Mannose-coupled AAV2: A second-generation AAV vector for increased retinal gene therapy efficiency.

Inherited retinal diseases are a leading and untreatable cause of blindness and are therefore candidate diseases for gene therapy. Recombinant vectors derived from adeno-associated virus (rAAV) are currently the most promising vehicles for in vivo therapeutic gene delivery to the retina. However, there is a need for novel AAV-based vectors with greater efficacy for ophthalmic applications, as underscored by recent reports of dose-related inflammatory responses in clinical trials of rAAV-based ocular gene therapies. Improved therapeutic efficacy of vectors would allow for decreases in the dose delivered, with consequent reductions in inflammatory reactions. Here, we describe the development of new rAAV vectors using bioconjugation chemistry to modify the rAAV capsid, thereby improving the therapeutic index. Covalent coupling of a mannose ligand, via the formation of a thiourea bond, to the amino groups of the rAAV capsid significantly increases vector transduction efficiency of both rat and nonhuman primate retinas. These optimized rAAV vectors have important implications for the treatment of a wide range of retinal diseases.

Novel chemical tyrosine functionalization of adeno-associated virus improves gene transfer efficiency in liver and retina.

Decades of biological and clinical research have led to important advances in recombinant adeno-associated viruses rAAV-based gene therapy gene therapy. However, several challenges must be overcome to fully exploit the potential of rAAV vectors. Innovative approaches to modify viral genome and capsid elements have been used to overcome issues such as unwanted immune responses and off-targeting. While often successful, genetic modification of capsids can drastically reduce vector yield and often fails to produce vectors with properties that translate across different animal species, such as rodents, non-human primates, and humans. Here, we describe a chemical bioconjugation strategy to modify tyrosine residues on AAV capsids using specific ligands, thereby circumventing the need to genetically engineer the capsid sequence. Aromatic electrophilic substitution of the phenol ring of tyrosine residues on AAV capsids improved the in vivo transduction efficiency of rAAV2 vectors in both liver and retinal targets. This tyrosine bioconjugation strategy represents an innovative technology for the engineering of rAAV vectors for human gene therapy.

Click-electrochemistry for the rapid labeling of virus, bacteria and cell surfaces.

Methods for direct covalent ligation of microorganism surfaces remain poorly reported, and mostly based on metabolic engineering for bacteria and cells functionalization. While effective, a faster method avoiding the bio-incorporation step would be highly complementary. Here, we used N-methylluminol (NML), a fully tyrosine-selective protein anchoring group after one-electron oxidation, to label the surface of viruses, living bacteria and cells. The functionalization was performed electrochemically and in situ by applying an electric potential to aqueous buffered solutions of tagged NML containing the viruses, bacteria or cells. The broad applicability of the click-electrochemistry method was explored on recombinant adeno-associated viruses (rAAV2), Escherichia coli (Gram-) and Staphyloccocus epidermidis (Gram + ) bacterial strains, and HEK293 and HeLa eukaryotic cell lines. Surface electro-conjugation was achieved in minutes to yield functionalized rAAV2 that conserved both structural integrity and infectivity properties, and living bacteria and cell lines that were still alive and able to divide.

Hexavalent thiofucosides to probe the role of the Aspergillus fumigatus lectin FleA in fungal pathogenicity.

Aspergillus fumigatus is a pathogenic fungus infecting the respiratory system and responsible for a variety of life-threatening lung diseases. A fucose-binding lectin named FleA which has a controversial role in A. fumigatus pathogenesis was recently identified. New chemical probes with high affinity and enzymatic stability are needed to explore the role of FleA in the infection process. In this study, we developed potent FleA antagonists based on optimized and non-hydrolysable thiofucoside ligands. We first synthesized a set of monovalent sugars showing micromolar affinity for FleA by isothermal titration calorimetry. The most potent derivative was co-crystallized with FleA to gain insights into the binding mode in operation. Its chemical multimerization on a cyclodextrin scaffold led to an hexavalent compound with a significantly enhanced binding affinity (Kd = 223 ± 21 nM) thanks to a chelate binding mode. The compound could probe the role of bronchial epithelial cells in a FleA-mediated response to tissue invasion.