Subretinal delivery of adeno‐associated virus serotype 2 results in minimal immune responses that allow repeat vector administration in immunocompetent mice

Background

Adeno‐associated virus serotype 2 (AAV2) vectors show considerable promise for ocular gene transfer. However, one potential barrier to efficacious long‐term therapy is the development of immune responses against the vector or transgene product.

Methods

We evaluated cellular and humoural responses in mice following both single and repeated subretinal administration of AAV2, and examined their effects on RPE65 and green fluorescent protein transgene expression.

Results

Following subretinal administration of vector, splenocytes and T‐cells from draining lymph nodes showed minimal activation following stimulation by co‐culture with AAV2. Neutralizing antibodies (NAbs) were not detected in the ocular fluids of any mice receiving AAV2 or in the serum of mice receiving a lower dose. NAbs were present in the serum of a proportion of mice receiving a higher dose of the vector. Furthermore, no differences in immunoglobulin titre in serum or ocular fluids against RPE65 protein or AAV2 capsid between treated and control mice were detected. Histological examination showed no evidence of retinal toxicity or leukocyte infiltration compared to uninjected eyes. Repeat administration of low‐dose AAV.hRPE65.hRPE65 to both eyes of RPE65^?/? mice resulted in transgene expression and functional rescue, but re‐administration of high‐dose AAV2 resulted in boosted NAb titres and variable transgene expression in the second injected eye.

Conclusions

These data, which were obtained in mice, suggest that, following subretinal injection, immune responses to AAV2 are dose‐dependent. Low‐dose AAV2 is well tolerated in the eye, with minimal immune responses, and transgene expression after repeat administration of vector is achievable. Higher doses lead to the expression of NAbs that reduce the efficacy of repeated vector administration. Copyright © 2009 John Wiley & Sons, Ltd.

     

Environmental Enrichment Enhances Episodic-Like Memory in Association with a Modified Neuronal Activation Profile in Adult Mice

Although environmental enrichment is well known to improve learning and memory in rodents, the underlying neuronal networks'' plasticity remains poorly described. Modifications of the brain activation pattern by enriched condition (EC), especially in the frontal cortex and the baso-lateral amygdala, have been reported during an aversive memory task in rodents. The aims of our study were to examine 1) whether EC modulates episodic-like memory in an object recognition task and 2) whether EC modulates the task-induced neuronal networks. To this end, adult male mice were housed either in standard condition (SC) or in EC for three weeks before behavioral experiments (n = 12/group). Memory performances were examined in an object recognition task performed in a Y-maze with a 2-hour or 24-hour delay between presentation and test (inter-session intervals, ISI). To characterize the mechanisms underlying the promnesiant effect of EC, the brain activation profile was assessed after either the presentation or the test sessions using immunohistochemical techniques with c-Fos as a neuronal activation marker. EC did not modulate memory performances after a 2 h-ISI, but extended object recognition memory to a 24 h-ISI. In contrast, SC mice did not discriminate the novel object at this ISI. Compared to SC mice, no activation related to the presentation session was found in selected brain regions of EC mice (in particular, no effect was found in the hippocampus and the perirhinal cortex and a reduced activation was found in the baso-lateral amygdala). On the other hand, an activation of the hippocampus and the infralimbic cortex was observed after the test session for EC, but not SC mice. These results suggest that the persistence of object recognition memory in EC could be related to a reorganization of neuronal networks occurring as early as the memory encoding.     

High Resolution Ultrasound and Photoacoustic Imaging of Orthotopic Lung Cancer in Mice: New Perspectives for Onco-Pharmacology

ObjectivesWe have developed a relevant preclinical model associated with a specific imaging protocol dedicated to onco-pharmacology studies in mice.ResultsThis study demonstrates the ability of ultrasound imaging to detect and monitor the in vivo orthotopic lung tumor growth by high resolution ultrasound imaging. This approach enabled us to characterize key biological parameters such as oxygenation, perfusion status and vascularization of tumors.ConclusionSuch an experimental approach has never been reported previously and it would provide a nonradiative tool for assessment of anticancer therapeutic efficacy in mice. Considering the absence of ultrasound propagation through the lung parenchyma, this strategy requires the implantation of tumors strictly located in the superficial posterior part of the lung.