Bioengineered Hybrid Rep 2/6 Gene Improves Encapsulation of a Single-Stranded Expression Cassette into AAV6 Vectors.

The production of clinical-grade recombinant adeno-associated viral (AAV) vectors for gene therapy trials remains a major hurdle in the further advancement of the gene therapy field. During the past decades, AAV research has been predominantly focused on the development of new capsid modifications, vector-associated immunogenicity, and the scale-up vector production. However, limited studies have examined the possibility to manipulate non-structural components of AAV such as the Rep genes. Historically, naturally isolated, or recombinant library-derived AAV capsids have been produced using the AAV serotype 2 Rep gene to package ITR2-flanked vector genomes. In the current study, we mutated four variable amino acids in the conservative part of the binding domain in AAV serotype 6 Rep to generate a Rep2/6 hybrid gene. This newly generated Rep2/6 hybrid had improved packaging ability over wild-type Rep6. AAV vectors produced with Rep2/6 exhibited similar in vivo activity as standard AAV6 vectors. Furthermore, we show that this Rep2/6 hybrid also improves full/empty capsid ratios, suggesting that Rep bioengineering can be used to improve the ratio of fully encapsulated AAV vectors during upstream manufacturing processes.

Extracellular vesicles enhance pulmonary transduction of stably associated adeno-associated virus following intratracheal administration.

Adeno-associated virus (AAV) vector has shown multiple clinical breakthroughs, but its clinical implementation in inhaled gene therapy remains elusive due to difficulty in transducing lung airway cells. We demonstrate here AAV serotype 6 (AAV6) associated with extracellular vesicles (EVs) and secreted from vector-producing HEK-293 cells during vector preparation (EVAAV6) as a safe and highly efficacious gene delivery platform for inhaled gene therapy applications. Specifically, we discovered that EVAAV6 provided markedly enhanced reporter transgene expression in mucus-covered air-liquid interface (ALI) cultures of primary human bronchial and nasal epithelial cells as well as in mouse lung airways compared to standard preparations of AAV6 alone. Of note, AAV6 has been previously shown to outperform other clinically tested AAV serotypes, including those approved by the FDA for treating non-lung diseases, in transducing ALI cultures of primary human airway cells. We provide compelling experimental evidence that the superior performance of EVAAV6 is attributed to the ability of EV to facilitate mucus penetration and cellular entry/transduction of AAV6. The tight and stable linkage between AAV6 and EVs appears essential to exploit the benefits of EVs given that a physical mixture of individually prepared EVs and AAV6 failed to mediate EV-AAV6 interactions or to enhance gene transfer efficacy.

Tumor antigen-loaded AAV vaccine drives protective immunity in a melanoma animal model.

We previously described therapeutic opportunities provided by capsid- and expression cassette-optimized adeno-associated virus serotype 6 (AAV6) vectors to suppress tumor growth in both solid and metastatic mouse models by using artificial ovalbumin (OVA) immunogen. In the current study, we further elucidated the mechanism of function of a novel AAV-based vaccine loaded with the melanoma tumor-associated antigens premelanosome protein gp100, tyrosinase (Tyr), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (TRP2). We showed that the AAV6-based vaccine creates cellular and humoral antigen-specific responses, while antigen expression at the site of vaccine injection was temporal, and the clearance of antigen coincided with T cell infiltration. Our data revealed the superior protective immune response of optimized AAV6-TRP1 compared with other self-antigens in a disease-free mouse model. We further assessed the ability of AAV6-TRP1 to protect animals from metastatic spread in the lungs and to extend animal survival by inhibiting solid tumor growth. Flow cytometry-based analysis indicated significant infiltration of CD8+ T cells and natural killer (NK) cells in the tumor site, as well as changes in the polarization of intratumoral macrophages. Altogether, our data strongly support the use of optimized AAV vectors for cancer vaccine development.

Evaluating the Emotion Ontology through use in the self-reporting of emotional responses at an academic conference.

BACKGROUND: We evaluate the application of the Emotion Ontology (EM) to the task of self-reporting of emotional experience in the context of audience response to academic presentations at the International Conference on Biomedical Ontology (ICBO). Ontology evaluation is regarded as a difficult task. Types of ontology evaluation range from gauging adherence to some philosophical principles, following some engineering method, to assessing fitness for purpose. The Emotion Ontology (EM) represents emotions and all related affective phenomena, and should enable self-reporting or articulation of emotional states and responses; how do we know if this is the case? Here we use the EM 'in the wild' in order to evaluate the EM's ability to capture people's self-reported emotional responses to a situation through use of the vocabulary provided by the EM. RESULTS: To achieve this evaluation we developed a tool, EmOntoTag, in which audience members were able to capture their self-reported emotional responses to scientific presentations using the vocabulary offered by the EM. We furthermore asked participants using the tool to rate the appropriateness of an EM vocabulary term for capturing their self-assessed emotional response. Participants were also able to suggest improvements to the EM using a free-text feedback facility. Here, we present the data captured and analyse the EM's fitness for purpose in reporting emotional responses to conference talks. CONCLUSIONS: Based on our analysis of this data set, our primary finding is that the audience are able to articulate their emotional response to a talk via the EM, and reporting via the EM ontology is able to draw distinctions between the audience's response to a speaker and between the speakers (or talks) themselves. Thus we can conclude that the vocabulary provided at the leaves of the EM are fit for purpose in this setting. We additionally obtained interesting observations from the experiment as a whole, such as that the majority of emotions captured had positive valence, and the free-form feedback supplied new terms for the EM. AVAILABILITY: EmOntoTag can be seen at http://www.bioontology.ch/emontotag; source code can be downloaded from http://emotion-ontology.googlecode.com/svn/trunk/apps/emontotag/and the ontology is available at http://purl.obolibrary.org/obo/MFOEM.owl.