Zachary Fitzpatrick

In Vivo Selection Yields AAV-B1 Capsid for Central Nervous System and Muscle Gene Therapy

Adeno-associated viral (AAV) vectors have shown promise as a platform for gene therapy of neurological disorders. Achieving global gene delivery to the central nervous system (CNS) is key for development of effective therapies for many of these diseases. Here we report the isolation of a novel CNS tropic AAV capsid, AAV-B1, after a single round of in vivo selection from an AAV capsid library. Systemic injection of AAV-B1 vector in adult mice and cat resulted in widespread gene transfer throughout the CNS with transduction of multiple neuronal subpopulations. In addition, AAV-B1 transduces muscle, β-cells, pulmonary alveoli, and retinal vasculature at high efficiency. This vector is more efficient than AAV9 for gene delivery to mouse brain, spinal cord, muscle, pancreas, and lung. Together with reduced sensitivity to neutralization by antibodies in pooled human sera, the broad transduction profile of AAV-B1 represents an important improvement over AAV9 for CNS gene therapy.

Extracellular Vesicles as Enhancers of Virus Vector–Mediated Gene Delivery

Extracellular vesicles (EVs) being released from two adjacent adeno-associated virus serotype 1 (AAV1)-producing 293T cells are shown by electron microscopy. We have shown that AAV vectors can associate with EVs and enter the media. Furthermore, we have recently reported that EV-associated AAV has robust gene delivery and antibody evasion properties in vivo.

Enhanced liver gene transfer and evasion of preexisting humoral immunity with exosome-enveloped AAV vectors

Results from clinical trials of liver gene transfer for hemophilia demonstrate the potential of the adeno-associated virus (AAV) vector platform. However, to achieve therapeutic transgene expression, in some cases high vector doses are required, which are associated with a higher risk of triggering anti-capsid cytotoxic T-cell responses. Additionally, anti-AAV preexisting immunity can prevent liver transduction even at low neutralizing antibody (NAb) titers. Here, we describe the use of exosome-associated AAV (exo-AAV) vectors as a robust liver gene delivery system that allows the therapeutic vector dose to be decreased while protecting from preexisting humoral immunity to the capsid. The in vivo efficiency of liver targeting of standard AAV8 or AAV5 and exo-AAV8 or exo-AAV5 vectors expressing human coagulation factor IX (hF.IX) was evaluated. A significant enhancement of transduction efficiency was observed, and in hemophilia B mice treated with 4 × 1010 vector genomes per kilogram of exo-AAV8 vectors, a staggering ∼1 log increase in hF.IX transgene expression was observed, leading to superior correction of clotting time. Enhanced liver expression was also associated with an increase in the frequency of regulatory T cells in lymph nodes. The efficiency of exo- and standard AAV8 vectors in evading preexisting NAbs to the capsid was then evaluated in a passive immunization mouse model and in human sera. Exo-AAV8 gene delivery allowed for efficient transduction even in the presence of moderate NAb titers, thus potentially extending the proportion of subjects eligible for liver gene transfer. Exo-AAV vectors therefore represent a platform to improve the safety and efficacy of liver-directed gene transfer.

Influence of Pre-existing Anti-capsid Neutralizing and Binding Antibodies on AAV Vector Transduction

Pre-existing immunity to adeno-associated virus (AAV) is highly prevalent in humans and can profoundly impact transduction efficiency. Despite the relevance to AAV-mediated gene transfer, relatively little is known about the fate of AAV vectors in the presence of neutralizing antibodies (NAbs). Similarly, the effect of binding antibodies (BAbs), with no detectable neutralizing activity, on AAV transduction is ill defined. Here, we delivered AAV8 vectors to mice carrying NAbs and demonstrated that AAV particles are taken up by both liver parenchymal and non-parenchymal cells; viral particles are then rapidly cleared, without resulting in transgene expression. In vitro, imaging of hepatocytes exposed to AAV vectors pre-incubated with either NAbs or BAbs revealed that virus is taken up by cells in both cases. Whereas no successful transduction was observed when AAV was pre-incubated with NAbs, an increased capsid internalization and transgene expression was observed in the presence of BAbs. Accordingly, AAV8 vectors administered to mice passively immunized with anti-AAV8 BAbs showed a more efficient liver transduction and a unique vector biodistribution profile compared to mice immunized with NAbs. These results highlight a virtually opposite effect of neutralizing and binding antibodies on AAV vectors transduction.

