![]() CAPTO CORE 400 FULLAgain, all of these strategies are working towards the end goal of increasing DNA replication within the production cell, improving packaging (or the number of full capsids that are produced in a system), and increasing the viral particle (VP) or capsid protein ratio in order to create a product that is more infectious.Īdditional recent research has focused on modulating gene expression within the cell line to create a more favorable environment for AAV production. Furthermore, research focused on codon optimization of both the helper and AAV replication sequences is ongoing. Another strategy employed to improve control of the production cascade is modifying the amount of the replication proteins that are present, either through modifying the start codons, or using alternative constitutive or inducible promoters to better control which of those Rep proteins are expressed, and the timing of their expression within the cell. One of the first modifications typically introduced to upstream bioprocesses is modifying the ratios of the plasmids or vector components that are going into the cell, with the goal of identifying the ideal amount needed for each specific serotype or Cis sequence to increase production of full capsids. Many of these begin with modifying the replication helper or capsid sequences. Multiple strategies have been employed to improve both AAV production and product quality. While research has continued to improve overall recombinant AAV production, the titers that are reported from the above systems are generally observed to be 5 to 10 times lower in productivity per cell than wild-type AAV, indicating that there are still learnings to be gleaned from the wild-type virus to help drive improvements in recombinant production systems. Furthermore, both adherent and suspension platforms are frequently employed in this production process. ![]() In addition, there are multiple cell types that are utilized, including HEK293, BHK, HeLa and insect Sf9 cells. ![]() In some production systems, these sequences are packaged into recombinant viruses such as herpes, adenovirus, or baculovirus, which then transduce the production cell to initiate the production cascade. The most frequently used is plasmid transfection, where 2 to 3 different plasmids and DNA sequences are transfected into the cell. The ratios of these different viral and cellular helper proteins, as well as the AAV2 replication protein, help to dictate the overall number of particles packaged, the number of particles that contain DNA, and often the integrity or completeness of the genome that’s packaged within these capsids.Ĭurrently, multiple methods are utilized to deliver each of these helper components and replication and capsid sequences into the cell. ![]() The replicated DNA sequences are then packaged into the AAV capsid and harvested from the cells and/or supernatant through the purification process. These proteins are critical in AAV production, assisting in multiple functions including limiting replication of the packaging cell, expressing viral capsid proteins, and increasing production of the Cis DNA sequences. When these helper virus sequences are stressed, they trigger expression of both cellular factors critical in AAV production and activate the four different AAV Rep proteins (48, 52, 68, 78).
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