Methods for Producing Populations of High Titer Recombinant Adeno-Associated Virus (rAAV) Lacking Prokaryotic Sequences are disclosed.

A method for reducing heterogeneity of a virus preparation may include generating virus ions from the virus preparation, repeatedly increasing at least one of a temperature and an incubation period at the increased temperature of at least one of the virus preparation and the generated virus ions, measuring mass-to-charge ratios and charge magnitudes of at least some of the generated virus ions at each increase of the at least one of the temperature and the incubation period, determining a mass spectrum at each increase of the at least one of the temperature and the incubation period based on values of the respective mass-to-charge ratios and charge magnitudes, and determining, based on the mass spectrums, optimum ones of the temperature and the incubation period which together minimize, or at least reduce, a heterogeneity of the virus preparation without aggregation of virus capsids in the virus preparation.

Nucleic acids encoding fusion proteins that contain an unwanted antigen and a leader sequence for cell secretion are described. Also described are expression cassettes, vectors, cells, and cell lines containing the nucleic acids, as well as methods of using the nucleic acids to treat autoimmune, allergic and other diseases and disorders, such as multiple sclerosis.

A method of preparing a recombinant adeno-associated virus (rAAV) including: (1) preparing a shuttle plasmid and a corresponding recombinant bacmid including a baculovirus genome, where the shuttle plasmid includes at least an rAAV gene of interest flanked by inverted terminal repeats (ITR-GOI) integrated with a heterologous functional gene fragment, and the recombinant bacmid includes an expression cassette of functional protein components necessary for assembly of the rAAV; (2) integrating the rAAV ITR-GOI and the expression cassette of functional protein components by using the shuttle plasmid and the recombinant bacmid, to yield a recombinant bacmid including a recombinant baculovirus genome; and (3) transfecting, with the recombinant bacmid, a host cell line.

The present invention relates to a nucleic acid sequence comprising a nucleotide of interest and a tryptophan RNA-binding attenuation protein (TRAP) binding site, and optionally a Kozak sequence, wherein said TRAP binding site overlaps the Kozak sequence and/or the ATG start codon of the nucleotide of interest. The present invention further relates to a nucleic acid sequence comprising a nucleotide of interest and a Kozak sequence, wherein said Kozak sequence comprises a portion of a tryptophan RNA-binding attenuation protein (TRAP) binding site. The present invention further relates to a nucleic acid sequence comprising a nucleotide of interest and TRAP binding site wherein the TRAP binding site comprises a portion of the start codon ATG of said nucleotide of interest or wherein the ATG start codon comprises a portion of the TRAP binding site. The present invention further relates to a nucleic acid sequence comprising a nucleotide of interest, a binding site for tryptophan RNA-binding attenuation protein (TRAP), a multiple cloning site and a Kozak sequence, wherein said multiple cloning site is overlapping with or located downstream to the 3′ KAGN2-3 repeat of the TRAP binding site and upstream of the Kozak sequence.

A two-step chromatography purification scheme is described which selectively captures and isolates the genome-containing rAAV vector particles from the clarified, concentrated supernatant of a rAAV production cell culture. The process utilizes an affinity capture method performed at a high salt concentration followed by an anion exchange resin method performed at high pH to provide rAAV vector particles which are substantially free of rAAV intermediates.

A method for reducing a contaminating DNA content of a preparation containing AAV capsids and contaminating DNA, comprising the steps of a) Performing an extraction of DNA with a solid phase bearing positive charges at its surface said solid phase is contacted with the preparation at a pH of 7.0±1.0, and a salt concentration of 10 mM to 200 mM yielding a first fraction, (b) Diafiltering the first fraction by a first tangential flow filtration to obtain a second fraction, (c) Treating the second fraction with DNase, (d) Diafiltering the DNase treated second fraction obtained by step c) by a second tangential flow, (e) filtration to a buffer with pH of 7.0±1.0, and a salt concentration of 10 mM to 20 mM to yield a third fraction, and optionally (f) Concentrating the third fraction by tangential flow filtration before supplemental chromatography.

In accordance with the invention, provided herein are methods for purifying recombinant adeno-associated (rAAV) vector particles.

A method for the systemic delivery of a polypeptide within a subject is provided by creating genetically modified skin cells via topical introduction of a genetically engineered virus which delivers a nucleic acid encoding a therapeutic polypeptide for expression by the skin cells, wherein the expressed therapeutic polypeptide is secreted by the skin cells and is introduced into the circulatory system of the subject.

The present invention provides a polyploid adeno-associated virus (AAV) capsid, wherein the capsid comprises capsid protein VP1, wherein said capsid protein VP1 is from one or more than one first AAV serotype, wherein said capsid protein VP2 is from one or more than one first AAV serotype and capsid protein VP3, wherein said capsid protein VP3 is from one or more than one second AAV serotype and wherein at least one of said first AAV serotype is different from at least one of said second AAV serotype and is different from at least one of said third AAV serotype, in any combination.