605. Residual Cesium Chloride in AAV Vectors Purified by CsCl Gradient Centrifugation Does Not Cause Obvious Inflammation or Retinal Degeneration in C57Bl6/J Mice Following Subretinal Injection
Authors: Karen Guerin, Vivian Choi, Jorge Aranda, John Demirs, Hui Li, Junzheng Yang, Niem Nguyen, Steve Bottega, Bruce Jaffee, Ted Dryja, Seshidhar Police
Journal: Molecular Therapy (2015)

Cesium chloride density gradient ultracentrifugation is the conventional method of purification for adeno-associated viral vectors. This method is often preferred because it allows for good separation of full and empty vector particles, and because it can be adjusted to purify any AAV serotype with minimal optimization. AAV vectors purified by CsCl gradient centrifugations have been evaluated in many preclinical studies both in small and large animals, and in clinical studies for ophthalmology disease indications. Most CsCl is removed at the end of the purification process when buffer exchange is done by dialysis, but the level of residual CsCl has not been reported. Furthermore, there has been little or no evaluation of the potential toxicity of residual CsCl in the final AAV product. The purpose of this work was to measure the amount of residual CsCl in purified AAV vectors and investigate its potential toxicity following subretinal injection in C57Bl6/J mice.CsCl in 20 purified AAV vectors was measured by inductively coupled plasma mass spectrometry. The amount of CsCl delivered with a subretinal injection of 1×109 vector genomes was calculated for each vector based on a 1-μ1 injection volume and the vector's titer. CsCl levels were found to be in the range of 0.3 ng to 5 ng per vector dose. To determine whether CsCl is toxic at this level, we performed a dose-response study in which our formulation buffer was spiked with known amounts of CsCl, ranging from 0-100 ng per injection. We used fundus imaging and microglial cell counts to evaluate inflammatory responses and H&E staining of paraffin-embedded eyes for histopathology assessment 6 weeks post injection. The lowest level of residual CsCl measured in our AAV vector preps was on the order of 0.3 ng in a dose of 1×109vg. Extended buffer exchanges did not result in complete removal of CsCl from the vector preparations, suggesting that CsCl may be associated with the AAV particles. A dose-response study with CsCl-purified AAV vectors was carried out to determine whether residual CsCl in the presence of AAV could be detrimental. Null AAV2 and AAV8 vectors were tested at three different doses (1×108 vg, 5×108 vg, 1×109 vg/eye). No evidence of inflammation or damage to the retina or RPE could be seen on the fundus images. These observations were confirmed by the histopathology assessment and a count of the number of microglial cells in the retina and posterior eyecup. We did not see evidence of inflammation, even at the highest dose of 100 ng of CsCl. On H&E-stained sections retinal thinning was seen only at the inferred injection sites, and this observation was consistent across all dose groups. Finally we tested a null vector purified by affinity chromatography, a CsCl-free process. As in our previous studies we found no evidence of inflammation or retinal damage. In conclusion we did not find evidence that residual CsCl from an AAV purification process is toxic to the retina following subretinal injection.