digested with excess Pad and separated from the linkers on an LMP agarose gel. After religation, DNAs were transformed into competent DH5a and colonies analysed. Ten clones were selected that showed a deletion of approximately the desired length and these were further analysed by T-track sequencing (T7 sequencing kit, Pharmacia Biotech) . Two clones were found with the Pad linker inserted just downstream of the rITR. After digestion with Pad, clone #2 has 28 bp and clone #8 has 27 bp attached to the ITR. pWE/Ad.Aflll-rlTR (ECACC deposit P97082116) Cosmid vector pWE15 (Clontech) was used to clone larger Ad5 inserts. First, a linker containing a unique Pad site was inserted in the EcoRI sites of pWE15 creating pWE.pac. To this end, the double stranded Pad oligo as described for pBr/Ad.Aflll-BamHI was used but now with its EcoRI protruding ends. The following fragments were then isolated by electro- elution from agarose gel: pWE.pac digested with Pad, pBr/AflII-Bam digested with Pad and BamHI and pBr/Ad.Bam- rITR#2 digested with BamHI and Pad. These fragments were ligated together and packaged using 1 phage packaging extracts (Stratagene) according to the manufacturers protocol. After infection into host bacteria, colonies were grown on plates and analysed for presence of the complete insert. pWE/Ad.Aflll-rlTR contains all adenovirus type 5 sequences from bp 3534 (Aflll site) up to and including the right ITR (missing the most 3 ' G residue) . pBr/Ad.HTR-Sal (9.4) (ECACC deposit P97082115) Adeno 5 wt DNA was treated with Klenow enzyme in the presence of excess dNTPs and subsequently digested with Sail. Two of the resulting fragments, designated left ITR-Sal(9.4) and Sal (16.7) -right ITR, respectively, were isolated in LMP agarose (Seaplaque GTG) . pBr322 DNA was digested with EcoRV and Sail and treated with phosphatase (Life Technologies) . The vector fragment was isolated using the Geneclean method (BIO 101, Inc.) and ligated to the Ad5 Sail fragments. Only the ligation with the 9.4 kb fragment gave colonies with an insert . After analysis and sequencing of the cloning border a clone was chosen that contained the full ITR sequence and extended to the Sail site at bp 9462. pBr/Ad.lITR-Sal(16.7) (ECACC deposit P97082118) pBr/Ad.HTR-Sal (9.4) is digested with Sail and dephosphorylated (TSAP, Life Technologies) . To extend this clone up to the third Sail site in Ad5 , pBr/Ad. Cla-Bam was linearised with BamHI and partially digested with Sail. A 7.3 kb Sail fragment containing adenovirus sequences from 9462- 16746 was isolated in LMP agarose gel and ligated to the Sail-digested pBr/Ad. HTR-Sal (9.4) vector fragment. pWE/Ad.Aflll-EcoRI pWE.pac was digested with Clal and 5' protruding ends were filled using Klenow enzyme. The DNA was then digested with Pad and isolated from agarose gel. pWE/Aflll-rlTR was digested with EcoRI and after treatment with Klenow enzyme digested with Pad . The large 24 kb fragment containing the adenoviral sequences was isolated from agarose gel and ligated to the Clal-digested and blunted pWE.pac vector using the Ligation Express kit from Clontech. After transformation of Ultracompetent XLIO-Gold cells from Stratagene, clones were identified that contained the expected insert. pWE/AflII-EcoRI contains Ad5 sequences from bp 3534-27336. Construction of new adapter plasmids The absence of sequence overlap between the recombinant adenovirus and El sequences in the packaging cell line is essential for safe, RCA-free generation and propagation of new recombinant viruses. The adapter plasmid pMLPI.TK (figure. 1) is an example of an adapter plasmid designed for use according to the invention in combination with the improved packaging cell lines of the invention. This plasmid was used as the starting material to make a new vector in which nucleic acid molecules comprising specific promoter and gene sequences can be easily exchanged. First, a PCR fragment was generated from pZipΔMo+PyFlOl (N ) template DNA (described in PCT/NL96/00195) with the following primers: LTR-1: 5'-CTG TAC GTA CCA GTG CAC TGG CCT AGG CAT GGA AAA ATA CAT AAC TG-3 ' and LTR-2 : 5 ' -GCG GAT CCT TCG AAC CAT GGT AAG CTT GGT ACC GCT AGC GTT AAC CGG GCG ACT CAG TCA ATC G-3 ' . Pwo DNA polymerase (Boehringer Mannheim) was used according to manufacturers protocol with the following temperature cycles: once 5' at 95°C; 3' at 55°C; and 1' at 72°C, and 30 cycles of 1 ' at 95°C, 1' at 60°C, 1' at 72°C, followed by once 10' at 72°C. The PCR product was then digested with BamHI and ligated into pMLPIO (Levrero et al . , 1991) vector digested with PvuII and BamHI, thereby generating vector pLTRlO. This vector contains adenoviral sequences from bp 1 up to bp 454 followed by a promoter consisting of a part of the Mo-MuLV LTR having its wild-type enhancer sequences replaced by the enhancer from a mutant polyoma virus (PyFlOl) . The promoter fragment was designated L420. Next, the coding region of the murine HSA gene was inserted. pLTRlO was digested with BstBI followed by Klenow treatment and digestion with Ncol . The HSA gene was obtained by PCR amplification on pUC18-HSA (Kay et al . , 1990) using the following primers: HSA1 , 5 ' -GCG CCA CCA TGG GCA GAG CGA TGG TGG C-3' and HSA2 , 5 ' -GTT AGA TCT AAG CTT GTC GAC ATC GAT CTA CTA ACA GTA GAG ATG TAG AA-3 ' . The 269 bp amplified fragment was subcloned in a shuttle vector using the Ncol and LO LO to to H μ> LΠ O LΠ o LΠ o LΠ TJ ι-3 Ω o 0 Φ PJ 0 ^ • CO LQ Φ 3 Φ Hj μ- 0 fu Φ rt 1 Hi μ- tr fu 0 ) rt 0 03 Φ Φ 0 Pi td Hi H to Hi *< Pi Φ CO Φ Ω rt 0 Φ Φ 3 3 rt fj 1 Φ μ- Pi ø rt fυ ø J Hj 0 Φ Φ rt Ω Hi 0 fu 0 Pi u Φ H- J μ- 0 0 ø 0 £ P> < μ- 0 μ- ø rt Hj LQ 0 fu 03 Ω Φ 0 Φ 03 0 0 CQ 0 rt <^ Hi μ- 0 H Ω 0 rt 03 03 φ CD PJ Hi ≤ Φ H- rt TJ " Hi Φ rt TJ ø J fu Φ Hi Φ 0 Φ a) An adapter construct containing the expression cassette with the gene of interest linearised with a restriction enzyme that cuts at the 3 ' side of the overlapping adenoviral genome fragment, preferably not containing any pBr322 vector sequences, and b) A complementing adenoviral genome construct pWE/Ad.AfIII- rlTR digested with Pad. These two DNA molecules are further purified by phenol/ chloroform extraction and EtOH precipitation. Co-transfection of these plasmids into an adenovirus packaging cell line, preferably a cell line according to the invention, generates recombinant replication deficient adenoviruses by a one-step homologous recombination between the adapter and the complementing construct (figure. 