Virus vector-mediated genetic modification of brain tumor stromal cells after intravenous delivery

The malignant primary brain tumor, glioblastoma (GBM) is generally incurable. New approaches are desperately needed. Adeno-associated virus (AAV) vector-mediated delivery of anti-tumor transgenes is a promising strategy, however direct injection leads to focal transgene spread in tumor and rapid tumor division dilutes out the extra-chromosomal AAV genome, limiting duration of transgene expression. Intravenous (IV) injection gives widespread distribution of AAV in normal brain, however poor transgene expression in tumor, and high expression in non-target cells which may lead to ineffective therapy and high toxicity, respectively. Delivery of transgenes encoding secreted, anti-tumor proteins to tumor stromal cells may provide a more stable and localized reservoir of therapy as they are more differentiated than fast-dividing tumor cells. Reactive astrocytes and tumor-associated macrophage/microglia (TAMs) are stromal cells that comprise a large portion of the tumor mass and are associated with tumorigenesis. In mouse models of GBM, we used IV delivery of exosome-associated AAV vectors driving green fluorescent protein expression by specific promoters (NF-κB-responsive promoter and a truncated glial fibrillary acidic protein promoter), to obtain targeted transduction of TAMs and reactive astrocytes, respectively, while avoiding transgene expression in the periphery. We used our approach to express the potent, yet toxic anti-tumor cytokine, interferon beta, in tumor stroma of a mouse model of GBM, and achieved a modest, yet significant enhancement in survival compared to controls. Noninvasive genetic modification of tumor microenvironment represents a promising approach for therapy against cancers. Additionally, the vectors described here may facilitate basic research in the study of tumor stromal cells in situ.Graphical abstract

595. Enhanced Liver Transduction and Efficient Protection from Pre-Existing Neutralizing Antibodies with Exosome-Associated AAV8 Vectors

Adeno-associated virus (AAV) based vectors are ideal tools for in vivo gene transfer. The excellent safety profile and the highly efficient targeting of hepatocytes in vivo have been demonstrated in both preclinical studies and clinical trials. Although AAV vectors are not strongly immunogenic, they can give rise to both cellular and humoral immune responses. Additionally, as a result of exposure to wild-type AAV, an important fraction of humans harbor pre-existing neutralizing antibodies against the viral capsid, which can prevent successful transduction by AAV vectors. This pose a serious obstacle to the widespread use of AAV vectors in gene therapy. Recently, we have shown that AAV can associate with exosomes (exo-AAV) ultracentrifuged media samples from 293T producer cells. Exosome-associated AAV1, 2, and 9 serotype capsids were tested and found to have enhanced transduction and antibody evasion capabilities compared to conventional AAV vectors. With the goal of developing an enhanced vector for liver directed gene therapy, we sought to characterize the in vivo transduction and biodistribution profile of exo-AAV8 compared to conventional cell-lysated harvested, iodixanol-gradient purified AAV8 vectors purified from the same preparation. The efficiency of liver targeting of conventional AAV8 vs. exo-AAV8 expressing human factor IX (F.IX) under the control of liver specific promotor were tested in naive and pre-immunized animals. C57BL/6 mice (n=5/group) were passively immunized with intravenous human immunoglobulin (IVIg) intraperitoneally, followed 24h later by the intravenous administration of either conventional AAV8 or exo-AAV8 vectors expressing coagulation factor IX (F.IX, 5×1010 vg/mouse). exo-AAV8 completely shielded the capsid vector from neutralizing antibodies at IVIg doses between 0.5mg and 2mg/mouse (NAb titer ~ 1:3.16), resulting in equivalent levels of F.IX transgene expression to naive animals treated with conventional AAV8 vector. At the highest IVIg dose tested (8mg/mouse), residual levels of F.IX transgene expression were about 30% of naive animals treated with conventional AAV8 vectors. We next evaluated the efficiency of exo-AAV8 vs. AAV8 vectors in naive animals. In male mice, no statistically significant difference in F.IX transgene expression levels was observed between the two vector types; also indicated by the similar pattern of vector genome biodistribution. Interestingly, female mice (in which the efficiency of liver transduction with AAV is extremely low compared with male animals) treated with exo-AAV8 showed a dramatic increase in transgene expression, comparable to that of male mice receiving conventional AAV8 vectors. In conclusion, exo-AAV8 vectors present an enhanced liver transduction profile compared with conventionally purified AAV vectors, both in naive female animals and in animals carrying anti-capsid neutralizing antibodies.

