Investigador Principal: ANTONIO RODRÍGUEZ

CALCINEURIN AS A THERAPEUTIC TARGET

The calcium-sensitive phosphatase calcineurin, CN (also called protein phosphatase 2B) is implicated in many eukaryotic activation and developmental programmes, including lymphocyte activation, heart-valve morphogenesis, angiogenesis, and neural and muscle development.

Figure 1. Overall architecture of Calcineurin. Space-filling representation of the L-shaped structure of the catalytic subunit, CnA, bound to the regulatory subunit, CnB (shown in orange). CnA contains a phosphatase domain (shown in yellow) and a regulatory region, linked by a short sequence known as linker (shown in grey). The regulatory region contains a CnB-binding helical domain (shown in purple), a calmodulin binding region (CaMBR), and an autoinhibitory loop (shown in red). Residues corresponding to CaMBR and the C-terminal region are not visible in the crystals.

 

The importance of this phosphatase is graphically illustrated by the observation that the immunosuppressive actions of the microbial drugs Cyclosporin A (CsA) and FK506 arise from their inhibition of CN. As substrates of calcineurin, transcription factors of the NFAT (Nuclear Factor of Activated T-cells) family play an essential role in lymphocyte activation, and it follows that their function is also inhibited by CsA and FK506. Although the use of these drugs has been crucial for the success of organ transplantation, their therapeutic use is associated with severe side effects. Our current research interest is focused on a detailed analysis of the CN specific sequences involved in the interaction with the two CN-binding sites of NFAT, PxIxIT and LxVP motifs. A better knowledge of the specificity and regulation of NFAT-calcineurin interactions may lead to the development of more specific inhibitors of calcineurin. Reagents that exploit the selective interaction of CN with NFAT would be of use in dissecting important biological processes in which the CN/NFAT pathway plays a key role.

 

Figure 2. The Calcineurin-NFAT signalling pathway. An increase of intracellular calcium levels activates the cellular phosphatase Calcineurin (CN) through its interaction with Calmodulin (CaM). Activated CN is able to dephosphorylate NFAT (Nuclear Factor of Activtated T-cells), and allows the nuclear translocation of this transcription factor. In the nucleus, NFAT binds to specific DNA motifs within the promoter of numerous genes and induce their transcription.

 

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.

 

GENE THERAPY AS AN ALTERNATIVE FOR INFLAMMATORY DISEASE TREATMENT

For the last decades there has been a significant 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 half-lives 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 anti-inflammatory agents under these inducible systems in animal models resembling human diseases where inflammation is involved such as cancer and Alzheimer's disease.

 

Figure 3. Drawing showing the aims of this project. Inducible Lentivector (LV) systems will be employed to express anti-inflammatory agents in animal models of Alzheimer's and cancer.

 

SERUM 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.