John Demirs

AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice.

Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 nullizygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year.

ADIPOR1 is essential for vision and its RPE expression is lost in the Mfrp rd6 mouse

The knockout (KO) of the adiponectin receptor 1 (AdipoR1) gene causes retinal degeneration. Here we report that ADIPOR1 protein is primarily found in the eye and brain with little expression in other tissues. Further analysis of AdipoR1 KO mice revealed that these animals exhibit early visual system abnormalities and are depleted of RHODOPSIN prior to pronounced photoreceptor death. A KO of AdipoR1 post-development either in photoreceptors or the retinal pigment epithelium (RPE) resulted in decreased expression of retinal proteins, establishing a role for ADIPOR1 in supporting vision in adulthood. Subsequent analysis of the Mfrprd6 mouse retina demonstrated that these mice are lacking ADIPOR1 in their RPE layer alone, suggesting that loss of ADIPOR1 drives retinal degeneration in this model. Moreover, we found elevated levels of IRBP in both the AdipoR1 KO and the Mfrprd6 models. The spatial distribution of IRBP was also abnormal. This dysregulation of IRBP hypothesizes a role for ADIPOR1 in retinoid metabolism.

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

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 vectors 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.