Novel compositions and methods for production of recombinant virus

Abstract

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.

Description

TECHNICAL FIELD OF THE INVENTION [0001] 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,...

AI Technical Summary

Benefits of technology

[0023] Alternatively, if a recombinant retrovirus is desired, the carrier vector or vectors would comprise 1) an embedded recombinant viral genome comprising the retroviral LTRs and the transgene of interest driven from the retroviral LTRs or from a heterologous promoter, and 2) DNA sequences encoding any one or a combination of gag, pol and env for the functions of replication and encapsidation of the retrovirus not supplied in the host cell. In a preferred embodiment, all of the required elements to produce a recombinant virus in a particular host cell are contained on a single carrier vector because the use of a single carrier vector having all functions not supplied by a host cell increases the efficiency of transduction, and can be more easily scaled for industrial production of the embedded recombinant virus.

[0031] This invention thus has many advantages over current methods for manufacturing recombinant viruses. These advantages include: (1) the nonmammalian virus backbone permits insertion of large DNA sequences without compromising the efficiency of recombinant virus production; (2) sequences normally toxic to mammalian cells (e.g., AAV rep, VSV-G, retroviral envelope proteins, eukaryotic regulatory proteins, etc.) are not expressed in substantial amounts from their mammalian regulatory sequences in the nonmammalian host cell of the nonmammalian carrier vector and thus can be tolerated by the nonmammalian carrier vector during the course of its replication in the nonmammalian host cell; (3) nonmammalian viruses do not replicate in mammalian cells, precluding contamination of the final eukaryotic vector stocks with the nonmammalian carrier vector; (4) in some embodiments no helper viruses are necessary, with the result that the final recombinant virus preparation is essentially free of helper virus; (5) frequency of wildtype virus production due to homologous or non-homologous recombination is minimized; and (6) the methods of the present invention are particularly suitable to large scale production of recombinant viruses which are themselves replication-deficient. Additionally, nonmammalian viruses are not normally pathogenic to mammalian cells, may be propagated in serum free media, and may be grown to a high titer. Other features and advantages of the invention will be apparent from the following drawings, the description of the invention and its preferred embodiments, and the examples described herein.

[0033] In another embodiment, the invention includes a method of producing replication-deficient recombinant viral vector lysates and stocks that are free of helper or other contaminating virus. In a preferred embodiment, the method is one which is easily scaled for industrial production of recombinant viral vectors. In another preferred embodiment, the method is one in which a high titer of recombinant viral vector lysates and stocks is achieved.

Problems solved by technology

For instance, in Type I diabetes mellitus, the P 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.

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.

However, progress towards establishing AAV as a transducing vector for the delivery of DNA in the form of a desired transgene has been slow for a variety of reasons.

However, because AAV requires a particular genome packaging size, addition of a transgene results in deletion of necessary gene functions for rep and cap.

The disadvantage of this method is that the rAAV vector stock is contaminated with helper virus, which is labor-intensive and difficult to separate from the helper virus, and co-transfection of two plasmids along with infection by a helper virus is inefficient and cannot be easily scaled up for industrial production of rAAV.

The disadvantage of this method is that a triple transfection is also inefficient and is difficult to scale up.

The disadvantage of this method is that wild-type Ad may be produced, which must be separated from the rAAV vector before use in a patient.

Thus, current methods of producing recombinant AAV are incapable of yielding the high amounts of essentially homogeneous virus for pharmaceutical compositions needed for the treatment of a large number of patients in a easily scaled industrial production.

While baculovirus has been used for expressing particular proteins in a mammalian cell, see U.S. Pat. No. 5,731,182, baculovirus has not been used to produce pharmaceutical compositions of replication-deficient recombinant virus using an easily scaled industrial process.

In addition, the methods disclosed in U.S. Pat. No. 5,731,182 do not result in production of an altogether distinct, essentially homogeneous recombinant virus, at high titers.

Current viral production methods include costly and time consuming purification and concentration steps, and are incapable of producing sufficient recombinant virus for pharmaceutic applications.

Current production methods result in contaminating helper virus which must be inactivated and / or removed from the final products prior to pharmaceutical application.

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