Provided herein are pseudotyped recombinant lyssavirus particles and methods for their use in delivering a transgene into a target cell. Also provided are packaging systems and methods of using the packaging systems to produce pseudotyped recombinant lyssavirus particles.

Provided herein are recombinant negative-strand RNA virus genomes (e.g., recombinant rabies virus genomes) and recombinant negative-strand RNA viruses (e.g., recombinant rabies viruses) and methods for their use in delivering a guide RNA and, optionally, a transgene, into a target cell. Also provided are packaging systems and methods of using the packaging systems to produce recombinant negative-strand RNA viruses.

Provided herein are recombinant rabies virus genomes and recombinant rabies viruses and methods for their use in delivering a transgene into a target cell. Also provided are packaging systems and methods of using the packaging systems to produce recombinant rabies viruses.

The present invention provides a closed system to propagate virus-infected cells without the effect of shear force, while providing quicker access to nutrients than is available conventionally. This system design allows for a high density of infected cell growth to increase the virus yields and to maintain homogeneity of the contents of the main container. The system further provides a nuclease to degrade the cellular DNA prior for purification of the virus or viral components. As the system is designed for maximum containment at low risk, the live virus can be a hazardous virus such as a Bio-safety Level 3 (BSL 3), BSL 4 or BSL5 virus.

This application discloses a method for purifying pDNA, particularly pDNA that that can be used to produce RNA, the RNA preferably encoding a therapeutic or immunogenic peptide or polypeptide. The pDNA can be grown in a bacteria such as E. coli by culturing or fermenting bacteria containing the plasmid and obtaining and purifying the pDNA. The present method allows the pDNA to be obtained in high yield and with high purity. In one embodiment of the invention, the level of all non-pDNA materials can be significantly reduced by the process. In some embodiments, the ratio of supercoiled plasmid DNA (scDNA) to non-supercoiled pDNA (non-scDNA, such as open circular plasmid DNA (ocDNA)) can be increased by one or more process steps that separate or allow for separation of scDNA and ocDNA or process steps that increase the amount of scDNA to ocDNA.