Targeting aav capsids, methods of manufacturing and using same
AssigneeREGENXBIO Inc.
InventorsElad Firnberg, Andrew Mercer
Life of this patent
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Abstract
The present invention relates to recombinant adeno-associated viruses (rAAVs) having either wild-type capsid proteins or capsid proteins engineered to be atropic or have limited tropism which may be further engineered by insertion of a DARPin targeting element that confers and/or enhances desired properties, particularly increased transduction in CNS or muscle cells or other target tissue relative to a rAAV having a reference capsid.
Claims74
A method of producing a recombinant AAV (rAAV) particle, wherein the rAAV particle comprises a capsid and an artificial genome, wherein the capsid comprises at least one rAAV capsid protein comprising a DARPin which is displayed on the surface of the capsid, wherein the method comprises:
- culturing a cell comprising one or more polynucleotides, wherein the one or more polynucleotides comprise:
- (a) one or more polynucleotides encoding VP1, VP2 and VP3 proteins;
- wherein at least one polynucleotide encodes a VP3-DARPin protein, which comprises a VP3 protein having a DARPin inserted within VR-IV or VR-VIII of the VP3 protein:
- (b) a polynucleotide encoding afunctional rep gene;
- (c) a polynucleotide comprising the artificial genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to a regulatory control element which directs expression of the gene product in a target cell;
- and
- (d) one or more polynucleotides encoding sufficient helper functions to permit packaging of the artificial genome into the AAV capsid protein under conditions which permit packaging of the genome into the AAV capsid:
- wherein the cell is cultured under conditions that allow production of the recombinant AAV (rAAV) particle.
A method of producing a recombinant AAV (rAAV) particle, wherein the rAAV particle comprises a capsid and an artificial genome, wherein the capsid comprises at least one rAAV capsid protein comprising a DARPin which is displayed on the surface of the capsid, wherein the method comprises:
- culturing a cell comprising one or more polynucleotides, wherein the one or more polynucleotides comprise:
- (a) one or more polynucleotides encoding VP1, VP2 and VP3 proteins;
- wherein at least one polynucleotide encodes a VP1-DARPin protein, which comprises a VP1 protein having a DARPin inserted within VR-IV or VR-VIII of the VP1 protein:
- (b) a polynucleotide encoding afunctional rep gene;
- (c) a polynucleotide comprising the artificial genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to a regulatory control element which directs expression of the gene product in a target cell;
- and
- (d) one or more polynucleotides encoding sufficient helper functions to permit packaging of the artificial genome into the AAV capsid protein under conditions which permit packaging of the genome into the AAV capsid;
- wherein the cell is cultured under conditions that allow production of the recombinant AAV (rAAV) particle.
A method of producing a recombinant AAV (rAAV) particle, wherein the rAAV particle comprises a capsid and an artificial genome, wherein the capsid comprises at least one rAAV capsid protein comprising a DARPin which is displayed on the surface of the capsid, wherein the method comprises:
- culturing a cell comprising one or more polynucleotides, wherein the one or more polynucleotides comprise:
- (a) one or more polynucleotides encoding VP1, VP2 and VP3 proteins;
- wherein at least one polynucleotide encodes a VP2-DARPin protein, which comprises a VP2 protein having a DARPin inserted within or at the VR-IV or VR-VIII of the VP2 protein;
- (b) a polynucleotide encoding afunctional rep gene;
- (c) a polynucleotide comprising the artificial genome comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to a regulatory control element which directs expression of the gene product in a target cell;
- and
- (d) one or more polynucleotides encoding sufficient helper functions to permit packaging of the artificial genome into the AAV capsid protein under conditions which permit packaging of the genome into the AAV capsid;
- wherein the cell is cultured under conditions that allow production of the recombinant AAV (rAAV) particle.
A recombinant AAV capsid protein comprising an insertion of a DARPin which targets a cell surface molecule and wherein the DARPin is flanked on at least one end by a linker, wherein a recombinant AAV particle incorporating the recombinant AAV capsid protein has cell transduction activity.
Dependencies are inferred from the claim text.