Optimization of Dexamethasone Administration for Maintaining Global Transduction Efficacy of Adeno-Associated Virus Serotype 9.

Glucocorticoids have been commonly used in clinic for their anti-inflammatory and immunosuppressive effects, and it has been proposed that they be used to prevent liver toxicity when systemic administration of adeno-associated virus (AAV) vectors is needed in patients with central nervous system diseases and muscular disorders. Glucocorticoids also enable modulation of vascular permeability. First, this study investigated the impact of dexamethasone on AAV vascular permeability after systemic injection. When a low dose of AAV9 was injected into mice treated with dexamethasone, global transduction and vector biodistribution were not significantly different in most tissues, other than the liver and the heart, when compared to control mice. When AAV9 vectors were used at a high dose, both the transgene expression and the AAV vector genome copy number were significantly decreased in the majority of murine tissues. However, no effect on global transduction was observed when dexamethasone was administered 2 h after AAV vector injection. The study on the kinetics of AAV virus clearance demonstrated that dexamethasone slowed down the clearance of AAV9 in the blood after systemic application. The mechanism study showed that dexamethasone inhibited the enhancement of AAV9 vascular permeability mediated by serum proteins. The findings indicate that dexamethasone is able to inhibit the vascular permeability of AAV and compromise the therapeutic effect after systemic administration of AAV vector. In conclusion, this study provides valuable information for the design of future clinical studies when glucocorticoids are needed to be compatible with the systemic administration of AAV vectors in patients with central nervous system and muscular diseases.

Cryoprecipitate augments the global transduction of the adeno-associated virus serotype 9 after a systemic administration.

Adeno-associated virus (AAV) vectors have been successfully used for transgene delivery in clinical trials. A systemic administration of AAV vectors has been proposed in order to achieve global transduction, which requires that the AAV vector is capable of crossing the blood vessels. It has been demonstrated that serum proteins are able to directly interact with AAV virions to enhance liver transduction. In this study, we investigate whether the serum proteins have the potential to increase the capacity of AAV to diffuse through the endothelial cells and deliver the transgene into the whole body. First, we found that the direct interaction of serum with AAV9 virions increased the epithelial cell permeability of AAV9 in vitro. Several serum proteins with a potential effect on AAV vascular permeability have been identified from mass spectrometry analysis, including fibrinogen, fibronectin, von Willebrand factor (vWF), platelet factor 4, alpha-1-acid glycoprotein, and plasminogen. The incubation of these serum proteins with AAV9 enhanced the global transduction in mice after a systemic administration. To apply these findings in clinical practice, we demonstrated that the clinical product cryoprecipitate (mainly containing fibrinogen and vWF) augmented AAV9 global transduction. The mechanism study revealed that cryoprecipitate slowed down the clearance of AAV9 vectors in the blood so that the AAV9 vectors had sufficient time to travel to the peripheral organs. In summary, the results from this study suggests that serum proteins interact with AAV virions and enhance the AAV9 vascular permeability for global transduction, and, more importantly, cryoprecipitate can be immediately applied for clinical patients who need the systemic administration of AAV vectors for global transduction.

Application of polyploid adeno-associated virus vectors for transduction enhancement and neutralizing antibody evasion.

Adeno-associated virus (AAV) vectors have been used successfully in clinical trials for patients with hemophilia or blindness, but pre-existing neutralizing antibodies (Nab) are common in the general population and exclude many patients from clinical trials. Exploration of effective strategies to enhance AAV transduction and escape from Nab activity is still imperative. Previous studies have shown the compatibility of capsids from AAV serotypes and homology of recognition sites of AAV Nab located on different capsid subunits from one virion. In this study, we co-transfected AAV2 and AAV8 helper plasmids at different ratios (3:1, 1:1 and 1:3) to assemble haploid capsids and study both their transduction efficiency and Nab escape activity. After muscular injection, all of the haploid viruses induced higher transduction than their parental AAV vectors (2- to 9-fold over AAV2), with the highest of these being the haploid vector AAV2/8 3:1. After systemic administration, a 4-fold higher transduction in the liver was observed with haploid AAV2/8 1:3 than that with AAV8 alone. We then packaged the therapeutic factor IX cassette into haploid AAV2/8 1:3 capsids and injected them into FIX knockout mice via the tail vein. Higher FIX expression and improved phenotypic correction were achieved with the haploid AAV2/8 1:3 virus vector when compared to that of AAV8. Additionally, the haploid virus AAV2/8 1:3 was able to escape AAV2 neutralization and did not increase capsid antigen presentation capacity when compared to AAV8. To improve the Nab evasion ability of the haploid virus, we produced the triploid vector AAV2/8/9 by co-transfecting AAV2, AAV8 and AAV9 helper plasmids at a ratio of 1:1:1. After systemic administration, a 2-fold higher transduction in the liver was observed with the triploid vector AAV2/8/9 than that with AAV8. Nab analysis demonstrated that the triploid AAV2/8/9 vector was able to escape Nab activity from mouse sera immunized with parental serotypes. These results indicate that polyploid viruses might potentially acquire advantages from parental serotypes for enhancement of AAV transduction and evasion of Nab recognition without increasing capsid antigen presentation in target cells. Polyploid AAV vectors can be generated from any AAV serotype, whether natural, rational, library derived or a combination thereof, providing a novel strategy that should be explored in future clinical trials in patients with neutralizing antibodies.