4) . Alternatively, in stead of pWE/Ad.Aflll-rlTR other fragments can be used, e.g., pBr/Ad. Cla-Bam digested with EcoRI and BamHI or pBr/Ad.AfIII -BamHI digested with Pad and BamHI can be combined with pBr/Ad.Sal-rlTR digested with Sail. In this case, three plasmids are combined and two homologous recombinations are needed to obtain a recombinant adenovirus (figure. 5) . It is to be understood that those skilled in the art may use other combinations of adapter and complementing plasmids without departing from the present invention. A general protocol as outlined below and meant as a non- limiting example of the present invention has been performed to produce several recombinant adenoviruses using various adapter plasmids and the Ad.Aflll-rlTR fragment. Adenovirus 2 packaging cells (PER.C6) were seeded in ~25 cm flasks and the next day when they were at ~80% confluency, transfected with a mixture of DNA and lipofectamine agent (Life Techn.) as described by the manufacturer. Routinely, 40 μl lipofectamine, 4 μg adapter plasmid and 4 μg of the complementing adenovirus genome fragment Aflll- rITR (or 2 μg of all three plasmids for the double homologous recombination) are used. Under these conditions transient transfection efficiencies of ~50% (48 hrs post transfection) are obtained as determined with control transfections using a pAd/CMV-LacZ adapter. Two days later, cells are passaged to 2 ~80 cm flasks and further cultured. Approximately five (for the single homologous recombination) to eleven days (for the double homologous recombination) later a cytopathogenic effect (CPE) is seen, indicating that functional adenovirus has formed. Cells and medium are harvested upon full CPE and recombinant virus is released by freeze-thawing. An extra 2 amplification step in an 80 cm flask is routinely performed to increase the yield since at the initial stage the titers are found to be variable despite the occurrence of full CPE. After amplification, viruses are harvested and plaque purified on PER.C6 cells. Individual plaques are tested for viruses with active transgenes . Besides replacements in the El region it is possible to delete or replace (part of) the E3 region in the adenovirus because E3 functions are not necessary for the replication, packaging and infection of the (recombinant) virus. This creates the opportunity to use a larger insert or to insert more than one gene without exceeding the maximum package size (approximately 105% of wt genome length) . This can be done, e.g., by deleting part of the E3 region in the pBr/Ad.Bam- rlTR clone by digestion with Xbal and religation. This removes Ad5 wt sequences 28592-30470 including all known E3 coding regions. Another example is the precise replacement of the coding region of gpl9K in the E3 region with a polylinker allowing insertion of new sequences. This, 1) leaves all other coding regions intact and 2) obviates the need for a heterologous promoter since the transgene is driven by the E3 promoter and pA sequences, leaving more space for coding sequences . To this end, the 2.7 kb EcoRI fragment from wt Ad5 containing the 5 ' part of the E3 region was cloned into the EcoRI site of pBluescript (KS ) (Stratagene) . Next, the Hindlll site in LO LO to to μ> μ> LΠ O LΠ o LΠ o LΠ Recombinant viruses that are both El and E3 deleted are generated by a double homologous recombination procedure as described above for El-replacement vectors using a plasmid- based system consisting of: a) an adapter plasmid for El replacement according to the invention, with or without insertion of a first gene of interest, b) the pWE/Ad.Aflll-EcoRI fragment, and c) the pBr/Ad.Bam-rITRΔgpl9K plasmid with or without insertion of a second gene of interest. In addition to manipulations in the E3 region, changes of (parts of) the E4 region can be accomplished easily in pBr/Ad.Bam-rlTR. Generation and propagation of such a virus, however, in some cases demands complementation in trans . Example 2 : Generation of adenovirus serotype 5 based viruses with chimaeric fiber proteins The method described infra to generate recombinant adenoviruses by co-transfection of two, or more separate cloned adenovirus sequences . One of these cloned adenovirus sequences was modified such that the adenovirus serotype 5 fiber DNA was deleted and substituted for unique restriction sites thereby generating ϊtemplate clones! which allow for the easy introduction of DNA sequences encoding for fiber protein derived from other adenovirus serotypes. Generation of adenovirus template clones lacking DNA encoding for fiber The fiber coding sequence of adenovirus serotype 5 is located between nucleotides 31042 and 32787. To remove the adenovirus serotype 5 DNA encoding fiber we started with construct pBr/Ad.Bam-rlTR. First a Ndel site was removed from this construct. For this purpose, pBr322 plasmid DNA was digested with Ndel after which protruding ends were filled using Klenow enzyme. This pBr322 plasmid was then re-ligated, digested with Ndel and transformed into E. coli DH5α. The obtained pBr/ΔNdel plasmid was digested with Seal and Sail and the resulting 3198 bp vector fragment was ligated to the 15349 bp Seal-Sail fragment derived from pBr/Ad.BamrlTR, resulting in plasmid pBr/Ad.Bam-rlTRΔNdel which hence contained a unique Ndel site . Next a PCR was performed with oligonucleotides NY-up: 5'- CGA CAT ATG TAG ATG CAT TAG TTT GTG TTA TGT TTC AAC GTG-3 ' And NY-down: 5 ' -GGA GAC CAC TGC CAT GTT-3 ' (figure 6) . During amplification, both a Ndel (bold face) and a Nsil restriction site (underlined) were introduced to facilitate cloning of the amplified fiber DNAs. Amplification consisted of 25 cycles of each 45 sec. at 94°C, 1 min. at 60°C, and 45 sec. at 72°C. The PCR reaction contained 25 pmol of oligonucleotides NY-up or NY-down, 2mM dNTP, PCR buffer with 1.5 mM MgCl 2 , and 1 unit of Elongase heat stable polymerase (Gibco, The Netherlands) . One-tenth of