Methods and systems of PCR-based recombinant adeno-associated virus manufacture

1. A method of manufacturing amplicon polynucleotides containing the sequence motif [ITR-cargo-ITR] via the polymerase chain reaction (PCR), said method comprising:

performing a PCR amplification reaction on a desired template DNA sequence containing a [ITR-cargo-ITR] DNA sequence motif, wherein the ITR is the wild type AAV ITR,

wherein the PCR amplification reaction uses a PCR primer pair such that the 3′ terminus of both the forward and reverse PCR primers overlap only about the last 2-8 bases of the A/A′ ITR sequences and the 5′ terminus of both the forward and reverse PCR primers extend into about 20-35 bases of the flanking sequences,

wherein PCR cycling parameters comprise a combined annealing/extension step at a temperature greater than 70° C.,

wherein the PCR amplification reaction contains one or more osmolytes, wherein an osmolyte is betaine at a concentration of between 0.01 and 1M,

wherein the placement of the primers, the combined annealing/extension step, and betaine destabilize the ITR fold,

thereby producing a plurality of amplicon polynucleotides containing the desired [ITR-cargo-ITR] DNA sequence motif.

2. The method of claim 1 , wherein the flanking regions have been designed for high-affinity PCR primer binding.

3. The method of claim 1 , wherein the concentration of betaine used in the PCR amplification reaction is between 0.5 and 0.75M.

4. The method of claim 1 wherein the [ITR-cargo-ITR] sequence is derived from a plasmid.

5. The method of claim 1 wherein the [ITR-cargo-ITR] sequence is not derived from a plasmid.

6. The method of claim 1 wherein PCR cycling parameters further comprises an additional annealing/extension time at a temperature greater than 70° C. for a duration longer than 3 minutes.

7. A method of manufacturing sequence verified amplicon polynucleotides containing the DNA sequence motif [ITR-cargo-ITR] via the polymerase chain reaction (PCR), said method comprising:

performing a PCR amplification reaction on a desired template DNA sequence containing a [ITR-cargo-ITR] DNA sequence motif, wherein the ITR is the wild type AAV ITR,

wherein the PCR amplification reaction uses a PCR primer pair such that the 3′ terminus of both the forward and reverse PCR primers overlap only about the last 2-8 bases of the A/A′ ITR sequences and the 5′ terminus of both the forward and reverse PCR primers extend into about 20-35 bases of the flanking sequences,

wherein PCR cycling parameters comprise a combined annealing/extension step at a temperature greater than 70° C., wherein the PCR amplification reaction contains one or more osmolytes, wherein an osmolyte is betaine at a concentration of between 0.01 and 1M, wherein the placement of the primers, the combined annealing/extension step, and betaine destabilize the ITR fold;

producing a plurality of amplicon polynucleotides containing the desired [ITR-cargo-ITR] DNA sequence motif;

pooling a representative sample of said amplicon polynucleotides containing the desired [ITR-cargo-ITR] DNA sequence motif; and

subjecting said pool representative sample of said amplicon polynucleotides containing the desired [ITR-cargo-ITR] DNA sequence motif to next generation sequencing (NGS).

8. The method of claim 7 , wherein the concentration of betaine used in the PCR amplification reaction is between 0.5 and 0.75M.

9. The method according to claim 1 , wherein the ITR is the wild type AAV ITR (SEQ ID NO: 4).

10. The method according to claim 1 , wherein the 3′ terminus of both the forward and reverse PCR primers overlap only the last 2 bases of the A/A′ ITR sequences.

11. The method according to claim 7 , wherein the ITR is the wild type AAV ITR (SEQ ID NO: 4).

12. The method according to claim 7 , wherein the 3′ terminus of both the forward and reverse PCR primers overlap only the last 2 bases of the A/A′ ITR sequences.