311. AAV8 Vexosome Vectors Enhance Cell Transduction In Vitro and Outperform Conventional AAV8 Vectors in Liver-Transduction In Vivo in the Presence of Anti-AAV Neutralizing Antibodies

Adeno-associated virus vectors based on serotype 8 (AAV8) have demonstrated superior efficiency of transduction of hepatocytes in vivo in animal models and in humans. Recently, we described extracellular vesicle (exosomes) -associated AAV vectors termed vexosomes (vAAV). We tested vAAV1, 2, and 9 and found them to enhance transduction and antibody evasion capabilities compared to conventional AAV vectors.With the goal of developing an enhanced vector for liver gene therapy, we sought to characterize the in vitro and in vivo transduction, biodistribution, and immunogenicity profile of vAAV8 vectors compared to conventional AAV8 vectors purified from the same preparation.Several cell lines were transduced with conventional AAV8 or vAAV8 vectors expressing luciferase, at multiplicity of infection (MOI) ranging from 250 to 25000, and luciferase expression was measured after an overnight incubation. In HeLa and HEK293 cells, levels of luciferase up to 1 log higher were observed with vAAV8 compared with AAV8 vectors, with detectable levels of luciferase at MOIs as low as 250. In hepatocyte cell lines, the magnitude of enhancement was about 3-fold, with lower max relative light unit signal measured compared to the other cell lines. Notably, although at low levels, transduction with vAAV8 vectors was also observed in a Jurkat T cell line, in which no transduction was observed with conventional AAV8 vectors.We then evaluated the efficiency of liver targeting in vivo with this vector system using the secreted factor IX (F.IX) transgene. Conventional or vAAV8 vectors expressing human F.IX under the control of a liver-specific promoter were administered i.v. to C57BL/6J male mice at 5×1010 vg/mouse. In naive animals, no difference in F.IX transgene expression levels was observed between the two vector types; similarly, vector genome were found at similar levels in liver, spleen, muscle, lungs, heart, and kidneys. In these animals, i.v. vector administration also resulted in identical levels of anti-AAV antibody formation. We next evaluated the effect of vAAV8 on liver transduction in the presence of antibodies. Animals were passively immunized with IVIg intraperitoneally (0.5, 5.0, and 15 mg/mouse) followed by administration of vector (5×1010 vg) intravenously 24 hours later. At a neutralizing antibody titer of~1:10 (0.5mg IVIg), vAAV8 vector-injected mice had levels of expression identical to those in naive animals. At higher titers (>1:100, 5.0 mg), low levels of residual expression of F.IX were measure with vAAV8 vectors and no expression was measured with conventional AAV. No expression was detected with either AAV8 or vAAV8 vectors in animals immunized with the highest dose of IVIg.In conclusion, vAAV8 vectors have unique transduction characteristics that may be exploited in vitro to transduce various cell types. In vivo, these vectors showed identical levels of liver transduction in naive male mice and provided some level of protection from antibody-mediated vector neutralization.