the PCR product was run on an agarose gel which demonstrated that the expected DNA fragment of ± 2200 bp was amplified. This PCR fragment was subsequently purified using Geneclean kit system (BiolOl Inc.). Then, both the construct pBr/Ad.Bam-rlTRΔNdel as well as the PCR product were digested with restriction enzymes Ndel and Sbfl. The PCR fragment was subsequently cloned using T4 ligase enzyme into the Ndel and Sbfl digested pBr/Ad.Bam- rITRΔNdel, generating pBr/Ad. BamRΔFib. This plasmid allows insertion of any PCR amplified fiber sequence through the unique Ndel and Nsil sites that are inserted in place of the removed fiber sequence. Viruses can be generated by a double homologous recombination in packaging cells described infra using an adapter plasmid, construct pBr/Ad.Aflll-EcoRI digested with Pad and EcoRI and a pBr/Ad.BamRΔFib construct in which heterologous fiber sequences have been inserted. To increase the efficiency of virus generation, the construct pBr/Ad.BamRΔFib was modified to generate a Pad site flanking the right ITR. Hereto, pBr/Ad.BamRΔFib was digested with 00 © o -1 H υ o n TJ- LO o o O H ro DNA encoding fiber protein was previously unknown and was therefore aligned with known sequences from other subgroup members to determine homology i.e. sequence divergence . Of the 51 human serotypes known to date, all fiber sequences, except for serotypes 1, 6, and 26, have been amplified and sequenced. The protein sequences of the fiber from different adenovirus serotypes is given in figure 7. Generation of fiber chimaeric adenoviral DNA constructs All amplified fiber DNAs as well as the vector (pBr/Ad.BamRΔ Fib) were digested with Ndel and Nsil. The digested DNAs was subsequently run on a agarose gel after which the fragments were isolated from the gel and purified using the Geneclean kit (BiolOl Inc) . The PCR fragments were then cloned into the Ndel and Nsil sites of pBr/AdBa RΔFib, thus generating pBr/AdBamRFibXX (where XX stands for the serotype number of which the fiber DNA was isolated) . So far the fiber sequence of serotypes 5/ 7/ 8/ 9/ 10/ 11/ 12/ 13/ 14/ 16/ 17/ 19/ 21/ 24/ 27/ 28/ 29/ 30/ 32/ 33/ 34/ 35/ 36/ 37/ 38/ 40-S/ 40-L/ 41-S/ 42/45/ 47/ 49/ 51 have been cloned into pBr/AdBamRFibXX. From pBr/AdBamRFibXX (where XX is 5/ 8/ 9/ 10/ 11/ 13/ 16/ 17/ 24/ 27/ 30/ 32/ 33/ 34/ 35/ 38/ 40-S/ 40- L/ 45/ 47/ 49/ 51) an 6 kb Avrll fragment encompassing the fiber sequence was isolated via gelelectrophoresis and Geneclean. This Avrll fragment was subsequently cloned in plasmid pBr/Ad.Bam-rlTR.pac (see example 1) which was digested to completion with Avrll and dephosphorylated as described previously, leading to the generation of the plasmid pBr/Ad.Bam-rlTR.pac . fibXX. This plasmid was subsequently used to generate a cosmid clone with a modified fiber using the constructs pWE.pac, pBr/AflII-Bam and pBr/Ad.Bam-rlTR.pac. fibXX. This cosmid cloning resulted in the formation of construct pWE/Ad.Aflll-rlTR/FibXX (where XX stands for the serotype number of which the fiber DNA was isolated) . Generation of pAd5/L420.HSA, pAd5/Clip and pAd5/Clipsal pMLPI.TK was used to make a new vector in which nucleic acid molecules comprising specific promoter and gene sequences can be easily exchanged. First, a PCR fragment was generated from pZipΔMo+PyFlOl (N ~ ) template DNA (described in PCT/NL96/00195) with the following primers: LTR-1: 5 ' -CTG TAC GTA CCA GTG CAC TGG CCT AGG CAT GGA AAA ATA CAT AAC TG-3 ' and LTR-2 : 5 ' -GCG GAT CCT TCG AAC CAT GGT AAG CTT GGT ACC GCT AGC GTT AAC CGG GCG ACT CAG TCA ATC G-3 ' . Pwo DNA polymerase (Boehringer Mannheim) was used according to manufacturers protocol with the following temperature cycles: once 5' at 95°C; 3' at 55°C; and 1' at 72°C, and 30 cycles of 1' at 95°C, 1' at 60°C, 1' at 72°C, followed by once 10' at 72°C. The PCR product was then digested with BamHI and ligated into pMLPIO (Levrero et al . , 1991; Gene 101, 195-202) digested with PvuII and BamHI, thereby generating vector pLTRlO. This vector contains adenoviral sequences from bp 1 up to bp 454 followed by a promoter consisting of a part of the Mo-MuLV LTR having its wild-type enhancer sequences replaced by the enhancer from a mutant polyoma virus (PyFlOl) . The promoter fragment was designated L420. Sequencing confirmed correct amplification of the LTR fragment however the most 5 ' bases in the PCR fragment were missing so that the PvuII site was not restored. Next, the coding region of the murine HSA gene was inserted. pLTRlO was digested with BstBI followed by Klenow treatment and digestion with Ncol. The HSA gene was obtained by PCR amplification on pUC18-HSA (Kay et al . , 1990; J. Immunol. 145, 1952-1959) using the following primers: HSA1 , 5' -GCG CCA CCA TGG GCA GAG CGA TGG TGG C-3 ' and HSA2 , 5 ' -GTT AGA TCT AAG CTT GTC GAC ATC GAT CTA CTA ACA GTA GAG ATG TAG AA-3 ' . The 269 bp amplified fragment was subcloned in a shuttle vector using the Ncol and Bglll sites. Sequencing confirmed incorporation of the correct coding sequence of the HSA gene, but with an extra TAG insertion directly following the TAG stop codon. The coding region of the HSA gene, 00 T o © o -J rrj υ CQ rrj TJ Φ 03 -H CJ X Φ 0 Φ A 4-J CQ r*ι «* 0 P • H 4-J (Tj CJ -H r-i Pi - T2J u < H Φ • A rH 4-J rrj 03 01 Pi 0 -H -H rH f> TJ U 0 ^^ rH LO o CJ J O 0 < -H Pi LO o LO O LO o LO H rH CN CN n ro Generation of pAd5ClipLacZ, pAd5Clip.Luc, pAd5Clip.TK and pAd5Clipsal .Luc The adapter plasmid pAd5/Clip. LacZ was generated as follows: The E.coli LacZ gene was amplified from the plasmid pMLP.nlsLacZ (EP 95-202 213) by PCR with the primers 5 * GGGGTGGCCAGGGTACCTCTAGGCTTTTGCAA and 5 ' GGGGGGATCCATAAACAAGTTCAGAATCC . The PCR reaction was performed Ex Taq (Takara) according to the suppliers protocol at the following amplification program: 5 minutes 94°C, 1 cycle; 45 seconds 94°C and 30 seconds 60°C and 2 minutes 72°C, 5 cycles; 45 seconds 94°C and 30 seconds 65°C and 2 minutes 72°C, 25 cycles; 10 minutes 72; 45 seconds 94°C and 30 seconds 60°C and 2 minutes 72°C, 5 cycles, I cycle. The PCR product was subsequently digested with Kpnl and BamHI and the digested DNA fragment was ligated into Kpnl/BamHI digested pcDNA3 (Invitrogen) , giving rise to