510152025303540455055DescriptionFIELD OF THE INVENTION[0001] The invention relates to the field of medicine, more in particular to the field of biotechnology, wherein genedelivery vehicles based on replication-defective recombinant adenoviruses are used in medicaments.BACKGROUND OFTHE INVENTION[0002] Adenovirusesare presently widely usedand tested in medical applications. Typically, recombinant adenovirusesare made replication-defective by deletion ofone or more genes thatare essential for replication fromthe viral backbone,rendering the virus incapable of replicating ina host cell. Generally, recombinant adenoviruses are made replication-defective by removal of most if not all ofthe early region-1 (E1) part ofthe viral genome. Such viruses can be producedin so-called packaging cell systems, whereina cell is used that has the E1 of an adenovirus stably incorporated into itscellular genome, thereby providingthe missing, but for replication essential, E1 function tothe virus replication machinery.Examples of packaging cells that are used in the art are the human kidney-derived293 cells, 911 cells, and the humanretinoblast-derived PER.C6 cells.[0003] It was recognized in the field of adenoviral vectors that many individuals around the world become infectedwith many ofthe generally studied and produced recombinant adenovirus serotypes, exemplified by Ad2 and Ad5. Itwas found that onlya relative small percentage of individuals were infected witha number of serotypes, that, for thatreasons were referred to as ’rare’ serotypes. Examples of such rare serotypes are Ad1 1, Ad34, Ad35, Ad48, Ad49andAd50. Recombinant vectors based on such viruses encounter titers of neutralizing antibodies only ina small percentageof human beings around the world, thereby making such vectors useful in the application of vaccines or gene therapymedicaments, in which the presence of neutralizing antibodies is unwanted. Also in prime-boost set ups in vaccine-related therapies it is preferred to use recombinant adenoviral vectors that do not encounter neutralizing antibodies inthe host, for instance due toa previous infection, ora preceding prime-vaccination.[0004] In the field of adenoviral technology, one has always looked for ways to incorporatea large variety of transgenesor nucleic acids encoding antigens. Ata certain stage, itwas realizedthatthe capacity ofthe adenoviral vector to becomepackaged ina packaging system, was limited. The size of the genome that can actually be packaged is limited. It isgenerally believed that for instance for Ad5, the genome cannot be larger than 105% of its wild type size. Also in thelow-neutralized vector based on Ad35, the maximum size of the genome is around 105% to 106% (the Ad35 geneencoding the fiber is smaller than theAd5 fiber gene).[0005] Already in the early days of recombinant adenoviral technology, one realized that certain parts of the viralgenome could be left out, without affecting the virus, the replication and the packaging thereof. One ofthe main regionsthat appeared to be redundant for many ofthe applications in gene therapy and/or vaccination, was the early region3(E3) inthe viral backbone. One ofthe major accomplishments inthe field of adenoviral technologywas the manufacturingofa recombinant vector in whicha transgenewas incorporated, which transgenewas expressed fromthe viral backbone,after infection ofthe virus in cells (be it in vitro or in vivo). Typically, the transgene was placed in an expression cassettecomprisinga promoter, followed by the transgene and terminated bya poly-adenylation (pA) signal. The promoterwaseither adenoviral derived, such as the Major Late Promoter, orthe E1 promoter, that originally was present to expressthe E1 proteins. Also heterologous promoters were applied, such as the Rous Sarcoma Virus (RSV) promoter ortheCytomegalovirus (CMV) promoter, the latter being preferred because of its high expression levels. The expressioncassettes typically comprise multiple cloningsites enablingone to clone different kinds of transgenes, or antigen-encodingnucleic acids. Also, the pA signal is generally heterologous tothe backbone vector, where the SV40 andthe BovineGrowth Hormone (BGH) pA signals are preferred.[0006] The deletion ofthe E3 region enabled one to clone larger nucleic acids of interest into the viral genome, as itsimply provided more space and thus, the possibility to get packaged. This system was further developed into settingswhere almost all adenoviral DNA was removed, in so-called minimal vectors, but since many ofthe essential componentsof the adenovirus could not be stably integrated into the genome of packaging cells (generally due to toxicity issues),such minimal vectors requirethe co-infection of helper viruses, making those systems not highlysuitable for large-scaleproduction, which is required when one intends to produce vaccines or gene therapy products for worldwide use.[0007] It was then realized that for certain particular applications, it would be helpful to incorporate multiple antigenicdeterminants, as some diseases are not treated by justone antigenic response, orone gene of interest in gene therapy.For instance, infectious diseases suchas malariaare characterized in thatthe parasite that causesthe infection, proceedsthrough multiple stages in its life-cycle, being present in different compartments within the infected host during thosedifferent stages. It is known that different antigens are expressed during different phases of that life cycle. It wouldtherefore be beneficial to attack the parasite not only when it is present in for instance the liver stage, but also when itis floating around in the bloodstream during the pre-liver stage. The vaccination against sucha parasite would benefit2
