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
1992-09-21
2002-08-13
Eyler, Yvonne (Department: 1812)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C435S069100, C435S252300, C435S252800, C530S351000, C530S324000, C536S023520
Reexamination Certificate
active
06432677
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of recombinant DNA technology, to means and methods utilizing such technology in the discovery of a broad class of non-human animal interferons and to the production thereof and to the various products of such production and their uses.
More particularly, the present invention relates to the isolation and identification of DNA sequences encoding non-human animal interferons and to the construction of recombinant DNA expression vehicles containing such DNA sequences operably linked to expression-effecting promoter sequences and to the expression vehicles so constructed. In another aspect, the present invention relates to host culture systems, such as various microorganism and vertebrate cell cultures transformed with such expression vehicles and thus directed in the expression of the DNA sequences referred to above. In yet other aspects, this invention relates to the means and methods of converting the novel end products of such expression to entities, such as pharmaceutical compositions, useful for the prophylactic or therapeutic treatment of non-human animals. In addition, this invention relates to various processes useful for producing said DNA sequences, expression vehicles, host culture systems and end products and entities thereof and to specific and associated embodiments thereof.
The present invention arises in part from the discovery of the DNA sequence and deduced amino acid sequence encoding a series of bovine alpha interferons, including the 3′- and 5′-flanking sequences thereof facilitating their in vitro linkage into expression vehicles. These, in turn, enable the development of the means and methods for producing, via recombinant DNA technology, sufficient amounts of non-human animal interferons, so as to enable, in turn, the determination of their biochemical properties and bioactivity, making possible their efficient production for commercial/biological exploitation.
The publications and other materials hereof used to illuminate the background of the invention, and in particular cases, to provide additional details respecting its practice are hereby incorporated herein by this reference, and for convenience, are numerically referenced by the following text and respectively grouped in the appended bibliography.
BACKGROUND OF THE INVENTION
A. Non-human Animal Interferons
Interferon components have been isolated from tissues of various phylogenetic species lower than human (1,2,3). Activity studies conducted with these interferons have demonstrated varying degrees of antiviral effects in the requisite host animal (3,4,5,6). It also has been demonstrated that these interferons are not always species specific. For example, preparations of bovine interferons isolated from tissues, had antiviral activity on monkey and human cells (7). Likewise, human interferons have been found active in various cells of phylogenetically lower species (see 7).
This species interactivity is doubtless due to a high degree of homologous conservation, both in amino acid composition and sequence, amongst the interferons. However, until now, this explanation remained theoretical because the amounts and purities of non-human animal interferons that have been obtainable were insufficient to carry out unambiguous experiments on the characterization and biological properties of the purified components versus several of their human counterparts (8,9,10,11,12).
In any event, despite these low amounts and purities, a causal connection between interferon and anti-viral activity in the requisite animal host has been established. Thus, the production of non-human animal interferons in high yields and purities would be very desirable in order to initiate and successfully conduct animal bioassay experiments leading toward commercial exploitation in the treatment of animals for viral infections and malignant and immunosuppressed or immunodeficient conditions. In addition, the production of isolated non-human animal interferon species would enable their characterization, both physical and bioactive, and thus provide a basis for categorization and consequential comparative studies with counterpart human interferon species (see 8 to 20).
The studies done with non-human animal interferons, until the present invention, being restricted to the use of rather crude preparations, due to their very low availability, nevertheless suggest very important biological functions. Not only have the class of non-human animal interferons a potent associated therapeutic antiviral activity, but also potential as a prophylactic adjunct with vaccine and/or antibiotic treatment, clearly pointing to very promising clinical and commercial candidates.
It was perceived that the application of recombinant DNA technology would be a most effective way of providing the requisite larger quantities of non-human animal interferons necessary to achieve clinical and commercial exploitation. Whether or not the materials so produced would include glycosylation which is considered characteristic of native derived material, they would probably exhibit bioactivity admitting of their use clinically in the treatment of a wide range of viral, neoplastic, and immunosuppressed conditions or diseases in non-human animals.
B. Recombinant DNA Technology
Recombinant DNA technology has reached the age of some sophistication. Molecular biologists are able to recombine various DNA sequences with some facility, creating new DNA entities capable of producing copious amounts of exogenous protein product in transformed microbes. The general means and methods are in hand for the in vitro ligation of various blunt ended or “sticky” ended fragments of DNA, producing potent expression vehicles useful in transforming particular organisms, thus directing their efficient synthesis of desired exogenous product. However, on an individual product basis, the pathway remains somewhat tortuous and the science has not advanced to a stage where regular predictions of success can be made. Indeed, those who portend successful results without the underlying experimental basis, do so with considerable risk of inoperability.
The plasmid, an extrachromosomal loop of double-stranded DNA found in bacteria and other microbes, often times in multiple copies per cell, remains a basic element of recombinant DNA technology. Included in the information encoded in the plasmid DNA is that required to reproduce the. plasmid in daughter cells (i.e., an origin of replication) and ordinarily, one or more phenotypic selection characteristics such as, in the case of, bacteria, resistance to antibiotics, which permit clones of the host cell containing the plasmid of interest to be recognized and preferentially grown in selective media. The utility of plasmids lies in the fact that they can be specifically cleaved by one or another restriction endonuclease or “restriction enzyme”, each of which recognizes a different site on the plasmid DNA. Thereafter heterologous genes or gene fragments may be inserted into the plasmid by endwise joining at the cleavage site or at reconstructed ends adjacent to the cleavage site. Thus formed are so-called replicable expression vehicles. DNA recombination is performed outside the cell, but the resulting “recombinant” replicable expression vehicle, or plasmid, can be introduced into cells by a process known as transformation and large quantities of the recombinant vehicle obtained by growing the transformant. Moreover, where the gene is properly inserted with reference to portions of the plasmid which govern the transcription and translation of the encoded DNA message, the resulting expression vehicle can be used to actually produce the polypeptide sequence for which the inserted gene codes, a process referred to as expression.
Expression is initiated in a region known as the promoter which is recognized by and bound by RNA polymerase. In the transcription phase of expression, the DNA unwinds, exposing it as a template for initiated synthesis of messenger RNA from the DNA sequen
Capon Daniel J.
Goeddel David V.
Agarwal Atulya R.
Andres Janet L.
Eyler Yvonne
Genentech Inc.
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