Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Patent
1986-06-04
1990-09-18
Warren, Charles F.
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
435320, 4353171, 435 691, 935 32, 935 57, C12N 700, C12N 1500, C12P 2100
Patent
active
049578650
DESCRIPTION:
BRIEF SUMMARY
The stable introduction of genetic material into organisms generally involves the accomplishment of this transfer in precursor cells having high potentialities for multiplication and differentiation. These precursor cells can either be representative of a very special differentiation pathway, such as, for example, cells of haematopoietic origin, or they can be totipotent such as germ cells. In all cases, the population of target cells for the transfer is in a tiny minority in the tissues in question.
The stable introduction of genetic sequences in these cells hence requires the use of transfer techniques, the yield of which should be high. Classical methods for introducing purified DNA fragments into cells in culture give rise to low levels of incorporation.
The use of compound vectors containing viral genomes have enabled a distinct improvement to be achieved in the incorporation of an exogenous sequence into a cell in culture.
The present invention relates to new vectors of this type, and especially gene transfer vectors derived from retroviruses, in particular avian erythroblastosis retrovirus.
Retroviruses owe to their special structure the capacity to be integrated readily in the form of a provirus in the genomic DNA of the cells which they infect. The structural elements which provide for this integration are carried at both ends of the provirus and are known as "long terminal repeat" or LTR.
This ease of insertion of the retroviruses within the genome of the cells which they infect has led to the idea of using them as a potential vector for transferring genetic material.
The genome of avian erythroblastosis retrovirus (AEV) contains two onc genes known as v-erbA and v-erbB, the expressions of which are responsible for the oncogenic power of this retrovirus.
In a first embodiment of the invention, the oncogenic power of the AEV virus was used as a selection marker and its capacity for insertion was used for transferring to the cells in culture an exogenous gene inserted in this retrovirus.
In a second embodiment of the invention, the oncogenic power of the AEV virus is eliminated and the marker gene is a foreign gene, for example a gene for resistance to an antibiotic such as G418.
A brief study of the characteristics of retroviruses will facilitate a better understanding of the invention.
The genome of a retrovirus which is not replication-defective (FIG. 1) is composed of an RNA molecule possessing, in the direction of the transcription (5'.fwdarw.3') an identical short sequence at each end, known as R (between 21 and 80 bases). This is followed, in order, by a single sequence known as U5 (from 70 to 80 bases), a tRNA binding site (TBS, approximately 20 bases), and a non-coding sequence ("leader" sequence). The RNA molecule continues with a region coding for three genes, the translation products of which are essential for the replication of the virus, and which are gag (virion structural proteins), pol (reverse polymerase) and env (envelope). The genome terminates, in order, with a non-coding sequence, a purinerich sequence (PU), a single sequence known as U3 (from 200 to 450 bases), and finally the R sequence. The repeat end sequences (R) or single sequences (U5 and U3) peculiar to retroviruses appear to be rather well conserved in this group of viruses and contain the signals involved in the control of the expression of the viral genome.
Some retroviruses, such as AEV, which are responsible for neoplastic transformations giving rise to leukaemias or tumours, owe their transforming power to the presence of special "onc" sequences in their genome. These onc sequences are cellular in origin, and have been integrated in the viral genome following recombinations with the genome of the cells which are hosts for the virus. In most of these viruses, this integration is accompanied by the complete or partial loss of gag, pol and env genes. These viruses are consequently replication-defective. In order to replicate, these viruses require the presence in the same host cell of a functional helper virus.
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Benchaibi Miloud
Chambonnet Frederique
Flamant Frederic
Nigon Victor
Poncet Didier
Chambers Jasemine C.
Institut National de la Recherche Agronomique (INRA)
Rowland Bertram I.
Warren Charles F.
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