pcDNA3.nlsLacZ . Next, the plasmid pAd5/Clip was digested with Spel. The large fragment containing part of the 5 ' part CMV promoter and the adenoviral sequences was isolated. The plasmid pcDNA3.nlsLacZ was digested with Spel and the fragment containing the 3 'part of the CMV promoter and the lacZ gene was isolated. Subsequently, the fragments were ligated, giving rise to pAd/Clip.LacZ . The reconstitution of the CMV promoter was confirmed by restriction digestion. The adapter plasmid pAd5/Clip.Luc was generated as follows: The plasmid pCMV.Luc (EP 95-202 213) was digested with Hindlll and BamHI. The DNA fragment containing the luciferase gene was isolated. The adapter plasmid pAd5/Clip was digested with Hindlll and BamHI, and the large fragment was isolated. Next, the isolated DNA fragments were ligated, giving rise to pAd5/Clip. Luc . The adapter pClipsal.Luc was generated in the same way but using the adapter pClipsal digested with HIII and BamHI as vector fragment. Likewise, the TK containing HIII-BamHI fragment from pCMV.TK (EP 95-202 213) was inserted in pClipsal to generate pAd5/Clip .TK. The presence of the Sail site just upstream of the left ITR enables liberation of vector sequences from the adeno insert . Removal of these vector sequences enhances frequency of vector generation during homologous recombination in PER.C6. Generation of recombinant adenovirus chimaeric for fiber protein To generate recombinant Ad 5 virus carrying the fiber of serotype 12, 16, 28, 40 -L, 51, and 5, three constructs, pCLIP.Luc, pWE/AdAflll-Eco and pBr/AdBamrlTR.pac/fibXX (XX = 12, 16, 28, 40-L, 51, and 5) were transfected into adenovirus producer cells. To generate recombinant Ad 5 virus carrying the fiber of 5/ 7/ 8/ 9/ 10/ 11/ 12/ 13/ 14/ 16/ 17/ 19/ 21/ 24/ 27/ 28/ 29/ 30/ 32/ 33/ 34/ 35/ 36/ 37/ 38/ 40-S/ 40-L/ 41-S/ 42/45/ 47/ 49/ 51, two constructs pCLIP.Luc and pWE/Ad.Aflll-rlTR/FibXX were transfected into adenovirus producer cells. For transfection, 2 μg of pCLIP.Luc, and 4 μg of both pWE/AdAflll-Eco and pBr/AdBamrlTR.pac/fibXX (or in case of cosmids: 4 μg of pCLIP.Luc plus 4 μg of pWE/Ad.AfIII- rlTR/FibXX) were diluted in serum free DMEM to 100 μl total volume. To this DNA suspension 100 μl lx diluted lipofectamine (Gibco) was added. After 30 minutes at room temperature the DNA-lipofectamine complex solution was added to 2.5 ml of serum-free DMEM which was subsequently added to 2 6 a T25 cm tissue culture flask. This flask contained 2x10 PER.C6 cells that were seeded 24-hours prior to transfection. Two hours later, the DNA-lipofectamine complex containing medium was diluted once by the addition of 2.5 ml DMEM supplemented with 20% foetal calf serum. Again 24 hours later the medium was replaced by fresh DMEM supplemented with 10% foetal calf serum. Cells were cultured for 6-8 days, subsequently harvested, and freeze/thawed 3 times. Cellular debris was removed by centrifugation for 5 minutes at 3000 rpm room temperature. Of the supernatant (12.5 ml) 3-5 ml was 2 used to infect again infect PER.C6 cells (T80 cm tissue culture flasks) . This re-infection results in full cytopathogenic effect (CPE) after 5-6 days after which the adenovirus is harvested as described above. Example 3 : Production, purification, and titration of fiber chimaeric adenoviruses Of the supernatant obtained from transfected PER.C6 cells typically 10 ml was used to inoculate a 1 litre fermentor which contained 1 - 1.5 x 10 6 cells/ ml PER.C6 that were specifically adapted to grow in suspension. Three days after inoculation, the cells were harvested and pelleted by centrifuging for 10 min at 1750 rpm at room temperature. The chimaeric adenoviruses present in the pelleted cells were subsequently extracted and purified using the following downstream processing protocol. The pellet was dissolved in 50 ml 10 mM NaP0 4 " and frozen at -20°C. After thawing at 37°C, 5.6 ml deoxycholate (5% w/v) was added after which the solution was homogenated. The solution was subsequently incubated for 15 minutes at 37°C to completely crack the cells. After homogenising the solution, 1875 μl (1M) MgCl 2 " was added and 5 ml 100% glycerol . After the addition of 375 μl DNase (10 mg/ ml) the solution was incubated for 30 minutes at 37°C. Cell debris was removed by centrifugation at 1880xg for 30 minutes at room temperature without the brake on. The supernatant was subsequently purified from proteins by loading on 10 ml of freon. Upon centrifugation for 15 minutes at 2000 rpm without brake at room temperature three bands are visible of which the upper band represents the adenovirus. This band was isolated by pipetting after which it was loaded on a Tris/HCl (1M) buffered caesiumchloride blockgradient (range: 1.2 to 1.4 gr./ml). Upon centrifugation at 21000 rpm for 2.5 hours at 10°C the virus was purified from remaining protein and cell debris since the virus, in contrast to the other components, does not migrate into the 1.4 gr./ ml caesiumchloride solution. The virus band is isolated after which a second purification using a Tris/ HC1 (1M) buffered continues gradient of 1.33 gr./ml of caesiumchloride is performed. After virus loading on top of this gradient the virus is centrifuged for 17 hours at 55000 rpm at 10°C. Subsequently the virus band is isolated and after the addition of 30 μl of sucrose (50 w/v) excess caesiumchloride is removed by three rounds of dialysis, each round comprising of 1 hour. For dialysis the virus is transferred to dialysis slides (Slide-a-lizer, cut off 10000 kDa, Pierce, USA) . The buffers used for dialysis are PBS which are supplemented with an increasing concentration of sucrose (round 1 to 3 : 30 ml, 60 ml, and 150 ml sucrose (50% w/v)/ 1.5 litre PBS, all supplemented with 7.5 ml 2% (w/v) CaMgCl 2 ) . After dialysis, the virus is removed from the slide-a-lizer after which it is aliquoted in portions of 25 and 100 μl upon which the virus is stored at -85°C. To determine the number of virus particles per millilitre, 100 μl of the virus batch is run on an high pressure liquid chromatograph (HPLC) . The adenovirus is bound to the column (anion exchange) after which it is eluted using a NaCl gradient (range 300-600 mM) . By determining the area