Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-02-16
2003-07-01
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S235100, C435S325000, C435S320100
Reexamination Certificate
active
06586208
ABSTRACT:
BACKGROUND OF THE INVENTION
The production of desirable genetically engineered products can be less than efficient because the gene product itself or accessory molecules required for the proper production of the genetically engineered product of interest can be toxic to the host cell. For example, the rep proteins of adeno-associated virus (AAV), which are necessary for the integration and replication of the AAV genome in the host cell, often are toxic to the host cell and to, for example, the helper adenovirus.
Other examples of gene products which are used commonly in the art of genetic engineering and which are toxic to host cells are the E4 ORF6 protein of adenovirus, the VSV-G gene product of vesicular stomatitis virus and the HIV tat gene product.
SUMMARY OF THE INVENTION
The first object of the instant invention is to provide a method for expressing a recombinant gene product in a host cell under conditions that minimize exposure of the host cell to any foreign elements or products thereof, whether endogenous or not, that may be toxic to that host cell. The method relies on the use of a termination codon introduced into the coding sequence of a gene encoding a toxic protein and a sufficient amount of complementing suppressor tRNA's to overcome the presence of the termination codon engineered into the coding sequence of the gene encoding the toxic gene product, as well as releasing factors, termination factors and so on that normally interact with termination codons, to regulate expression of the toxic gene product. The toxic protein may be the product of interest or may be a protein required for efficient expression of the desired gene product of interest. The genes coding for the molecules that are toxic to the host cell are configured to contain one or more termination codons. Multiple toxic proteins can be manipulated in that fashion in a single cell. Also, multiple toxic proteins may be regulated by a single species of suppressor tRNA by introducing a termination codon at the same amino acid residue in the plural toxic proteins. Sufficient amounts of the suppressor tRNA's are made available in the host cell, for example, by transfecting the host cell with adenoviral vectors carrying the tRNA coding sequence.
Yet another object of the instant invention is to provide a method for minimizing the exposure of a host cell to the toxic adeno-associated virus (AAV) rep and cap proteins by introducing termination codons into the coding sequence of, for example, a rep gene, and using sufficient amounts of complementing suppressor tRNA's to overcome the presence of the termination mutation and endogenous termination factors, to therefore regulate the expression of that rep gene carrying the termination codon. The suppressor tRNA's can be targeted to one, two, three or four of the rep proteins. Moreover, the suppressor tRNA's can be directed to any amino acid codon. A suitable target is serine that appears in all four rep proteins. In that way, only a single species of suppressor tRNA would be required to regulate the expression of the four rep proteins.
Yet another object of the instant invention is to provide specific nucleic acid constructs, cells, vectors and the like for regulating the expression of toxic gene products in a host cell, such as the rep protein in the production of recombinant AAV.
Those and other objects have been achieved by the development of materials and methods for regulating the expression of toxic gene products that limit the establishment and productivity of a host cell in the production of recombinant gene products. For example, termination codons can be engineered into the coding sequence of a gene that encodes a toxic gene product. Then a suppressor tRNA which recognizes the termination codon and carries an aminoacyl group and thus incorporates an amino acid into the growing polypeptide instead of resulting in termination of translation is used. The suppressor tRNA complements the termination mutant and enables translation to occur when expression of the foreign gene product is desired. A suitable target is the rep protein of AAV that is toxic to the host cell. Yet another suitable target is the cap protein of AAV.
The instant method can be applied to regulate a number of genes which encode products which are toxic to host cells and which are commonplace in the art of recombinant nucleic acid technology.
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Virology,vol. 184, pp. 310-318, (1991). Smuda et al. “Adeno-Associated Viruses Having Nonsense Mutations in the Capsid Genes: Growth in Mammalian Cells Containing an Inducible Amber Suppressor.”
Virology,vol. 171, pp. 239-247, (1989). Chejanovsky et al. “Replication of a Human Parvovirus Nonsense Mutant in Mammalian Cells Containing an Inducible Amber Suppressor.”
J. Virology,vol. 70, No. 1, pp. 559-565, (1996). Yeh et al. “Efficient Dual Transcomplementation of Adenovirus E1 and E4 Regions from a 293-Derived Cell Line Expressing a Minimal E4 Functional Unit.”
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Donahue Brian A.
Hardy Stephen F.
Snyder Richard O.
Cell Genesys Inc.
Judge, Esq. Linda R.
Kelber Steven B.
Mosher Mary E.
Piper Rudnick LLP
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