Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Patent
1998-04-13
2000-12-05
Schwartzman, Robert A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
435 914, 4353201, 435462, 536 231, C12Q 170, C12P 1934, C12N 1587, C12N 1563, C07H 2104
Patent
active
061564974
DESCRIPTION:
BRIEF SUMMARY
This invention relates to the generation of adenoviral vectors. More particularly, this invention relates to the generation of adenoviral vectors directly from polynucleotide constructs such as plasmids, whereby a site-specific recombinase mediates transfer of an adenoviral ITR with a terminal protein bound thereto to the plasmid, thereby enabling the plasmid to replicate efficiently as if it were an adenovirus. This invention also relates to adenoviral vectors that are devoid completely of adenoviral backbone genes and to adenoviral vectors that transfer retroviral vector sequences, as well as the genes for retroviral structural proteins, including gag, pol, and env.
The term "adenoviral vector," as used herein, means a gene transfer vehicle which is an adenoviral particle containing recombinant adenoviral DNA that incorporates a desired DNA sequence to be transferred into an appropriate cell.
BACKGROUND OF THE INVENTION
Adenovirus genomes are linear, double-stranded DNA molecules about 36 kilobase pairs long. Each extremity of the viral genome has a short sequence known as the inverted terminal repeat (or ITR), which is necessary for viral replication. The well-characterized molecular genetics of adenovirus render it an advantageous vector for gene transfer. The knowledge of the genetic organization of adenoviruses allows substitution of large fragments of viral DNA with foreign sequences. In addition, recombinant adenoviruses are structurally stable and no rearranged viruses are observed after extensive amplification.
Adenoviruses thus may be employed as delivery vehicles for introducing desired genes into eukaryotic cells, whereby the adenovirus delivers such genes to eukaryotic cells by binding cellular receptors, internalizing via coated pits, disrupting endosomes, and releasing particles to the cytoplasm followed by nuclear translocation and molecular expression of the adenovirus genetic program.
In general, in order to construct an adenoviral vector including a heterologous gene, or transgene, a plasmid is prepared which contains the transgene expression cassette and some adenoviral sequences, usually from the left end, or 5' end, of the virus. The plasmid then is co-transfected with DNA containing most of the adenoviral sequence, and homologous recombination occurs, yielding plaques on the adenoviral producer cells, such as, for example, 293 cells. The plaques are picked, amplified, screened, and scaled up further. Such process may take several months or more. If one desires to change the vector backbone, the process then involves further manipulation. If a single adenoviral gene is to be deleted from the adenoviral backbone, the first step is to generate a packaging cell that expresses the gene in trans. Next, a new plasmid is generated which encodes a portion of the adenoviral backbone with the adenoviral gene deleted. This plasmid is co-transfected with DNA encoding the remainder of the adenovirus. Homologous recombination then occurs, and adenoviral plaques are generated, screened, and scaled up, thus yielding a virus with an altered backbone. This virus then is used to generate a vector with a transgene by homologous recombination with the transgene-encoding plasmid as hereinabove described. The generation of such an adenoviral vector may take one year or more.
SUMMARY OF THE INVENTION
The present invention provides a cell and a method for generating adenoviral vectors directly from polynucleotides, such as plasmids, without the need to obtain, screen, or cycle up plaques. The present invention saves a substantial amount of time and enables vectors with transgene and/or backbone gene changes to be made and tested very rapidly. In particular, the present invention provides for the generation of an adenoviral vector from a polynucleotide, such as a plasmid, through the recombinase-mediated transfer of an adenoviral ITR with a covalently attached adenoviral terminal protein to the plasmid. According to the present invention, a high concentration of adenoviral vector can be obtained directl
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Genetic Therapy, Inc.
Lillie Raymond J.
Olstein Elliot M.
Sandals William
Schwartzman Robert A.
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