under the virus peak the number of virus particles can be calculated. To determine the number of infectious units (IU) per ml present in a virus batch, titrations are performed on 911 cells. For this purpose, 4xl0 4 911 cells are seeded per well of 96-well plates in rows B, D, and F in a total volume of 100 μl per well. Three hours after seeding the cells are attached to the plastic support after which the medium can be removed. To the cells a volume of 200 μl is added, in duplicate, containing different dilutions of virus (range: 10 2 times diluted to 2xl0 9 ) . By screening for CPE the highest virus dilution which still renders CPE after 14 days is considered to contain at least one infectious unit. Using this observation, together with the calculated amount of virus volume present in these wells renders the number of infectious units per ml of a given virus batch. The production results i.e. virus particles per ml and IU per ml 2P8 or those chimaeric adenoviruses that were produced so far, are shown in table 3. Example 4: Presence of Ad5 Receptor molecules on human cells To investigate the importance of the presence of CAR on target cells for infection with chimaeric adenoviruses, a panel of human cell lines and primary cells were tested for the presence and/ or absence of CAR, MHC class I, and integrins (αvβ3 , αvβ5) . For this purpose, 1x10 s target cells or were transferred to tubes (4 tubes per cell type) designed for flow cytometry. Cells were washed once with PBS/ 0.5% BSA after which the cells were pelleted by centrifugation for 5 minutes at 1750 rpm at room temperature. Subsequently, 10 μl of a 100 times diluted α v β3 antibody (Mab 1961, Brunswick chemie, Amsterdam, The Netherlands) , a 100 times diluted antibody α v β5 (antibody (Mab 1976, Brunswick chemie, Amsterdam, The Netherlands) , or 2000 times diluted CAR antibody was a kind gift of Dr. Bergelson, Harvard Medical School, Boston, USA (Hsu et al) was added to the cell pellet after which the cells were incubated for 30 minutes at 4°C in a dark environment. After this incubation, cells were washed twice with PBS/0.5% BSA and again pelleted by centrifugation for 5 minutes at 1750 rpm room temperature. To label the cells, 10 μl of rat anti mouse IgGl labelled with phycoerythrin (PE) was added to the cell pellet upon which the cells were again incubated for 30 minutes at 4°C in a dark environment. Finally the cells were washed twice with PBS/0.5% BSA and analysed on a flow cytometer. The results of flow cytometric analysis of these experiments are shown in table 4. These results show that human erythroid leukemia cells (K562, ATCC: CCL-243), human primary fibroblasts (GM09503) , human primary smooth muscle cells, and primary human synoviocytes do not express detectable levels of the CAR receptor. In contrast, human lung carcinoma cells (A549, ATCC: CCL-1185) , human lymphoblast cells (SupTl (B and T cell hybrid, ATCC, CRL-1991) , and human liver cells (HEPG2, ATCC, HB8065) express high amounts of CAR protein. Human lymphoblast cells (CEM, ATCC: CRL-1992) , primary human umbilical vein endothelial cells (HUVEC) , and human primary chorion villi express low amounts of CAR protein. Example 5: Infection of CAR negative cells with fiber chimaeric adenovirus Several of the cell types described in example 4, i.e. A549, K562, GM09503, SupTl , chorion villi, and HepG2 were infected with a panel of chimaeric adenoviruses. This panel consists of adenovirus serotype 5 (subgroup C) , and of adenovirus serotype 5 containing the fiber of serotypes 16 and 51 (subgroup B) , of 28, 32, and 49 (subgroup D) , of 12 (subgroup A) , and of 40 (40-S and/or 40-L: subgroup F) . For this 5 purpose target cells are seeded at a concentration of 10 cells per well of 6-well plates in 2 ml Dulbecco ' s modified Eagles medium (DMEM, Life Technologies, The Netherlands) supplemented with 10% Foetal calf serum. Twenty-four hours later the medium is replaced by fresh medium containing the different chimaeric adenoviruses at an increasing MOI of 0, 10, 50, 250, 1250, 2500, 5000 (MOI based on virus particles per millilitre) . Approximately 2 hours after the addition of virus the medium containing the virus is discarded, cells are washed once with PBS, and subsequently 2 ml of fresh medium (not containing virus) is added to each well. 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These experiments show if the fiber protein of members of the same adenovirus subgroup recognise the same adherence molecules on a cell membrane. Naturally, this approach is also used to investigate inter-subgroup variation for example usage of adherence molecules by subgroup D and B members 2) Interference studies with fiber protein derived peptides Peptides of 6-12 amino acids are synthetically synthesised which together form the complete knob domain of a fiber from a subgroup D, for example 49. Next, one or more peptides are mixed in various concentrations with the fiber 49 chimaeric adenovirus after which the mixture is added to the cells. Using this approach one or more peptides are identified which block, at a certain concentration, the infection of the fiber 49 chimaeric adenovirus. This peptide or these peptides are subsequently used to investigate whether the infection of other subgroup D members is blocked by addition of the peptide (s) and whether inhibition of infection occurs using the same concentration of peptide. Identical to the example for subgroup D described above peptides are synthesised using the knob domain of a member of subgroup A, B, C, E, and F. These experiments show not only which adherence molecules are used but also which part of the fiber protein is directly involved in binding to target cells. Naturally these peptides are also used to investigate inter-subgroup variation. 3) Interference studies with baculovirus produced recombinant knob proteins Of each adenovirus subgroup, the knob region of one member is amplified by PCR. The forward oligonucleotide hybridises to the final repeat of the shaft part of the fiber just upstream of the start of the knob protein. 