Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
2000-05-25
2004-09-21
Winkler, Ulrike (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant virus encoding one or more heterologous proteins...
C424S184100, C424S204100, C424S233100, C435S069100, C435S235100, C435S320100, C435S455000, C435S456000, C435S457000, C514S04400A, C536S023100, C536S023720
Reexamination Certificate
active
06793926
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to novel nonmammalian carrier vectors and viruses useful in the production of high titers of recombinant viruses which may contain foreign DNA inserts or which may be point-mutated or deleted viruses, and methods of producing those viruses. The nonmammalian carrier vector (“carrier vector”) is a chimeric vector which includes those portions of a nonmammalian virus backbone which allow replication in a nonmammalian host cell. The carrier vector includes various nucleic acid cassettes, which may include an embedded recombinant viral genome containing a desired transgene, components necessary for production of a replication-defective recombinant virus containing the transgene, and domains that permit the carrier vector to bind to mammalian cells. The invention also provides methods of producing high concentrations of recombinant virus as a substantially homogeneous preparation, compositions to produce the recombinant virus, and novel recombinant viruses.
BACKGROUND OF THE INVENTION
A recombinant virus carrying a foreign DNA insert may be used to deliver genes to cells, where the gene may be expressed, if desired, to permit production of recombinant proteins in vitro or in vivo, vaccination of human and non-human mammals, or treatment or amelioration of diseases or genetic defects in humans or non-human mammals. One may treat or ameliorate diseases or genetic defects by providing normal gene products, increased levels of gene products or by blocking endogenous production of a gene, whose expression would be deleterious to the cell or organism.
Methods for delivering an exogenous gene to a mammalian cell include the use of mammalian viral vectors, such as those which are derived from retroviruses, adenoviruses, herpes viruses, vaccinia viruses, polio viruses, adeno-associated viruses, hybrid viruses (e.g., hybrid adenovirus-AAV, see U.S. Pat. No. 5,856,152) and the like. Other methods include direct injection of DNA, biolistic administration of DNA, electroporation, calcium phosphate precipitation, as well as methods of administration which utilize ligand-DNA conjugates, liposome conjugates of DNA, polycation-DNA complexes or adenovirus-ligand-DNA conjugates.
A transgene is a nucleic acid encoding a protein of interest; it may be a gene to allow for genetic or drug selection, e.g., a gene conferring resistance to antibiotics, or a reporter gene allowing detection, e.g., by color in the case of the use of green fluorescent protein. Alternatively, the transgene may be one that is useful for corrective applications. For instance, a transgene may be a normal gene that replaces or augments the function of a patient's defective gene. The transgene may be one that counteracts the effects of a disease, such as introduction and expression of a gene that is distinct from the one that it replaces or augments, but which has the same function or compensates for the defective gene's function. The transgene may be a gene which blocks or represses the expression of a malfunctioning, mutated, or viral gene in the patient, thereby giving rise to a corrective effect. A transgene may also be used for immunization against various agents, by provoking an immunogenic response in an animal. Delivery of therapeutic transgenes to a patient thus effects a correction of a defect or prevention of disease. The transgene also may be one which is useful for production of proteins in vitro, such as for large-scale production of therapeutic proteins.
Appropriate genes for expression in the cell include, without limitation, those genes which are normally expressed in cells but whose products are produced in insufficient amounts. Alternatively, the appropriate gene for expression is one which expresses a normal gene product which replaces a defective gene product, encodes ribozymes or antisense molecules which repair or destroy mutant cellular RNAs expressed from mutated genes, or modifies or destroys viral RNAs. Transgenes used for production of proteins in vitro include proteins such as secreted factors, including hormones, growth factors and enzymes.
Many gene therapy methods involve supplying an exogenous gene to overcome a deficiency in the expression of a gene in a patient. Some of these deficiencies are congenital and are due to a mutation in a particular gene in all the cells of the patient. For instance, in cystic fibrosis, there are one or more mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) which prevents the CFTR protein from functioning properly. In other cases, a deficiency in gene expression is due to an accident or disease that occurs during the patient's life. For instance, in Type I diabetes mellitus, the &bgr; pancreatic islet cells, which produce insulin, are destroyed, such that patients with this disease can no longer synthesize insulin. In other cases, the endogenous gene may be structurally normal but is not produced in high enough quantities due to disease, medical treatment or other environmental conditions, or mutations in the regulatory elements of the endogenous gene. For example, there are a number of blood disorders, such as anemia, in which there is insufficient production of red blood cells, which may be treated with erythropoietin (EPO) or with a transgene encoding EPO. Transgenes may also be used for genetic immunization, i.e., to elicit an immune response to a pathogen in an animal, including humans. For instance, a transgene may include a sequence from a viral, bacterial or fungal pathogen, such as influenza virus, human immunodeficiency virus (HIV), or mycobacterium tuberculosis.
Certain methods are amenable to targeted delivery of the exogenous gene to specific tissues, such as liver tissue. One method of delivering genes to specific cells relies upon the function of a cell-specific receptor. The asialoglycoprotein receptor (ASGP-R), which is present on the surface of hepatocytes (Spiess et al., 1990, Biochem. 29:10009-10018), is a lectin which has affinity for the terminal galactose residues of glycoproteins, and has been used to target gene delivery to liver hepatocytes. For example, a DNA complex is bound to a ASGP-R on the cell surface, allowing subsequent endoyctosis by the liver hepatocyte.
Viruses that are commonly used in gene delivery applications are modified by replacing viral nucleic acid with a desired transgene. Frequently, DNA removed from the virus encodes proteins necessary for viral replication or encapsidation, in which case the recombinant virus containing a transgene is replication-deficient and will not replicate or encapsidate in the host. To permit replication and encapsidation, current methods recognize that those portions of DNA which have been deleted must be supplied by wild-type or modified viruses or by plasmids containing DNA encoding the required gene products. Supplying wild-type or modified virus may result in recombinant virus stocks contaminated with wild-type or modified virus. Supplying plasmids encoding the required gene products through cotransfection results in low efficiency of recombinant virus production, as well as recombination events which yield wild-type virus contaminants.
A number of different viruses have been used to deliver a transgene to mammalian cells. These viruses include retrovirus, hepatitis B virus (HBV), adenovirus, adeno-associated virus (AAV) and herpesvirus. AAV possesses unique features that make it attractive as a vector for delivering foreign DNA (i.e., a transgene) to cells, and various groups have studied the potential use of AAV in the treatment of disease states.
AAV is a parvovirus, the genome of which is about 4.7 kb in length, including 145 nucleotide inverted terminal repeats (ITRs). The AAV genome encodes two genes, rep and cap, each of which expresses a family of related proteins from separate open reading frames and produced as a result of alternative mRNA splicing. Rep polypeptides (rep78, rep68, rep52, and rep40) are involved in replication, rescue and integration of the AAV genome.
Chen Haifeng
Gonda Matthew A.
Rasty Siyamak
Genovo, Inc.
Morrison & Foerster / LLP
Winkler Ulrike
LandOfFree
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