Crucell Holland B.v.

Menzo Havenga

Ronald Vogels

Abraham Bout

Majid Mehtali

Maria Grazia Pau

Lennart Holterman

Stefan Kostense

Mieke Caroline Sprangers

Adenovirus serotypes differ in their natural tropism. The adenovirus serotypes 2, 4, 5 and 7 all have a natural affiliation towards lung epithelia and other respiratory tissues. In contrast, serotypes 40 and 41 have a natural affiliation towards the gastrointestinal tract. The serotypes described, differ in at least capsid proteins (penton-base, hexon), proteins responsible for cell binding (fiber protein), and proteins involved in adenovirus replication. This difference in tropism and capsid protein among serotypes has led to the many research efforts aimed at redirecting the adenovirus tropism by modification of the capsid proteins.

Serotype of adenovirus and uses thereof

The present invention provides methods and vector systems for the generation of chimeric recombinant adenoviruses. These hybrid adenoviruses contain a genome that is derived from different adenovirus serotypes. In particular, novel hybrid adenoviruses are disclosed with improved properties for gene therapy purposes. These properties include: a decreased sensitivity towards neutralizing antibodies, a modified host range, a change in the titer to which adenovirus can be grown, the ability to escape trapping in the liver upon in vivo systemic delivery, and absence or decreased infection of antigen presenting cells (APC) of the immune system, such as macrophages or dendritic cells. These chimeric adenoviruses thus represent improved tools for gene therapy and vaccination since they overcome the limitations observed with the currently used serotype subgroup C adenoviruses.

Chimeric adenoviruses

The present invention provides new uses of recombinant adenoviral vectors in vaccination regimens, such as prime/boost set-ups and subsequent vaccinations and applications for gene therapy. Moreover, the invention provides new assays to determine the best regimen for applying the most suitable recombinant viral vector in a vaccination or gene therapy setting.

Settings for recombinant adenoviral-based vaccines

The invention provides a gene delivery vehicle and a gene of interest comprising at least one Ad35 element or a functional equivalent thereof, responsible for avoiding or diminishing neutralizing activity against adenoviral elements by the host to which the gene is to be delivered. A functional equivalent/homologue of an Ad35 (element) includes an adenovirus (element) which, like adenovirus 35, encounters pre-existing immunity in less than about 10% of the hosts to which it is administered for the first time, or which is capable in more than about 90% of the hosts to which it is administered of avoiding or diminishing the immune response.

A packaging cell line capable of complementing recombinant adenoviruses based on serotypes from subgroup B, preferably adenovirus type 35. The cell line is preferably derived from primary diploid human cells transformed by adenovirus E1 sequences either operatively linked on one or two DNA molecules, the sequences operatively linked to regulatory sequences enabling transcription and translation of encoded proteins. Also, a cell line derived from PER.C6 that expresses functional Ad35-E1B sequences. The Ad35-E1B sequences are driven by the E1B promoter and terminated by a heterologous poly-adenylation signal. The new cell lines are useful for producing recombinant adenoviruses. The cell lines can be used to produce human recombinant therapeutic proteins such as human antibodies. In addition, the cell lines are useful for producing human viruses other than adenovirus such as influenza, herpes simplex, rotavirus, and measles.

Complementing cell lines

Adenovirus serotypes differ in their natural tropism. The adenovirus serotypes 2, 4, 5, and 7 all have a natural affiliation towards lung epithelia and other respiratory tissues. In contrast, serotypes 40 and 41 have a natural affiliation towards the gastrointestinal tract. The serotypes described, differ in at least capsid proteins (penton-base, hexon), proteins responsible for cell binding (fiber protein), and proteins involved in adenovirus replication. This difference in tropism and capsid protein among serotypes has led to the many research efforts aimed at redirecting the adenovirus tropism by modification of the capsid proteins.

A packaging cell line capable of complementing recombinant adenoviruses based on serotypes from subgroup B, preferably adenovirus type 35. The cell line is preferably derived from primary, diploid human cells transformed by adenovirus E1 sequences either operatively linked on one or two DNA molecules, the sequences operatively linked to regulatory sequences enabling transcription and translation of encoded proteins. Also, a cell line derived from PER.C6 that expresses functional Ad35 E1B sequences. The Ad35-E1B sequences are driven by the E1B promoter and terminated by a heterologous poly-adenylation signal. The new cell lines are useful for producing recombinant adenoviruses. The cell lines can be used to produce human recombinant therapeutic proteins such as human antibodies. In addition, the cell lines are useful for producing human viruses other than adenovirus such as influenza, herpes simplex, rotavirus, and measles.

The present invention relates to a gene delivery vehicle comprising a recombinant adenovirus having a tropism for a primary human chondrocyte. By efficiently transducing a nucleic acid of interest into a primary chondrocytes, the gene delivery vehicle is able to at least in part improve the counteraction of cartilage disease. In one embodiment the recombinant adenovirus comprises a deletion in the gene encoding for fiber protein, which is replaced by a nucleic acid sequence encoding at least part of a fiber protein of a B-type adenovirus.

Gene delivery vectors with cell type specificity for primary human chondrocytes

A packaging cell line capable of complemention recombinant adenoviruses based on serotypes from subgroup B, preferably adenovirus type 35. The cell line is preferably human embryonic kidney cells and primary human amniocytes) which are transformed by adenovirus E1 sequences either operatively linked on one DNA molecule or located on two separate DNA molecules, the sequences being operatively linked to regulatory sequences enabling transcription and translation of encoded proteins.

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Alphen aan den Rijn, Netherlands

Menzo Havenga