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TJ 03 0 rrj rrj Φ • 5 Oi SH rH rH 4H Xi CJ rH -H SH -H O 0 rl 4H rrj 4-J -H (TJ m (Tj -H ■ ri SH -H J XI Φ TJ Φ 03 r-i rrj 03 J CQ 4-1 4-J -H -H -H (Tj XI 4-J 0 > 03 υ rH 03 XI SH <H Φ 0 Pi φ Φ rrj -H Φ 0 -H 0 0 4-J 03 > 4-J 0 CQ • H P (Tj ■ H PH 4-J rrj 03 Φ -H rrj J -H 03 4-1 TJ 01 0 TJ rH X! 0 TJ -H φ CJ 03 P CQ (Tj -H 0 4-1 rrj 0 Pi -H rH SH Φ 4H 03 rrj 0 (Tj ■ ri Φ A 4-J 0 ■ ri r-H -H Φ rH 1 ^^ 4-J K TJ φ rH -H 0 XI M 0 Oi XI X -H SH ■ ri 0 (Tj Xi EH -H rrj Oi rl >1 CJ SH 0 (Tj Φ rrj Φ TJ r- J SH S Φ 0 φ -H -H 40 Φ U Pi 4H 4-J • H X 0 4-J ε I XI " CJ TJ N 0 Φ 03 rrj Φ 0 tn 4-J rl 4-J rrj rH H > ε 0 XI _ Oi . -H 03 0 r-i 1 φ 0 03 X rrj Φ rl XI 0 5 Φ Φ 0 01 (Tj r H Φ 4-> 03 Φ 0 SH ^^ SH Φ SH 0 XI SH 0 φ Φ A LD Φ Φ 4-J 4H J ■ 8 4-J -H X) SH P >-ι TJ • H -— - SH -H 0 03 0 En Φ 01 SH 1 SH Pi 1 0 03 -rl CO (Tj 0 4-J CO (Tj φ -H Φ Φ rrj xi 4H Pi 0 ^ - ^ X! -H 03 4-J 4H ø tn rH rrj tn TJ 03 03 -H rH rl TJ CQ φ . Φ >i 4-J rH rrj 0 0 CQ 0 4-J < • • to -H 0 TJ Pi (Tj Φ 4H ■ ri , (Tj 40 TJ Φ CQ 4-J r-i 0 SH rrj • 4H ■ ri 0 4-J CQ t^ 4-J rrj 40 tn 03 > 0 4-1 03 S 0 -rH 0 TJ Φ rrj 0 0 03 -H 0 J 0 φ 03 φ -H Pi rl CQ 0 0 Φ -H CQ (Tj Pi — Φ 4-J tr φ Oi rt Pi 0 Φ 0 > -H Φ X! -H 0 X ø Φ ■ H Φ 01 φ -H 0 TJ Φ xi 03 Φ Φ - CQ ■ ri 0 ,0 4 CQ 40 0 tr Φ 0 H > ε Pi rrj CJ rrj φ -H > 0 rt 13 rrj TJ rl Pi TJ 01 xi Φ rH 0 XI 0 rrj o 4-J rl (Tj <-i $ υ Φ 01 φ r-i SH P. o 0 -H φ 0 X! Φ -H XI φ -H 0 CJ Pi Pi φ Pi φ (Tj φ 4-J 0 0 Φ -i 0 TJ 4H rH Oi tn 40 u η X! 40 -H • H tn X! 4-J > 0 CJ U 4H CJ ε 0 CQ 0 CJ X) & tn -H ε XI ro Φ 0 4H rH rrj Pi -H g 4-1 Pi X SH φ TJ tr. Φ Φ 4-J 0 φ 0 rrj Φ 0 >1 rt Φ 0 0 ø X ) 0 • H -H 0 ^ ~. X! Φ X! Φ -H Φ ■ ri 0 XI 0 φ X! TJ ■ ri ε rl CJ X) rt -H CJ 4H rt 03 CJ CQ W id EH XI TJ 4H rλ Oi Pi ε • 5 Pi ε Φ 40 rrj CQ 4-J S LO O LO O LO o LO H r-i CN CN ro ro LO O LO o LO o LO rH H CN CN ro ro electrophoresis or Malditoff of a purified batch of adenovirus only. As a negative control for the above described experiments cells negative for infection with a chimaeric adenovirus carrying a fiber of a member of subgroup D can be used. Alternatively, cells which are highly transducible with chimaeric adenoviruses carrying the fiber protein of members of for example subgroup D, are first treated with peptidases after which the medium is incubated with adenoviruses bound to a plastic support. The above described examples encompasses the construction of recombinant adenoviral vectors chimaeric for the fiber protein which results in an altered infection hostrange. The alteration of the infection host range results in highly efficient infection of cells negative for the CAR protein which is the protein required by adenovirus serotype 5 for efficient infection. These vectors are generated for the purpose of gene transfer and recombinant DNA vaccines. These vectors are thus ideally suited for gene transfer to tissues, and/or organs of which de cells do not express detectable levels of CAR. Figure and table legends Table 1 : Association of human adenovirus serotypes with human disease. Table 2: Oligonucleotides and degenerate oligonucleotides used for the amplification of DNA encoding for fiber protein derived from alternative human adenovirus serotypes. Bold letters in oligonucleotides A-E represent an Ndel restriction site. Bold letters in oligonucleotides 1-6 and 8 represent an Nsil restriction site. Bold letters in oligonucleotide 7 represents a Pad restriction site. Table 3: Production results of fiber chimaeric adenoviruses. The number of virus particles per ml were determined using HPLC. The number of infectious units (IU) per millilitre were determined through titration on human 911 cells. For infection experiments, the number of virus particles per millilitre is taken from all chimaeric adenoviruses since IU/ ml reflects a receptor mediated process. Table 4: Flow cytometric results on expression of integrins α v β3 and α v β5, the Coxsacki adenovirus receptor (CAR) , and MHC class I on the membranes of human cell lines and human primary cells. A549: Human lung carcinoma cell line (ATCC, CCL-1185) . K562: Human erythroid leukemia (ATCC, CCL-243). SupTl: Human Lymphoblast hybrid B and T (ATCC, CRL-1991) . GM09503: Human primary fibroblasts . HEPG2 : Human liver carcinoma (ATCC, HB8065) . CEM: human lymphoblast cells (ATCC, CRL-1992) . HeLa : Human cervix carcinoma (ATCC, CCL-2) . Primary amniocytes and chorion villi cells were obtained from department of antropogenetics, Leiden, The Netherlands. Primary Smooth muscle cells, Human umbilical vein endothelial cells, and synoviocytes were obtained from TNO-PG, Leiden, The Netherlands. Shown is the percentage of cells expressing either molecule on their membrane. ND: not determined. 0% means undetectable expression of the molecule on the membrane of the cell using flow cytometry. 100% means high expression of the molecule on the cell membrane. Figure 1: Schematic presentation of adapter plasmid pMLPI.TK. Figure 2: Schematic presentation of adapter plasmid pAd/L420- HAS. Figure 3: Schematic presentation of adapter plasmid pAd5/CLIP Figure 4 : Schematic presentation of plasmid system which requires only one recombinational event to generate recombinant adenoviruses . Figure 5 : Schematic presentation of plasmid system which requires two recombinational events to generate recombinant adenoviruses . Figure 6: Schematic presentation of generation of plasmid pBr/AdBamRDeltaFib in which the Adenovirus type 5 fiber DNA is replaced by a short DNA stretch containing an unique Nsil site . Figure 7: Fiber protein sequences of adenovirus serotypes 8, 9, 13, 14,20, 23, 24, 25, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 42, 43, 44, 45, 46, 47, 48, 49, and 51. Bold letters represent part of the tail of adenovirus serotype 5. If bold letters not present it means that a PCR fragment was sequenced which does not contain the Ad5 tail. An X, present in the sequence means unidentified amino acid due to unidentified nucleotide. At the end of the sequence the stop codon of the fiber is presented by a dot. Figure 8: Transduction of human lung carcinoma cells (A549) with a panel of chimaeric adenoviruses carrying the fiber of adenovirus 12, 16, 17, 28, 32, 40-L, or 51. Adenovirus 5 served as reference. Cells were infected with increasing MOI based on virus