Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Recovery or purification
Reexamination Certificate
1999-10-13
2002-07-09
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Recovery or purification
C435S005000, C435S069100, C435S235100, C435S320100, C435S456000, C435S457000, C435S465000, C435S476000, C514S023000, C514S023000, C514S023000
Reexamination Certificate
active
06416992
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to systems for use in adeno-associated virus (AAV) vector production. More specifically, the invention relates to methods of producing recombinant AAV virions in cells grown in suspension cultures, cells grown in suspension cultures and then adhered to a substrate or in cells grown in culture systems which are able to produce large amounts of virions.
BACKGROUND
Scientists are continually discovering genes that are associated with human diseases such as diabetes, hemophilia and cancer. Research efforts have also uncovered genes, such as erythropoietin (which increases red blood cell production), that are not associated with genetic disorders but code for proteins that can be used to treat numerous diseases. However, despite significant progress in the effort to identify and isolate genes, a major obstacle facing the biopharmaceutical industry is how to safely and persistently deliver effective quantities of the expression products of these genes to patients.
Currently, the protein products of these genes are synthesized in cultured bacterial, yeast, insect, mammalian, or other cells and delivered to patients by intravenous injection. While intravenous injection of recombinant proteins has been successful, it suffers from several drawbacks. First, patients often require one or more intravenous administrations in a single day in order to maintain the necessary levels of the protein in the blood stream. Even then, the level of protein is not maintained at physiological levels—the level of the protein is usually abnormally high immediately following injection and far below optimal levels prior to injection. Second, the cost of daily administrations for many diseases is often prohibitive, ranging in the hundreds of thousands of dollars a year. As a result, many patients must do without medicinal protein. Third, intravenous administration often does not deliver the protein to the target cells, tissues or organs in the body. And, if the protein reaches its target, it is often diluted to nontherapeutic levels. Finally, the daily intravenous administration is inconvenient and severely restricts the patient's lifestyle, especially when the patient is a child.
These shortcomings have led to the development of gene therapy methods for delivering sustained levels of specific proteins into patients. These methods allow clinicians to introduce a nucleic acid coding for a gene of interest directly into a patient (in vivo gene therapy) or into cells isolated from a patient or a donor, which are then returned to the patient (ex vivo gene therapy). The introduced nucleic acid then directs the patient's own cells or grafted cells to produce the desired protein product. Gene delivery, therefore, obviates the need for daily injections. Gene therapy will also allow clinicians to select specific organs or cellular targets (e.g., muscle, blood cells, brain cells, etc.) for therapy.
DNA may be introduced into a patient's cells in several ways. There are transfection methods including chemical methods, such as calcium phosphate precipitation and liposome-mediated transfection, and physical methods such as electroporation. In general, transfection methods are not suitable for in vivo gene delivery. There are also methods that use recombinant viruses. Current viral-mediated gene delivery methods employ retrovirus, adenovirus, herpes virus, pox virus, and adeno-associated virus vectors.
One of the more promising viral system that has been used for gene delivery is adeno-associated virus (AAV). AAV is a parvovirus which belongs to the genus Dependovirus. AAV has several attractive features not found in other viruses. First, AAV can infect a wide range of host cells, including nondividing cells. Second, AAV can infect cells from different species. Third, AAV has not been associated with any human or animal disease and does not appear to alter the biological properties of the host cell upon integration. Indeed, it is estimated that 80-85% of the human population has been exposed to the virus. Finally, AAV is stable at a wide range of physical and chemical conditions which lends itself to production, storage and transportation requirements.
The AAV genome is a linear, single-stranded DNA molecule containing about 4681 nucleotides. The AAV genome generally comprises an internal nonrepeating genome flanked on each end by inverted terminal repeats (ITRs). The ITRs are approximately 145 base pairs (bp) in length. The ITRs have multiple functions, including as origins of DNA replication, and as packaging signals for the viral genome.
The internal nonrepeated portion of the genome includes two large open reading frames, known as the AAV replication (rep) and capsid (cap) genes. The rep and cap genes code for viral proteins that allow the virus to replicate and package into a virion. In particular, a family of at least four viral proteins are expressed from the AAV rep region, Rep 78, Rep 68, Rep 52, and Rep 40, named according to their apparent molecular wight. The AAV cap region encodes at least three proteins, VPI, VP2, and VP3.
AAV is a helper-dependent virus; that is, it requires co-infection with a helper virus (e.g., adenovirus, herpesvirus or vaccinia), in order to form AAV virions. In the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral genome inserts into a host cell chromosome, but infectious virions are not produced. Subsequent infection by a helper virus “rescues” the integrated genome, allowing it to replicate and package its genome into an infectious AAV virion. While AAV can infect cells from different species, the helper virus must be of the same species as the host cell. Thus, for example, human AAV will replicate in canine cells co-infected with a canine adenovirus.
AAV has been engineered to deliver genes of interest by deleting the internal nonrepeating portion of the AAV genome (i.e., rep and cap genes) and inserting a heterologous gene between the ITRs. The heterologous gene is typically functionally linked to a heterologous promoter (constitutive, cell-specific, or inducible) capable of driving gene expression in the patient's target cells under appropriate conditions. Termination signals, such as polyadenylation sites, can also be included.
To produce infectious rAAV virus containing the heterologous gene, a suitable producer cell line is transfected with a rAAV vector containing a heterologous gene. The producer cell is concurrently transfected with a second plasmid harboring the AAV rep and cap genes under the control of their respective endogenous promoters or heterologous promoters. Finally, the producer cell is infected with a helper virus, such as adenovirus, or helper virus genes, such as the adenovirus E1, E2A, E4 and VA RNA genes (WO 97/17458).
Once these factors come together, the heterologous gene is replicated and packaged as though it were a wild-type AAV genome. When a patient's cells are infected with the resulting rAAV virions, the heterologous gene enters and is expressed in the patient's cells. Because the patient's cells lack the rep and cap genes and the adenovirus helper genes, the rAAV cannot further replicate and package. Similarly, wild-type AAV cannot be formed.
Despite the promise of rAAV as a gene therapy tool, one of the shortcomings of the currently available technology is the inability to produce commercial scale viral titers.
Currently, high titer AAV virions are made by simultaneously transfecting a monolayer of producer cells (typically 293 cells) with an AAV vector and an AAV helper vector carrying the rep and cap genes using conventional transfection reagents (e.g., calcium phosphate) followed by infection with live adenovirus. Current protocols perform transfections in 10 cm plates (see, e.g., U.S. Pat. No. 5,173,414). Typically, in order to have a successful transfection, the monolayer must not be confluent. Alternatively, the production cell line is simultaneously transfected with an AAV vector, an AAV helper vector, an
Avigen, Inc.
Robins & Pasternak LLP
Stucker Jeffrey
Winkler Ulrike
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