Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1998-06-23
2003-09-02
Falk, Anne-Marie (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C424S093100, C424S093210, C424S093600, C424S093700, C435S320100, C435S455000, C514S04400A
Reexamination Certificate
active
06613319
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The field of this invention is gene expression. More particularly, this invention pertains to a process for increasing the circulating levels of gene products over an extended period of time.
BACKGROUND OF THE INVENTION
A large number of inherited and acquired serum protein deficiencies including hemophilia A, diabetes mellitus and the erythropoietin-responsive anemias are currently treated by repeated intravenous or subcutaneous injections of purified or recombinant proteins. Although largely effective, such therapies are both expensive and inconvenient. Moreover, in diseases such as hemophilia A, there is not sufficient recombinant protein available to allow a comprehensive program of prophylactic therapy. Given these problems, there has been considerable interest in developing novel gene-based therapies for such serum protein deficiencies. An initial series of studies demonstrated that skeletal myoblasts genetically modified in vitro could be reimplanted by intramuscular injection and would subsequently produce stable, physiological levels of recombinant proteins in the systemic circulation of adult immunocompetent mice. Subsequently, several groups have demonstrated the stable production of recombinant serum proteins following a single intramuscular (IM) injection of replication-defective adenovirus (RDAd) vectors. Despite these initial successes, both myoblast transplantation and IM injection of RDAd vectors have thus far been associated with problems that may preclude their widespread clinical application.
The studies reported to date have all been done on rodents such as mice. Those data may not reflect and may not be predictive of results in larger animals such as primates. It is well known in the art, for example, that physiological or therapeutic doses observed in rodents are not necessarily predictive of effective doses in larger mammals. Still further, the amount of vector needed in large mammals may preclude their utility. For example, the mass of vector needed in primates may be so large that their injection results in either adverse reactions to the injection (e.g., anaphylactic shock), generation of an immune response or secondary infection resulting from the use of large numbers of viral particles. Still further, the data from previous reports do not address the question of whether there is any correlation between the amount or dose of transforming vectors and increases in the levels of gene products. There continues to be a need in the art, therefore, for processes for increasing the circulating levels of gene products in large mammals such as primates.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a process of increasing the circulating levels of a gene product in the blood stream of a mammal for a period of time greater than about 30 days. The process includes the step of transforming muscle cells of the mammal with a polynucleotide that encodes the gene product, wherein the expression vector drives long-term expression of the polynucleotide.
A preferred mammal is an animal used for food such as a cow, domesticated animals such as dogs and cats and primates. A preferred primate is a human. A process of the present invention can be used to increase circulating levels of any gene product. Exemplary such gene products are RNA molecules, single-stranded DNA molecules and polypeptides. Polypeptides are particularly preferred. Especially preferred polypeptides are polypeptide hormones such as growth hormone and erythropoietin.
A process of the present invention can use any muscle such as smooth muscle, cardiac muscle and skeletal muscle. Cardiac and skeletal muscle are preferred. The use of skeletal muscle is most preferred.
Any suitable expression vector can be used in the present process. Exemplary and preferred such vectors are plasmids and replication defective adenoviral vectors. The muscle cells can be transformed either in vivo or ex vivo. When transformed in vivo, the expression vector is directly injected into a muscle mass of the mammal. When transformed ex vivo, muscle cells are removed from the mammal, transformed ex vivo and the transformed muscle cells reimplanted into the mammal.
A process of the present invention is useful for increasing the circulating levels of gene products over an extended period time. Using a process of this invention, those levels can be increased for periods of time ranging from greater than about 60, 90 or 120 days and even for as long as one year.
The safety and efficacy of IM injection of adenoviral vectors encoding Epo in both mice and non-human primates has been determined. In an initial series of experiments, the relationship between the dose of vector injected and the corresponding elevations in serum Epo levels and hematocrits in both species were studied. The results demonstrated that there is a threshold dose in both mice and monkeys (approximately 2.5-8×10
7
pfu/gm body weight) that is required to obtain long-term Epo expression and polycythemia. A single IM injection of mice with 10
9
pfu of vector resulted in elevations in hematocrits from control values of 49% to treated values of 81% which were stable for more than one year. Similarly, a single IM injection of a monkey with 4×10
11
pfu of an adenoviral vector encoding simian Epo (AdsEpo) resulted in elevations of hematocrits from control levels of 40% to treated levels of =70% which were stable for 84 days. IM injection of monkeys with vector was determined to be safe in that no abnormalities in chest X-rays, serum chemistries, hematologic or clotting profiles (except for elevated hematocrits) or organ pathology were seen during the 84 day time course of the experiment.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a process of increasing the circulating level of a gene product in the blood stream of a mammal for a period of time greater than about 30 days. The process includes the step of transforming muscle cells of the mammal with a polynucleotide that encodes the gene product, wherein the expression vector drives long-term expression of the polynucleotide.
As is well known in the art, gene products include polynucleotides such as DNA and RNA and polypeptides. As is also well known in the art, those gene products can be secreted from the cells where they are made into the extracellular fluid compartment of the organism. From there, those products diffuse into the blood. The process of the present invention can be used to increase the levels of those gene products in the blood over an extended period of time. A process of this invention is particularly useful in increasing the levels of polypeptide gene products. Preferably, the polypeptide is secretory product of a cell. Exemplary such products are cytokines, colony stimulating factors, nerve growth factors and the like. Exemplary and preferred such secretory products are polypeptide or protein hormones. Such hormones are well known in the art. Exemplary polypeptide hormones are insulin, glucagon, renin, parathyroid hormone, growth hormone, erythropoietin and the like.
A process of the present invention can be used to increase the circulating level of a gene product in any mammal. The process is particularly useful in large mammals such as domestic pets, those used for food production and primates. Exemplary large mammals are dogs, cats, horses, cows, sheep, deer and pig. Exemplary primates are monkeys, apes and humans. The use of the present process in humans is particularly preferred.
The present invention discloses that increased circulating levels of gene products can be realized by transforming muscles cells of the mammal with a polynucleotide that encodes that gene product. As is well known in the art, mammals contain three types of muscle cells: smooth muscle, cardiac muscle and skeletal muscle. Any one of these muscle types can be used in a present process. Because of the accessibility of large masses of cardiac and skeletal muscle, use of these muscle types is preferred. In an especially preferred embodi
Arch Development Corporation
Falk Anne-Marie
Hale and Dorr LLP
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