Defective adenoviruses and corresponding complementation lines

Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...

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4353201, 435456, 424 932, C12N 510, C12N 1586, C12N 1534

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060401745

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BRIEF SUMMARY
The invention relates to new defective adenoviral vectors permitting the transfer and expression of genes of interest to a host eukaryotic cell or organism, as well as to new complementation lines complementing in trans the essential viral functions which have been deleted from the genome of these recombinant adenoviruses. The invention is of very special interest for prospects of gene therapy, in particular in man.
Adenoviruses are DNA viruses which display a broad host range. They have been demonstrated in many animal species and many cell types. There are many serotypes which differ in particular in respect of their genome sequence. Most human adenoviruses are only marginally pathogenic and generally produce only benign symptoms.
The adenovirus enters the permissive host cell via a specific receptor, and it is then internalized and passes into endosomes. Their acidification contributes to a change in conformation of the virus and to its emergence into the cytoplasm. The viral DNA associated with certain viral proteins needed for the first steps of the replicative cycle then enters the nucleus of the infected cells, where its transcription is initiated by cellular enzymes. Replication of the adenoviral DNA takes place in the nucleus of the infected cells and does not require cell replication. Assembly of the new virions also takes place in the nucleus. In a first stage, the viral proteins assemble so as to form empty capsids of icosahedral structure, in which the adenoviral DNA is then encapsidated. The viral particles or virions are released from the infected cells and are capable of infecting other permissive cells.
The infectious cycle of the adenovirus takes place in 2 steps: adenoviral genome, and which permits production of the regulatory proteins participating in the replication and transcription of the viral DNA, and
In general terms, the adenoviral genome consists of a double-stranded linear DNA molecule approximately 36 kb in length which contains the sequences coding for more than 30 proteins. At each of its ends, a short inverted sequence of 100 to 150 nucleotides, depending on the serotypes, designated ITR (inverted terminal repeat), is present. ITRs are involved in the replication of the adenoviral genome. The encapsidation region of approximately 300 nucleotides is located at the 5' end of the genome immediately after the 5' ITR.
The early genes are distributed in 4 regions which are dispersed in the adenoviral genome, designated E1 to E4 (E denoting "early"). The early regions comprise at least-six transcription units which possess their own promoters. The expression of the early genes is itself regulated, some genes being expressed before others. Three regions, E1, E2 and E4, respectively, are essential to the viral replication. Thus, if an adenovirus is defective for one of these functions, that is to say if it cannot produce at least one protein encoded by one of these regions, this protein will have to be supplied to it in trans.
The E1 early region is located at the 5' end of the adenoviral genome, and contains 2 viral transcription units, E1A and E1B, respectively. This region codes for proteins which participate very early in the viral cycle and are essential to the expression of almost all the other genes of the adenovirus. In particular, the E1A transcription unit codes for a protein which transactivates the transcription of the other viral genes, inducing transcription from the promoters of the E1B, E2A, E2B and E4 regions.
The products of the E2 region, which also comprises two transcription units E2A and E2B, are directly involved in the replication of the viral DNA. This region governs, in particular, the synthesis of a 72 kDa protein which displays a strong affinity for single-stranded DNA, and of a DNA polymerase.
The E3 region is not essential to the replication of the virus. It codes for at least six proteins which appear to be responsible for inhibition of the host's immune response with respect to an adenovirus infection. In particular, the gp19 kDa glycoprotein appears

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