particles per cell: 10, 50, 250, 1250, 2500 (see legend on the right of graph) . Luciferase transgene expression is expressed as relative light units (RLU) per μg of protein. Figure 9 : Transduction of human erythroid leukemia cells (K562) with a panel of chimaeric adenoviruses carrying the fiber of adenovirus 12, 16, 28, 32, 40-S, 40-L, 49, or 51. Adenovirus 5 served as reference. Cells were infected with increasing MOI based on virus particles per cell: 10, 50, 250, 1250, 2500, 5000 (see legend on the right of graph) . Luciferase transgene expression is expressed as relative light units (RLU) per μg of protein. Error bars represent SD. Figure 10: Transduction of human primary fibroblasts (GM09503) with a panel of chimaeric adenoviruses carrying the fiber of adenovirus 12, 16, 28, 32, 40-L, 49, or 51. Adenovirus 5 served as reference. Cells were infected with increasing MOI based on virus particles per cell: 10, 50, 250, 1250, 2500, 5000 (see legend on the right of graph) . Luciferase transgene expression is expressed as relative light units (RLU) per μg of protein. Error bars represent SD . Figure 11: Transduction of human lymphoblast cells (SupTl) with a panel of chimaeric adenoviruses carrying the fiber of adenovirus 12, 16, 28, 32, 40-S, 40-L, 49, or 51. Adenovirus 5 served as reference. Cells were infected with increasing MOI based on virus particles per cell: 10, 50, 250, 1250, 2500, 5000 (see legend on the right of graph) . Luciferase transgene expression is expressed as relative light units (RLU) per μg of protein. Error bars represent SD. Figure 12: Transduction of human chorion villi cells with a panel of chimaeric adenoviruses carrying the fiber of adenovirus 12, 16, 28, 32, 40-L, 49, or 51. Adenovirus 5 served as reference. Cells were infected with increasing MOI based on virus particles per cell: 10, 50, 250, 1250, 2500, 5000 (see legend on the right of graph) . Luciferase transgene expression is expressed as relative light units (RLU) per μg of protein. Error bars represent SD. Figure 13: Transduction of human hepatic cells (HEPG2) with a panel of chimaeric adenoviruses carrying the fiber of adenovirus 12, 16, 28, 32, 40-S, 40-L, 49, or 51. Adenovirus 5 served as reference. Cells were infected with increasing MOI based on virus particles per cell: 10, 50, 250, 1250, 2500, 5000 (see legend on the right of graph) . Luciferase transgene expression is expressed as relative light units (RLU) per μg of protein. Error bars represent SD. REFERENCES Arnberg N. , Mei Y. and adell G. , 1997. Fiber genes of adenoviruses with tropism for the eye and the genital tract . Virology 227: 239-244. Bout A., 1997. Gene therapy, p. 167-182. In: D.J.A. Crommelin and R.D. Sindelar (ed.), Pharmaceutical biotechnology , Harwood Academic Publishers. Bout, A. 1996. Prospects for human Gene therapy. Eur. J. drug Met. And Pharma. 2, 175-179. Blaese et al . , Cancer Gene Ther., 2 (1995) : 291-297) . Brody and Crystal, Ann. N. Y. Acad. Sci . 716 (1994) : 90-101. 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Molecular characterization of the type-specific g-determinant located on the adenovirus fiber. Journal of Virology 71: 6576-6581. Francki, R.I.B., Fauquet, CM., Knudson, D.L. and Brown, F. (1991) Classification and nomenclature of viruses. Fifth report of the international Committee on taxonomy of viruses . Arch. Virol. Suppl . 2, 140-144 GahEry-SEgard H., Farace F., Godfrin D., Gaston J. , Lengagne R., Tursz T., Boulanger P. and Guillet J. , 1998. Immune response to recombinant capsid proteins of adenovirus in humans: antifiber and anti-penton base antibodies have a synergistic effect on neutralizing activity. Journal of Virology 72: 2388-2397. Gall J., Kass-Eisler A., Leinwand L. and Falck-Pedersen E., 1996. Adenovirus type 5 and 7 capsid chimera: fiber replacement alters receptor tropism without affecting primary immune neutralisation epitopes. Journal of Virology 70 (4) : 2116-2123. Greber, U.F., Willets, M. , Webster, P., and Helenius, A. (1993) . 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Schulick, A.H., Vassalli, C, Dunn, P.F., Dong, G. , Rade, J.J., Zamarron, C. and Dichek, D.A. (1997). Established immunity precludes adenovirus-mediated gene transfer inrat carotid arteries . Schnurr, D and Dondero, M.E. (1993) Two new candidate adenovirus serotypes Intervirol . 36, 79-83 Svensson, V. and Persson, R. (1984) . Entry of adenovirus 2 into Hela cells. J. Virol. 51, 687-694. Varga, M.J., Weibull, C, and Everitt, E. (1991). Infectious entry pathway of adenovirus type 2. J. Virol 65, 6061-6070. Wickham T.J., Carrion M.E. and Kovesdi I., 1995. Targeting of adenovirus penton base to new receptors through replacement of its RGD motif with other receptor-specific peptide motifs. Gene Therapy 2: 750-756. Wickham T.J., Segal, D.M. , Roelvink, P.W., Carrion M.E., Lizonova, A., Lee, G-M. , and Kovesdi, I. (1996). Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies. J. Virol. 70 (10), 6831-6838 Wickham, T.J., Mathias, P., Cherish, D.A. , and Nemerow, G.R. (1993) Integrins avb3 and avb5 promote adenovirus internalisation but not virus attachment. Cell 73, 309-319. Tables and figures Table 1 Syndrom Subgenus Serotype Respiratory illness A 31 B 3, 7,11,14,21,34,35,51 C 1, 2, 5, 6 D 39, 42-48 E 4 Keratoconjunctivitis (eye) B 11 D 8, 19, 37, 50 Hemorrhagic cystitis (Kidney) B 7,11, 14,16,21,34,35 And urogenital tract infections C 5 D 39, 42-48 Sexual transmission C 2 D 19,37 Gastroenteritis A 31 B 3 C 1,2,5 D 28 F 40,41 CNS disease A 12,31 B 3,7 C 2,5,6 D 32,49 Hepatitis A 31 C 1,2,5 Disseminated A 31 B 3,7,11,21 D 30, 43-47 None (???) A 18 D 9.10.13.1517.20.22-29.33.36.38 Table 2 Serotype Tail oligonucleotide Knob oligonucleotide A 1 8 B 2 9 B 2 12 E 3 16 C 4 19p B 2 28 B 2 32 B 2 36 B 2 37 B 2 40-1 D 5 40-2 D 6 41-S D 5 41-1 D 7 49 B 2 50 B 2 51 C 8 A: 5 - CCC GTG TAT CCA TAT GAT GCA GAC AAC GAC CGA CC- 3 ' B: 5 - CCC GTC TAC CCA TAT GGC TAC GCG CGG- 3 ' C: 5 - CCK GTS TAC CCA TAT GAA GAT GAA AGC- 3 ' D: 5 - CCC GTC TAC CCA TAT GAC ACC TYC TCA ACT C- 3 ' E: 5 - CCC GTT TAC CCA TAT GAC CCA TTT GAC ACA TCA GAC- 3 ' 1: 5 ' - CCG ATG CAT TTA TTG TTG GGC TAT ATA GGA - 3 ' 2 : 5 - CCG ATG CAT TYA TTC TTG GGC RAT ATA GGA - 3 ' 3 : 5 - CCG ATG CAT TTA TTC TTG GGR AAT GTA WGA AAA GGA - 3 ' 4: 5 - CCG ATG CAT TCA GTC ATC TTC TCT GAT ATA - 3 ' 5: 5 - CCG ATG CAT TTA TTG TTC AGT TAT GTA GCA - 3 ' 6: 5 - GCC ATG CAT TTA TTG TTC TGT TAC ATA AGA - 3 ' 7: 5' - CCG TTA ATT AAG CCC TTA TTG TTC TGT TAC ATA AGA A - 3 8: 5' - CCG ATG CAT TCA GTC ATC YTC TWT AAT ATA - 3 ' Table 3 Table 4

