Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1993-07-20
2004-02-24
Wilson, Michael C. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C424S093100, C424S093200, C424S093210
Reexamination Certificate
active
06696420
ABSTRACT:
The invention concerns a recombinant DNA including a nucleotide sequence coding for a specific polypeptide under the control of an adenovirus promoter, the vectors containing this recombinant DNA, the eucaryotic cells transformed by this recombinant DNA, the excretion products of these transformed cells and their applications, notably to the constitution of vaccines.
Human adenoviruses possess a long (around 36,000 bp) linear and double-stranded genome which codes for at least 30 proteins. The viral cycle, in the course of the infection of permissive cells, is divided into two phases, early and late. It is known that the four regions of the viral genome expressed in the early phase are called regions E1, E2, E3, and E4, whose respective positions in the whole genome are schematically represented in FIG.
1
. The E1 region, situated at the left end of the genome, is itself divided into two regions, E1A and E1B. The passage from the early phase to the late phase, marked by the replication of the viral DNA, is characterized by an abrupt change in the genetic program of the virus. The expression of certain early genes is repressed while the transcription of the late genes is accomplished principally via only one promoter, the major late promoter (FIG.
1
). In addition, a strong repression of protein synthesis of the host cell may be observed.
The genetic organization of type 2 or 5 human adenovirus (Ad2, Ad5) is sufficiently well known that their genome may be manipulated in vitro and its use as a vector for the expression of a foreign gene in an animal cell in culture has already been envisaged. Indeed it is known that the E3 region, which represents 6% of the genome, is not essential in vitro and may therefore be substituted in its entirety. The size of the foreign DNA fragment which it is possible to insert into the genome of these viruses, is large. In fact, the virus may encapsulate a genome whose length exceeds by 5% that of the wild genome.
Different vectors derived from adenoviruses of type 2 or 5 have therefore been constructed. In these recombinants, the foreign gene was expressed under the control of the major late promoter. This has permitted the obtaining in certain cases of a synthesis of the protein coded by a foreign gene at a level comparable to that of the late viral proteins. This being the case, it results from the preceding that the expression of the foreign gene under the control of the late promoter can only manifest itself in the late phase of the viral cycle.
The invention results from the observation that the promoter of the early region E1A of the genome of an adenovirus (hereafter designated simply as “E1A promoter”) could control in a particularly effective manner the expression of a heterologous gene (that is, foreign vis-a-vis the genes normally associated with it in the adenovirus) or more generally of a heterologous nucleotide sequence coding for a polypeptide sequence whose expression is sought. In other words, the E1A promoter acts like a strong promoter, and this more particularly when the E1A promoter combined with the heterologous coding sequence is inserted into a viral vector.
The invention therefore concerns in a general fashion a recombinant DNA for the transformation of eucaryotic cell lines, notably human or animal, chosen from among which are infectable by the adenoviruses or whose endogenous polymerases are likely to recognize the adenovirus promoters, this recombinant DNA being, in addition, modified by an insertion nucleic acid containing a nucleotide sequence coding for a polypeptide sequence whose expression in the said cell lines is sought. This recombinant DNA is more particularly characterized by the fact that the said insertion sequence is placed under the direct control of the early promoter of the E1A region of the genome of the adenovirus.
Preferably, this recombinant DNA is incorporated into a replicatable vector in the said cell lines or associated by genetic recombination with such a vector.
Being a viral vector, notably one derived from adenovirus, equally offers the advantages attached to the E1A region of the adenoviruses, namely that its expression is constitutive and permanent all during the viral cycle.
A particularly prefered form of the recombinant DNA according to the invention is characterized by the fact that it includes, ‘downstream’ of the insertion nucleic acid, in the direction of transcription, a defective adenovirus genome including nevertheless all of those of the essential sequences necessary to the replication of the corresponding adenovirus, which are normally situated ‘downstream’ of the genes normally under the direct control of the E1A early promoter in the said genome.
Advantageously, the defective adenovirus genome with which the recombinant DNA conforming to the invention is associated, is constituted of complete adenovirus genome, deprived however of the anterior part of the E1A region of the viral genome, notably of its 0-2.6% fragment (the percentage expressed relative to the total size of the adenovirus genome).
The recombinant DNAs of the invention, associated with the elements of vectors such as those which have been mentioned above, constitute in fact the vectors will again be the case where reference is made to “defective recombinant viruses”, when the elements of the vectors associated with the recombinant DNA of the invention will be derived from a defective genome of adenovirus. These defective recombinant viruses are advantageously used for the transformation of transformable cell lines from superior eucaryotes (notably of human or animal origin) themselves including a distinct sequence of nucleotides apt to complement the part of the genome of the adenovirus which is missing from the aforesaid vector, the said distinct sequence preferably being incorporated into the genome of the cells of the said cell line.
As a prefered example of such cell lines, one might mention the line 293, a human embryonic kidney line which contains, integrated into its genome, the first eleven percent of the left end of the genome of an Ad5. This permits the complementing of the defective recombinant viruses which have deletions of this region.
The use of these systems: defective recombinant virus vector—cells containing a sequence capable of complementing the defective recombinant viruses, is of a very particular interest, when the nucleotide sequence contained in the insertion nucleic acid of the recombinant DNA codes for a protein which, when it is expressed in the natural cellular host under the control of its natural promoter, is excreted into the culture medium of this natural cellular host.
The S gene of the genome of the virus of hepatitis B constitutes in this regard a nucleotide sequence of particular interest, arid this for several reasons. On the one hand, the product of the expression of the S gene in the cells which express it, HBsAg, is secreted into the cellular supernatant in the form of particles which are easy to detect and to quantify by radio-immunology, which permits a precise evaluation of the capacity of expression of the viral vector. On the other hand, the invention provides a recombinant viral vector permitting the study of the expression of the genes of the HBV at the level of transcription as well as that of translation, which is all the more interesting in that until now there had not existed a cell culture system capable of propagating the hepatitis B virus (HBV). And lastly, the cellular infection by the adenovirus-HBV recombinant virus illustrates particularly well the methodological basis of a process for the manufacture of a vaccine against a given pathologic agent (in this case the hepatitis B virus in the example under consideration). Another nucleotide sequence of the genome of the hepatitis B virus of particular interest is the S gene along with its pre-S2 region which codes for the HBs antigen and for a receptor of polymerized human serum albumen (pHSA) (25, 26).
It goes without saying that one may substitute for the S gene into the recombinant DNA, any other nucle
Ballay Annick
Levredo Massimo
Perricaudet Michel
Tiollais Pierre
Institut Pasteur
Wilson Michael C.
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