The present disclosure pertains to compositions and methods for the production of recombinant adeno-associated viruses (rAAVs).

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.

This invention relates to a process for producing a library of viruses, comprising first and second culturing steps. These steps aim to promote intra-species and inter-species recombination, respectively, between double-stranded DNA viruses of the same virus family.

The invention relates to a nucleic acid molecule encoding at least one AAV Rep polypeptide, wherein one or more of the AAV (p5), (p19) and (p40) promoters have been modified to reduce or eliminate expression of one or more of the Rep polypeptides, or the nucleic acid molecule does not encode functional (Rep52) or (Rep40) polypeptides, or the nucleic acid molecule does not encode a functional adenovirus inhibitor sequence. The invention also relates to a process for producing recombinant AAV vectors through the use of a 2-adenovirus system, wherein all of the genes required for AAV replication and packaging (i.e. an AAV rep sequence of the invention, AAV cap and the AAV transfer vector comprising a transgene) may be encoded within two adenoviruses.

Provided herein are engineered transcription factors for selective upregulation of SCN1a and uses thereof for treating diseases and disorders, such as, Dravet syndrome. Also provided are microRNA binding sites and uses thereof for selective expression in parvalbumin neurons.

The presently disclosed subject matter provides compositions and methods comprising improvements of a CRISPR system (e.g. CRISPR associated (Cas) 9 (CRISPR-Cas9, non-Cas9 CRISPR systems). Such compositions may comprise modifications to the H1 promoter region, addition of 5′UTR modifications, different orthologous sequences of the H1 promoter, novel compact bidirectional promoter sequences with both pol II and pol III activity, addition of Kozak consensus sequences, termination sequences, addition of conditional pol II/pol III bidirectional promoter expression, addition of a donor template sequence for correcting mutations, or combinations thereof. Other aspects of the invention relate to modifications to Cas9 through post-transcriptional cell-cycle regulation fusions, engineered partial target sites such that the nuclease can bind without DNA cleavage, auto-regulation sites, and N-terminal modifications to modulate half-life.

Disclosed herein are packaging cell lines, in which adenovirus (Ad) E1A is constitutively expressed, that also contain integrated AAV rep and cap genes. The packaging cell lines exhibit little to no expressed Rep protein until helper virus function, such as adenovirus (Ad) E4, E2A and/or VA RNA are provided by, for example, transduction of the cells with a virus, vector or plasmid, such as an Ad-AAV hybrid virus. The promoter driving expression of AAV rep gene can be positioned far enough upstream (5′) of the rep coding sequence that E1A is unable to activate the promoter, activate substantial transcription of the rep gene and in turn produce Rep protein. Introduction of helper virus function, such as E2A, E4 and/or VA RNA into these packaging cells is able to drive AAV rep gene transcription, subsequent Rep protein expression and production of rAAV vector particles.

The present invention relates to a process for producing a library of recombinant adeno-associated vims (AAV) particles which are encapsidated by a variety of different capsid polypeptides. Each recombinant AAV particle in the library comprises an AAV genome which comprises AAV ITRs flanking a first AAV cap gene encoding a first Cap polypeptide, wherein the particles in the library differ in the nucleotide sequences of their first AAV cap genes, and wherein each particle in the library is encapsidated by a Cap polypeptide which is encoded by the cap gene within its AAV genome. The library may be used to screen for recombinant AAV particles which have high specificity for cells of a target therapeutic tissue.

Expression constructs and methods for recombinant parvovirus production are disclosed. In some embodiments, the expression construct encodes (1) adenovirus E4 and E2a proteins, (2) parvovirus proteins necessary for the production of the recombinant parvovirus, and (3) a recombinant parvovirus genome, thus allowing production of the recombinant parvovirus by transfecting a host cell with a single expression construct.

Provided is an oncolytic virus expressing interferon. Specifically provided is an oncolytic adenovirus comprising a fusion protein expressing interferon, said oncolytic virus being capable of effectively suppressing tumors in vitro and in vivo.