Gene therapy as an alternative for inflammatory disease treatment

           For the last decades there has been a signifcant increase in the incidence of chronic inflammatory disease. This includes allergic conditions, cardiovascular diseases and autoimmune disorders such as neurodegenerative disease. Chronic inflammatory diseases are characterized by episodes of relapse and remission that often involve superposition of acute inflammation on top of the inflammation already present. Altering the cytokine network is a common therapeutic strategy in inflammatory diseases. Therapies based on natural cytokines are very promising as they are more effective, better tolerated, and more specific than pharmacological treatments. However, these treatments have several limitations such as expense, the need for repeated injections and unwanted side effects. Cytokines are expensive to produce, and have short halflives therefore requiring frequent administration. As they are systemically administered at high concentrations to achieve efficient local concentrations, they can affect other organs and tissues, often causing side-effects such as widespread immunosuppression. In addition, after stopping the treatment, there is usually a disease flare-up.Delivery by gene therapy may overcome many of these limitations as it can provide long term, safe and locally regulated gene expression. Development of gene therapy approaches for treating chronic inflammatory diseases is challenging as it requires the production of anti-inflammatory molecules at the diseased tissues only when they are needed. Studies of inflammatory flare-up reactions in animal models have shown the applicability and viability of local gene therapy in inflammatory diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA).
          In our lab we have generated several inflammation-regulated lentivector expression systems which are induced upon pro-inflammatory stimulation in vitro and in vivo. Our aim is to express IL-10 under these inducible systems in animal models resembling human diseases where inflammation is involved such as Alzheimer’s disease and cancer.


Serum complement protection of lentivectors

           The efficiency of the gene delivery and transfer depends on several factors, including the type of vector and the route of delivery. Among the most established viral vectors currently available, the VSV-G pseudotyped lentiviral system is a very promising tool: they can infect an expanded range of dividing and quiescent cells, provide long-term expression, can be concentrated to high titer, and their biosafety has been improved. However, serum inactivation of VSV-G pseudotyped lentivirus vectors is a significant barrier to the development of these otherwise highly efficient vectors for in vivo gene delivery.
          There are two kinds of experimental approaches to solve or minimize this problem: chemical and genetic modifications. Among chemical modifications, poly(ethylene) glycol (PEG) conjugation to VSV-G pseudotyped lentivirus vector protects the virus from serum inactivation, improving the transduction efficiency in vivo. However, it requires chemical manipulation for every batch production, adding difficulty to the whole process and variability to the production efficiency.
          Among the genetic modifications, a common approach is the expression of Complement Regulatory Proteins (CRP) in the envelope of the virus particles. The activation of the complement cascade leads to the formation of a transmembrane pore complex known as the membrane attack complex (MAC), which causes lysis of the viral particle. Complement activity is normally controlled by a large number of CRPs to prevent inflammation and unwanted tissue damage in the host. However, since the complement system is critical for innate immunity and plays an essential role in the inflammatory response, the inhibition of complement activation after the viral particle administration would reduce, at least partially, the immune response in the host.
          Our lab is exploring new genetic modifications to generate complement resistant lentivectors without affecting the host immune response. Our data will be weighted and compared with those already published in order to decide which strategy might be worth to employ for further pseudotyping. A protection from complement inactivation would mean a significant improvement in retrovirus/lentivirus-based gene transfer technology. Our results may help to optimize gene transfer after systemic administration of lentivirus-based vectors and reduce vector-associated toxicity by lowering the dose necessary for efficient gene transfer.

Calcineurin blocking nanobodies

          Angiogenesis, or the formation of new blood vessels out of pre-existing capillaries, is a sequence of events that is fundamental to many physiologic and pathologic processes such as cancer, ischemic diseases, and chronic inflammation. Among the pro-angiogenic factors, VEGF (Vascular Endothelial Growth Factor) is the major trigger of vasculogenesis and physiologic angiogenesis. Evidence has been gathered regarding the association between angiogenesis and inflammation in pathological situations. The gene repertoire induced by VEGF in endothelial cells surprisingly overlapped to 60% with the inflammatory repertoire. The data display that VEGF induces a gene repertoire, which includes an inherent inflammatory component possibly contributing to the cross-regulation of angiogenesis and inflammation. The capacity of VEGF-A to induce the inflammatory genes largely depended on activation of calcineurin (CN), since cyclosporine A (CsA) inhibited this induction. Therefore, CN is a good target to block angiogenesis in pathologies such as chronic inflammatory diseases and tumor angiogenesis. CN is a unique calcium- and calmodulin-dependent protein phosphatase that transmits calcium signals from the cytosol to the nucleus to regulate gene expression. This phosphatase is the common target of the widely used immunosuppressive drugs CsA and FK506. However, their therapeutic use is associated with severe side effects. There is, therefore a need to develop better, less toxic immunosuppressive agents.
          Antibodies (Abs) are glycoproteins that recognize with high affinity and specificity certain molecular targets (antigens). Their most common forms (IgGs) contain two polypeptide chains of different sizes, heavy (H) and light (L), forming a Y-shaped heterotetramer. Both chains are composed of repeats of Ig domains, the first of which presents more sequence variability among the different Abs and is called variable domain (VH or VL). The other Ig domains (CL, CH1, CH2, CH3, etc.) are called constant domains. The technologies of recombinant Abs (rAbs) have been developed mainly focusing in the expression of minimal Ab fragments containing V domains able to specifically bind to an antigen. Among the rAbs, there have been developed functional rAbs formed by just a single V domain (single domain Ab, sdAb). These sdAbs derives from a special class of antibodies that only possess heavy chains (heavy-chain-only Abs, HCAbs), that occurs naturally in camelids (camels, dromedaries, lamas, alpacas, etc.), in which the VH domain is solely responsible for binding to the antigen. These sdAbs, called VHH (heavy chain only VH), present a very similar sequence to the human VH3 subfamily, allowing their use in therapy and diagnosis in humans. In addition to its small size, among the advantages offered by the VHH are its high stability and solubility as well as the recognition, within the same antigen, of distinct epitopes than conventional Abs, being frequent these grooves and cavities of proteins, which means they can be used as inhibitors of enzymatic activities.
          Our goal within the ‘Angiobodies 2.0’ research network is to develop VHH antibodies (nanobodies) able to recognize and block the CN activity. These nanobodies might be useful for the inhibition of CN in order to block angiogenesis in pathologies such as chronic inflammatory diseases.