Provided herein are methods and compositions useful in the production of recombinant AAV (rAAV) in host producer cells, such as insect cells. In some embodiments, methods, uses and compositions are provided that comprise recombinant VP1 genes comprising modified Kozak sequences to express AAV VP1 proteins in amounts that are useful for producing infective rAAV particles. These infective rAAV particles may comprise a gene of interest.

Provided are compositions and methods for stabilizing a transfection cocktail containing DNA-cationic polymer complexes for an extended time, while maintaining high transfection efficiency. Such stabilized transfection cocktail can be used to generate transfected cells that can produce, for example, rAAV vectors on a large scale without impacting the key attributes of the virus production, such as, titer, DNA packaged rAAV particle fraction, and rAAV vector purification profile.

The present disclosure provides an automated method of producing viral vectors, utilizing engineered viral vector-producing cell lines, or packaging cells, within a fully-enclosed cell engineering system. Exemplary viral vectors that can be produced include lentivirus vectors, adeno-associated virus vectors, baculovirus vectors and retrovirus vectors.

The present invention is directed to viral vectors and methods of their production and use.

Methods for determining the relative abundance of viral capsid components in a sample of viral particles are disclosed. In embodiments, methods for determining the relative abundance of empty capsids, partially-full capsids and full capsids (e.g., containing a heterologous nucleic acid molecule) of adeno-associated virus are disclosed.

A recombinant adeno-associated virus (rAAV) comprising an AAV capsid and a vector genome comprising a frataxin gene is provided. Also provided is a composition containing an effective amount of rAAV to ameliorate symptoms of Freidreich’s ataxia, including, e.g., reduction in progression towards neurocognitive decline and/or cardiomyopathy.

Methods of purifying AAV particles using a chromatin-DNA nuclease (e.g., MNase), and compositions that include AAV particles and a chromatin-DNA nuclease (e.g., MNase) are described. Compositions and kits that include a chromatin-DNA nuclease (e.g., MNase) for the purification of AAV particles are also provided.

A vector system for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) the first vector comprises in a 5′ to 3′ direction: a promoter; an intron; a 5′ end portion of the transgene coding sequence (CDS); a splice donor sequence; and a first recombinogenic region; (b) the second vector comprises in a 5′ to 3′ direction: a second recombinogenic region; a splice acceptor sequence; and a 3′ end portion of the transgene CDS; wherein the 5′ end portion and the 3′ end portion together constitute the transgene CDS, and wherein the intron is not capable of homologous recombination with the splice donor sequence to excise the 5′ end portion of the transgene CDS.

In a first aspect, the present invention relates to a method for the in vitro generation of genetically engineered, linear, single-stranded nucleic acid fragments containing two viral inverted terminal repeat (ITR) sequences flanking a gene of interest (GOI). The method is based on a rolling circle amplification. In a further aspect, the present invention provides an isolated, linear, single-stranded nucleic acid comprising viral nucleic acid fragments obtainable by a method according to present invention. Further, a method for the production of recombinant virus particles, in particular, recombinant AAV particles (rAAV) based on the linear, single-stranded nucleic acid fragments is described herein. Moreover, a plasmid comprising specific nucleic acid sequence elements is provided.

The present invention relates to a separation method comprising: i) providing an aqueous solution comprising a target compound; ii) applying a separation step to the aqueous solution, thereby providing a plurality of fractions of the aqueous solution: iii) determining a concentration parameter indicating a concentration of the target compound in at least part of the fractions; iv) determining a nuclear magnetic resonance (NMR) parameter by applying an NMR measurement to the fractions, the NMR parameter indicating a nuclear magnetic spin relaxation in said at least part of the fractions; and v) determining a target parameter of said at least part of the fractions based on the concentration parameter and the nuclear magnetic resonance parameter. The present invention further relates to separation systems, uses, preparations, and methods related thereto.