Risk Mitigation of Immunogenicity: A Key to Personalized Retinal Gene Therapy.

Gene therapy (GT) for ocular disorders has advanced the most among adeno-associated virus (AAV)-mediated therapies, with one product already approved in the market. The bank of retinal gene mutations carefully compiled over 30 years, the small retinal surface that does not require high clinical vector stocks, and the relatively immune-privileged environment of the eye explain such success. However, adverse effects due to AAV-delivery, though rare in the retina have led to the interruption of clinical trials. Risk mitigation, as the key to safe and efficient GT, has become the focus of 'bedside-back-to-bench' studies. Herein, we overview the inflammatory adverse events described in retinal GT trials and analyze which components of the retinal immunological environment might be the most involved in these immune responses, with a focus on the innate immune system composed of microglial surveillance. We consider the factors that can influence inflammation in the retina after GT such as viral sensors in the retinal tissue and CpG content in promoters or transgene sequences. Finally, we consider options to reduce the immunological risk, including dose, modified capsids or exclusion criteria for clinical trials. A better understanding and mitigation of immune risk factors inducing host immunity in AAV-mediated retinal GT is the key to achieving safe and efficient GT.

Simultaneous determination of eight ß-lactam antibiotics in human plasma and cerebrospinal fluid by liquid chromatography coupled to tandem mass spectrometry.

Therapeutic drug monitoring of ß-lactam antibiotics is increasingly used for dose optimization in the individual patient to increase efficacy and reduce the risk of toxicity. The objective of this work is to develop and validate a fast and reliable method using liquid chromatography coupled to tandem mass spectrometric detection to quantify simultaneously amoxicillin, cloxacillin, cefazolin, cefotaxime, ceftazidime, cefepime, meropenem and piperacillin in plasma and cerebrospinal fluid (CSF). Sample clean-up included protein precipitation with acetonitrile followed by evaporation of the supernatant and reconstitution of the residue with mobile phase solvents. Eight deuterated ß-lactam antibiotics were used as internal standards. Chromatographic separation was performed on a C18 column (50 mm x 2.1 mm) using a binary gradient elution of water and acetonitrile both containing 0.1% (v/v) formic acid. The total run time was 8 min. The method was then used to perform therapeutic drug monitoring on 2221 patient plasma samples. 32 CSF samples were also analyzed. This method, with its simple sample preparation provides sensitive, accurate and precise quantification of the plasma and cerebrospinal fluid concentration of ß-lactam antibiotics and can be used for therapeutic drug monitoring.