Claims: Claims 1. A method for delivering a nucleic acid of interest to a host cell by means of a gene delivery vehicle based on adenoviral material, whereby said gene delivery vehicle delivers the nucleic acid to the host cell by associating with a binding site and/or a receptor present on CAR-negative cells, said binding site and/or receptor being a binding site and/or a receptor for adenovirus subgroups D and/or F. 2. Use of a gene delivery vehicle comprising a nucleic acid of interest and comprising adenoviral material involved in binding to a host cell, said material being from a subgroup D and/or F adenovirus, in delivering said nucleic acid of interest to a CAR-negative cell. 3. A gene delivery vehicle being a chimaera based on at least two adenoviruses, whereby a cell recognising element of said gene delivery vehicle is based on adenoviral material from a subgroup D and/or F adenovirus, which material confers the capability of infecting CAR negative cells. 4. A gene delivery vehicle according to claim 3, wherein said adenoviral material is based on a fiber, a penton and/or a hexon protein of a subgroup D and/or subgroup F adenovirus. 5. A gene delivery vehicle according to claim 3 or 4 , further comprising an element from adenovirus 35, responsible for at least partially avoiding an immune response against adenovirus 35. 6. A gene delivery vehicle according to any one of claims 3- 5, which comprises an element of adenovirus 16 or a functional analogue thereof, which element confers said virus with an enhanced capability to infect smooth muscle cells and/or synoviocytes . 7. A gene delivery vehicle according to any one of claims 3- 6, comprising a nucleic acid derived from an adenovirus. 8. A gene delivery vehicle according to any one of claims 3- 7, comprising a nucleic acid derived from at least two different adenoviruses. 9. A gene delivery vehicle according to claim 7 or claim 8, wherein said nucleic acid comprises at least one sequence encoding a capsid protein comprising at least a tissue tropism determining fragment of a subgroup D and/or subgroup F adenovirus capsid protein. 10. A gene delivery vehicle according to any one of claims 7- 9, wherein said nucleic acid derived from adenovirus is modified such that the capacity of said nucleic acid to replicate in a target cell has been reduced or disabled. 11. A gene delivery vehicle according to any one of claims 7- 10, wherein said nucleic acid derived from adenovirus is modified such that the capacity of a host immune system to mount an immune response against adenovirus proteins encoded by said nucleic acid derived from adenovirus has been reduced or disabled. 12. A gene delivery vehicle according to anyone of claims 7- 11, comprising a minimal adenovirus vector or an integrating adenovirus such as an Ad/AAV chimaeric vector, a retro- adenovirus or a transposon-adenovirus . 13. A gene delivery vehicle according to anyone of the claims 1-12, further comprising at least one non-adenovirus nucleic acid. 14. A gene delivery vehicle according to anyone of claims 7- 13, wherein said nucleic acid derived from adenovirus is produced by welding together through homologous recombination two nucleic acid molecules comprising partially overlapping sequences wherein said overlapping sequences allow essentially only one homologous recombination which leads to the generation of a physically linked nucleic acid comprising at least two functional adenovirus inverted terminal repeats, a functional encapsulation signal, a nucleic acid of interest, or functional parts, derivatives and/or analogues thereof . 15. A cell for the production of a gene delivery vehicle according to anyone of the claims 3-14, comprising means for the assembly of said gene delivery vehicle wherein said means includes a means for the production of an adenovirus capsid protein, wherein said capsid protein comprises at least a receptor and/or binding site binding fragment of a subgroup D and/or subgroup F adenovirus capsid protein. 16. A cell according to claim 15, wherein said cell is or is derived from a PER.C6 cell (ECACC deposit number 96022940) . 17. The use of a gene delivery vehicle according to anyone of the claims 1-14 as a pharmaceutical. 18. A receptor and/or a binding site for adenoviruses type D and/or F, present on or associated with CAR negative cells. 19. A receptor and/or a binding site according to claim 18, present on K562 cells, amniotic fluid cells and/or primary fibroblast cells. 20. A capsid protein derived from a subgroup D and/or a subgroup F adenovirus or a functional part, derivative and/or analogue thereof . 21. A capsid protein according to claim 20, wherein said protein is a fiber protein. 22. An isolate and/or recombinant nucleic acid encoding a capsid protein according to claim 20 or claim 21. 23. An isolate and/or recombinant nucleic acid according to claim 22, wherein said nucleic acid comprises a sequence as depicted in figure 7. Title: Infection with chimaeric adenoviruses of cells negative for the adenovirus serotype 5 Coxsacki adenovirus receptor (CAR) . Abstract The invention relates to the field of molecular genetics and medicine. The invention discloses a method for delivering a nucleic acid of interest to a host cell by means of a gene delivery vehicle based on adenoviral material, whereby said gene delivery vehicle delivers the nucleic acid to the host cell by associating with a binding site and/or a receptor present on CAR-negative cells, said binding site and/or receptor being a binding site and/or a receptor for adenovirus subgroups D and/or F.

Inventors: HAVENGA MENZO, VOGELS RONALD, HAVENGA, MENZO, VOGELS, RONALD
Applicants: HAVENGA MENZO, INTROGENE BV, VOGELS RONALD, HAVENGA, MENZO, INTROGENE B.V., VOGELS, RONALD
Application year: 2000
Main classification: C12N15
IPC codes: A61K48, C07K14, C12N5, C12N15