Dynamic Covalent Boronate Chemistry Accelerates the Screening of Polymeric Gene Delivery Vectors via In Situ Complexation of Nucleic Acids.

Gene therapy provides exciting new therapeutic opportunities beyond the reach of traditional treatments. Despite the tremendous progress of viral vectors, their high cost, complex manufacturing, and side effects have encouraged the development of nonviral alternatives, including cationic polymers. However, these are less efficient in overcoming cellular barriers, resulting in lower transfection efficiencies. Although the exquisite structural tunability of polymers might be envisaged as a versatile tool for improving transfection, the need to fine-tune several structural parameters represents a bottleneck in current screening technologies. By taking advantage of the fast-forming and strong boronate ester bond, an archetypal example of dynamic covalent chemistry, a highly adaptable gene delivery platform is presented, in which the polycation synthesis and pDNA complexation occur in situ. The robustness of the strategy entitles the simultaneous evaluation of several structural parameters at will, enabling the accelerated screening and adaptive optimization of lead polymeric vectors using dynamic covalent libraries.

A copper(ii) peptide helicate selectively cleaves DNA replication foci in mammalian cells.

The use of copper-based artificial nucleases as potential anticancer agents has been hampered by their poor selectivity in the oxidative DNA cleavage process. An alternative strategy to solve this problem is to design systems capable of selectively damaging noncanonical DNA structures that play crucial roles in the cell cycle. We designed an oligocationic CuII peptide helicate that selectively binds and cleaves DNA three-way junctions (3WJs) and induces oxidative DNA damage via a ROS-mediated pathway both in vitro and in cellulo, specifically at DNA replication foci of the cell nucleus, where this DNA structure is transiently generated. To our knowledge, this is the first example of a targeted chemical nuclease that can discriminate with high selectivity 3WJs from other forms of DNA both in vitro and in mammalian cells. Since the DNA replication process is deregulated in cancer cells, this approach may pave the way for the development of a new class of anticancer agents based on copper-based artificial nucleases.

Nanoparticle- and Microparticle-Based Vaccines against Orbiviruses of Veterinary Importance.

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are widespread arboviruses that cause important economic losses in the livestock and equine industries, respectively. In addition to these, another arthropod-transmitted orbivirus known as epizootic hemorrhagic disease virus (EHDV) entails a major threat as there is a conducive landscape that nurtures its emergence in non-endemic countries. To date, only vaccinations with live attenuated or inactivated vaccines permit the control of these three viral diseases, although important drawbacks, e.g., low safety profile and effectiveness, and lack of DIVA (differentiation of infected from vaccinated animals) properties, constrain their usage as prophylactic measures. Moreover, a substantial number of serotypes of BTV, AHSV and EHDV have been described, with poor induction of cross-protective immune responses among serotypes. In the context of next-generation vaccine development, antigen delivery systems based on nano- or microparticles have gathered significant attention during the last few decades. A diversity of technologies, such as virus-like particles or self-assembled protein complexes, have been implemented for vaccine design against these viruses. In this work, we offer a comprehensive review of the nano- and microparticulated vaccine candidates against these three relevant orbiviruses. Additionally, we also review an innovative technology for antigen delivery based on the avian reovirus nonstructural protein muNS and we explore the prospective functionality of the nonstructural protein NS1 nanotubules as a BTV-based delivery platform.

Production and Purification of Candidate Subunit Vaccines by IC-Tagging Protein Encapsulation.

Particulate material is more efficient in eliciting immune responses. Here we describe the production of micro- and nanospheres formed by protein muNS-Mi from avian reoviruses, loaded with foreign epitopes for their use as vaccines.

Chemical and thermal stabilization of CotA laccase via a novel one-step expression and immobilization in muNS-Mi nanospheres.

A methodology that programs eukaryotic or bacterial cells to encapsulate proteins of any kind inside micro/nanospheres formed by muNS-Mi viral protein was developed in our laboratory. In the present study such "in cellulo" encapsulation technology is utilized for immobilizing a protein with an enzymatic activity of industrial interest, CotA laccase. The encapsulation facilitates its purification, resulting in a cost-effective, one-step way of producing immobilized enzymes for industrial use. In addition to the ability to be recycled without activity loss, the encapsulated protein showed an increased pH working range and high resistance to chemical inactivation. Also, its activity was almost unaffected after 30 min incubation at 90 °C and 15 min at the almost-boiling temperature of 95 °C. Furthermore, the encapsulated laccase was able to efficiently decolorate the recalcitrant dye RB19 at room temperature.

Cross-protective immune responses against African horse sickness virus after vaccination with protein NS1 delivered by avian reovirus muNS microspheres and modified vaccinia virus Ankara.

African horse sickness virus (AHSV) is an insect-borne pathogen that causes acute disease in horses and other equids. In an effort to improve the safety of currently available vaccines and to acquire new knowledge about the determinants of AHSV immunogenicity, new generation vaccines are being developed. In this work we have generated and tested a novel immunization approach comprised of nonstructural protein 1 (NS1) of AHSV serotype 4 (AHSV-4) incorporated into avian reovirus muNS protein microspheres (MS-NS1) and/or expressed using recombinant modified vaccinia virus Ankara vector (MVA-NS1). The protection conferred against AHSV by a homologous MS-NS1 or heterologous MS-NS1 and MVA-NS1 prime/boost was evaluated in IFNAR (-/-) mice. Our results indicate that immunization based on MS-NS1 and MVA-NS1 afforded complete protection against the infection with homologous AHSV-4. Moreover, priming with MS-NS1 and boost vaccination with MVA-NS1 (MS-MVA-NS1) triggered NS1 specific cytotoxic CD8 + T cells and prevented AHSV disease in IFNAR (-/-) mice after challenge with heterologous serotype AHSV-9. Cross-protective immune responses are highly important since AHS can be caused by nine different serotypes, which means that a universal polyvalent vaccination would need to induce protective immunity against all serotypes.

IC-Tagging methodology applied to the expression of viral glycoproteins and the difficult-to-express membrane-bound IGRP autoantigen.

We have previously developed a methodology to produce protein microspheres (MS) that can be loaded with proteins of interest in living cells through their C or N-terminal tagging with the so-called IC-Tag. The IC-Tagging method has many applications ranging from the production of immobilized enzymes for industrial use to the production of subunit vaccines due to its intrinsic adjuvancy. Here we show the adaptation of the IC-Tagging to work inside the endoplasmic reticulum and bacteria, allowing us to produce properly modified viral glycoproteins. Additionally, we were able to express the Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), whose expression remained elusive to date possibly due to its toxicity when over-expressed. IGRP is an antigen of enormous pharmaceutical interest as it is specifically targeted during the autoimmune response taking place in both the Non-Obese Diabetic (NOD) mice and type 1 diabetes (T1D) patients leading to the destruction of insulin-producing beta cells.

Using IC-Tagging Methodology for Production and Purification of Epitope-Loaded Protein Microspheres for Vaccination.

Particulate material is more efficient in eliciting immune responses. Here we describe the production of microspheres formed by protein muNS-Mi from avian reoviruses, loaded with foreign epitopes by means of IC-Tagging, for their use as vaccines.