Adeno-associated virus (AAV) clade F vector and uses therefor
AssigneeThe Trustees of the University of Pennsylvania
InventorsJames M. Wilson, Qiang Wang, April Tepe, Kevin Turner
Life of this patent
Estimating the term…
Abstract
A recombinant adeno-associated virus (rAAV) vector comprising an AAVhu68 capsid produced in a production system comprising a nucleotide sequence of SEQ ID NO: 1, or a sequence at least 75% identical thereto which encodes SEQ ID NO:2. The AAVhu68 capsid comprises subpopulations of highly deamidated asparagine residues in asparagine-glycine pairs in the amino acid sequence of SEQ ID NO: 2. Also provided are compositions containing the rAAV and uses thereof. Additionally, rAAV having an engineered AAV capsid comprising at least one subpopulation of vp1 or vp2 proteins having a Val at amino acid position 157 with reference to the AAVhu68 vp1 numbering are provided.
Claims7
A method for purifying a rAAVhu68 vector comprising:
- (a) culturing a packaging host cell comprising
- (i) an AAVhu68 capsid nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:2 operably linked to regulatory sequences which direct expression of AAVhu68 VP1 capsid proteins, AAVhu68 VP2 capsid proteins and AAVhu68 VP3 capsid proteins, wherein said capsid proteins which assemble into an AAVhu68 capsid in the cells of the culture;
- (ii) a nucleic acid molecule comprising a vector genome for packaging into the AAVhu68 capsid, said vector genome comprising a 5′ AAV inverted terminal repeat (ITR), a non-AAV nucleic acid sequence encoding a gene product operably linked to regulatory control sequences, and a 3′ AAV ITR;
- (iii) a nucleic acid sequence comprising AAV rep functions and a nucleic acid sequence comprising helper functions for packaging of the vector genome into the AAVhu68 capsid;
- (b) harvesting vector-containing cells and/or culture media comprising rAAVhu68 viral particles and
- one or more of:
- concentrating the rAAVhu68 vector harvest, diafialtering the vector harvest, microfluidizing the vector harvest, nuclease digestion of the vector harvest, filtering the microfluidized intermediate, performing crude purification by chromatography, performing crude purification by ultracentrifugation, performing buffer exchange by tangential flow filtration, and/or formulation and/or filtration to prepare bulk rAAVhu68.
A method for separating recombinant adeno-associated virus hu68 (rAAVhu68) particles having packaged genomic sequences from genome-deficient AAVhu68 intermediates from an rAAVhu68 vector harvest comprising:
- (a) culturing a packaging host cell comprising
- (i) an AAVhu68 capsid nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:2 operably linked to regulatory sequences which direct expression of AAVhu68 VP1 capsid proteins, AAVhu68 VP2 capsid proteins and AAVhu68 VP3 capsid proteins, wherein said capsid proteins which assemble into an AAVhu68 capsid in the cells of the culture;
- (ii) a nucleic acid molecule comprising a vector genome for packaging into the AAVhu68 capsid, said vector genome comprising a 5′ AAV inverted terminal repeat (ITR), a non-AAV nucleic acid sequence encoding a gene product operably linked to regulatory control sequences, and a 3′ AAV ITR;
- (iii) a nucleic acid sequence comprising AAV rep functions and a nucleic acid sequence comprising helper functions for packaging of the vector genome into the AAVhu68 capsid;
- (b) harvesting vector-containing cells and/or culture media comprising rAAVhu68 viral particles and one or more of subjecting a suspension comprising the harvested recombinant AAVhu68 viral particles and AAVhu68 capsid intermediates to fast performance liquid chromatography, equilibrating a strong anion exchange resin at a pH of 10.0 to 10.4 for binding of the AAVhu68 viral particles and AAVhu68 intermediates;
- subjecting the resin to a salt gradient while monitoring eluate for ultraviolet absorbance at about 260 and about 280, and/or monitoring ultraviolet absorbance of the eluate for an inflection point in a ratio of A260/A280 and, collecting the full particles from the resulting fraction.
Dependencies are inferred from the claim text.