The present disclosure provides dual viruses capable of producing a primary virus and a secondary virus, and dual oncolytic viruses capable of producing a primary oncolytic virus and a secondary oncolytic virus.

The present invention includes methods and compositions for the production of high titer recombinant Adeno-Associated Virus (rAAV) in a variety of mammalian cells. The disclosed rAAV are useful in gene therapy applications. Disclosed methods based on co-infection of cells with two or more replication-defective recombinant herpes virus (rHSV) vectors are suitable for high-titer, large-scale production of infectious rAAV.

The present invention provides compositions and methods of use pertaining to rAAV-mediated delivery of therapeutically effective molecules for treatment of diseases such as Pompe disease. These compositions in combination with various routes and methods of administration result in targeted expression of therapeutic molecules in specific organs, tissues and cells.

The invention described herein provides a recombinant replication-defective vims derived from Herpesvirales order, wherein the virus is characterized by a complete deletion of a gene encoding ICP27, or a functional equivalent gene thereof. The invention also provides production cell lines for such recombinant replication-defective vims, wherein the cell lines have a coding sequence for ICP27 or a functional equivalent thereof, and wherein the coding sequence has no or minimal sequence overlap with the virus characterized by the complete deletion of the gene encoding ICP27. Method of using such recombinant replication-defective vims and production cell lines are also provided.

Provided herein are improved, cost-effective and environmentally friendly methods of production of recombinant AAV (rAAV) in embryonated avian eggs. Further provided herein is a provides embryonated avian eggs as novel host vehicles for high-yield production of rAAV, including both packaging and propagation. In particular, embryonated chicken eggs provide a novel expression vehicle for AAV of mammalian origin, irrespective of AAV serotype. The disclosed methods may comprise packaging of rAAV in embryonated avian eggs (e.g., chicken eggs) by inoculating an embryonated avian egg with a first nucleic acid vector comprising a transgene and a second nucleic acid vector comprising AAV rep and cap genes, incubating the egg, and isolating rAAV from the egg, wherein the AAV is of non-avian origin. Also provided are methods of purifying and propagating packaged rAAV in embryonated avian eggs or in avian embryonic fibroblasts.

Provided herein are methods of improving rAAV production in cells, the method comprising increasing the salt concentration in the media in which the cells are infected or transfected, cultured, or in which they produce AAV. Aspects of the disclosure relate to improved methods of rAAV production by co-infecting suspension adapted cells (e.g., suspension adapted HEK293 cells) with viruses that encode one or more AAV components for producing rAAV particles within the suspension adapted cells.

Provided herein are methods of improving rAAV production in cells, the method comprising increasing the salt concentration in the media in which the cells are infected or transfected, cultured, or in which they produce AAV. Aspects of the disclosure relate to improved methods of rAAV production by co-infecting suspension adapted cells (e.g., suspension adapted HEK293 cells) with viruses that encode one or more AAV components for producing rAAV particles within the suspension adapted cells.

The present invention includes methods and compositions for the production of high titer recombinant Adeno-Associated Virus (rAAV) in a variety of mammalian cells. The disclosed rAAV are useful in gene therapy applications. Disclosed methods based on co-infection of cells with two or more replication-defective recombinant herpes virus (rHSV) vectors are suitable for high-titer, large-scale production of infectious rAAV.

The present invention provides compositions and methods of use pertaining to rAAV-mediated delivery of therapeutically effective molecules for treatment of diseases such as Pompe disease. These compositions in combination with various routes and methods of administration result in targeted expression of therapeutic molecules in specific organs, tissues and cells.

Provided herein are methods of improving rAAV production in mammalian cells via the manipulation of a concentration of potassium chloride present in the media in which the mammalian cells are cultured and/or in which the rAAV particles are produced. The concentration of potassium chloride present in such media may be manipulated by, for example, supplementing a base medium with additional potassium chloride either before, at the same time as, or after the cells are contacted (e.g., infected) with one or more viral vectors (e.g., recombinant Herpes Simplex Virus (rHSV) vectors). Some embodiments of the present invention further contemplate supplementing the base medium with additional potassium chloride and sodium chloride in combination.