Systems for Amplification of AAV Rep and Cap Proteins

A vector system for providing amplification of expression of adenovirus associated virus (AAV) Rep and capsid proteins in a cell and production of recombinant AAV (rAAV) virions from the cell,

• (I) the vector system comprising:
• a first circular polynucleotide construct comprising a viral origin of replication, one or more promoters operably linked to a polynucleotide comprising a sequence encoding one or more AAV Rep proteins and to a polynucleotide comprising a sequence encoding one or more AAV capsid proteins;
• a second polynucleotide construct comprising a promoter operably linked to a polynucleotide comprising a sequence encoding one or more AAV helper proteins and/or VA RNAs;
• and
• a third polynucleotide construct comprising a polynucleotide comprising a sequence encoding a payload flanked by AAV inverted terminal repeats (ITRs),
• wherein either the second polynucleotide construct or the third polynucleotide construct further comprises a promoter operably linked to a polynucleotide comprising a sequence encoding a replicase compatible with the viral origin of replication or wherein the vector system comprises a fourth construct comprising a promoter operably linked to a polynucleotide comprising a sequence encoding a replicase compatible with the viral origin of replication, and
• wherein the replicase amplifies the first circular polynucleotide construct resulting in amplification of expression of AAV Rep and AAV cap protein in the cell;
• or
• (II) the vector system comprising:
• a first polynucleotide construct comprising a first recombination site, a viral origin of replication, one or more promoters operably linked to a polynucleotide comprising a sequence encoding one or more AAV Rep proteins and a polynucleotide comprising a sequence encoding one or more AAV capsid proteins, and a second recombination site;
• a second polynucleotide construct comprising a promoter operably linked to a polynucleotide comprising a sequence encoding one or more AAV helper proteins and/or VA RNA(s);
• and
• a third polynucleotide construct comprising a sequence encoding a payload flanked by AAV inverted terminal repeats (ITRs), wherein the first, second, or third polynucleotide construct further comprises a promoter operably linked to a polynucleotide comprising a sequence encoding a replicase compatible with the viral origin of replication and the first, second or third polynucleotide construct further comprises a promoter operably linked to a polynucleotide comprising a sequence encoding a recombinase, wherein the recombinase recombines the first and second recombination sites thereby producing a circular polynucleotide construct comprising the viral origin of replication, the one or more promoters operably linked to the polynucleotide comprising the sequence encoding one or more AAV Rep proteins and the polynucleotide comprising the sequence encoding one or more AAV capsid proteins, and wherein the replicase amplifies the first circular polynucleotide construct resulting in amplification of expression of AAV Rep and AAV cap protein in the cell.

A polynucleotide construct for inducibly amplifying expression of AAV Rep and cap proteins, the polynucleotide construct comprising:

• a first excisable sequence comprising a first recombination site, a viral origin of replication, one or more promoters operably linked to a first part of an AAV Rep coding region, a second excisable sequence comprising a third recombination site and a fourth recombination site flanking a sequence encoding a stop signal, a second part of the AAV Rep coding region, a promoter operably linked to a sequence encoding one or more AAV capsid proteins, a second recombination site, wherein the first, second, third, and fourth recombination sites are oriented in the same direction,
• wherein excision of the second excisable sequence by recombination of the third and fourth recombination sites by an inducible recombinase generates a complete AAV Rep coding region,
• wherein recombination of the first and second recombination sites results in excision of the first excisable sequence to form a circular polynucleotide construct comprising the viral origin of replication, the one or more promoters operably linked to a complete AAV Rep coding region encoding one or more AAV Rep proteins, the promoter operably linked to the sequence encoding the one or more AAV capsid proteins, and
• wherein replication of the circular polynucleotide results in amplification of expression of the one or more AAV Rep proteins and the one or more AAV capsid proteins.

A vector system for inducible amplification of expression of AAV Rep and cap proteins and for inducible production of rAAV, the vector system comprising:

• (i) a first polynucleotide construct comprising:
• a first excisable sequence comprising a first recombination site, a viral origin of replication, one or more promoters operably linked to a first part of an AAV Rep coding region, a second excisable sequence comprising a third recombination site and a fourth recombination site flanking a sequence encoding a stop signal, a second part of the AAV Rep coding region, a promoter operably linked to a sequence encoding one or more AAV capsid proteins, a second recombination site, wherein the first, second, third, and fourth recombination sites are oriented in the same direction,
• wherein excision of the second excisable sequence by recombination of the third and fourth recombination sites by a recombinase generates a complete AAV Rep coding region,
• wherein recombination of the first and second recombination sites results in excision of the first excisable sequence to form a circular polynucleotide construct comprising the viral origin of replication, the one or more promoters operably linked to a complete AAV Rep coding region encoding one or more AAV Rep proteins, the promoter operably linked to the sequence encoding the one or more AAV capsid proteins, and
• wherein replication of the circular polynucleotide construct results in amplification of expression of the one or more AAV Rep proteins and the one or more AAV capsid proteins;
• and
• (ii) a second polynucleotide construct comprising an inducible promoter operably linked to a sequence encoding a replicase.

A method for increasing production of rAAV virions from a cell, the method comprising:

• amplifying expression of AAV Rep and capsid proteins in the cell, wherein the amplifying comprises:
• increasing copy number of a polynucleotide construct comprising a sequence encoding one or more AAV Rep proteins and a sequence encoding one or more AAV cap proteins;
• introducing one or more CRISPR activators to amplify expression of the Rep/Cap genes;
• and/or
• introducing an agent to amplify expression of the Rep/Cap genes.

A vector system for inducible amplification of expression of a protein and for inducible production of the protein, the vector system comprising:

• (i) a first polynucleotide construct comprising:
• a first excisable sequence comprising a first recombination site, a viral origin of replication, a promoter operably linked to a first part of a a protein coding region, a second excisable sequence comprising a third recombination site and a fourth recombination site flanking a sequence encoding a stop signal, a second part of the protein coding region, a second recombination site, wherein the first, second, third, and fourth recombination sites are oriented in the same direction,
• wherein excision of the second excisable sequence by recombination of the third and fourth recombination sites by a recombinase generates a complete protein coding region,
• wherein recombination of the first and second recombination sites results in excision of the first excisable sequence to form a circular polynucleotide construct comprising the viral origin of replication, the promoter operably linked to a complete protein coding region, and
• wherein replication of the circular polynucleotide construct results in amplification of expression of the protein;
• and
• (ii) a second polynucleotide construct comprising an inducible promoter operably linked to a sequence encoding a replicase;
• optionally wherein the protein is a therapeutic protein, further optionally wherein the protein is an antibody or any fragment or derivative thereof.

A vector system for providing amplification of expression of a protein in a cell and production of the protein from the cell, the vector system comprising:

• a first polynucleotide construct comprising a first recombination site, a viral origin of replication, an inducible promoter operably linked to a polynucleotide comprising a sequence encoding the protein, and a second recombination site;
• a second polynucleotide construct comprising an inducible promoter operably linked to a polynucleotide comprising a sequence encoding for a recombinase;
• and
• wherein the first or second polynucleotide construct further comprises a promoter operably linked to a polynucleotide comprising a sequence encoding a replicase compatible with the viral origin of replication,
• wherein the recombinase recombines the first and second recombination sites thereby producing a circular polynucleotide construct comprising the viral origin of replication, the inducible promoter operably linked to the polynucleotide comprising the sequence encoding the protein, and wherein the replicase amplifies the first circular polynucleotide construct resulting in amplification of expression of the protein in the cell.