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As science progresses in its understanding of diseases and their treatment, advances have been made in the biotechnology used in disease therapy. Most gene therapy approaches utilise viral vectors to deliver genes of interest. However, multiple proteins are often involved in disease processes and there is often a need to efficiently deliver more than one gene. Researchers have employed several strategies to accomplish this goal. When designing vectors to express multiple genes, there are several factors that need to be taken into account, including cell type, the activity of the protein of interest and subcellular protein localisation. In most cases, it is ideal for each protein to be expressed at comparable levels, a leading issue with traditional strategies for multigene expression. This review describes some of the techniques that have been used to express multiple genes, and will focus on the use of 2A peptides or 2A peptide-like sequences in the design of multicistronic vectors that may alleviate some of these issues.

Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses, to generate multicistronic retroviral vectors with efficient translation of four cistrons. Using the T-cell receptor:CD3 complex as a test system, we show that a single 2A peptide-linked retroviral vector can be used to generate all four CD3 proteins (CD3epsilon, gamma, delta, zeta), and restore T-cell development and function in CD3-deficient mice. We also show complete 2A peptide-mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer-based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.

We thank Drs. Pandiyan and Lenardo for their interest in our study ([1][1]). Defining the mechanisms used by T regulatory cells (Tregs) to mediate suppression is an important but contentious topic ([2][2]). Our study specifically asked if T cells resistant to apoptosis mediated by the proapoptotic

Objectives Biologics are medication widely applied in management of inflammatory rheumatic diseases. The drugs were found to be effective but their application is associated with some disadvantages. Medication with biologics is relatively expensive, and in Poland, it is carried out in specialized centers. The study was designed to evaluate various aspects of satisfaction and dissatisfaction of the Polish patients treated with biologics. Methods Anonymous questionnaires were distributed in 23 Polish rheumatological centers involved in the treatment, 1231 questionnaires were used for analysis. Responses were received from 606 patients with rheumatoid arthritis (RA), 427 with ankylosing spondylitis (SA), 117 psoriatic arthritis (PS), and 62 adult patients with juvenile idiopathic arthritis (JIA) (in whom administration of the drugs had been introduced before they were 18-year-old), as well as 19 ones receiving the drugs due to other musculoskeletal disorders. The investigated group constituted about one-fifth of all rheumatic patients on biologics in Poland. Results The main reasons for satisfaction were positive influence of the treatment on family life (41% RA, 41% SA, 41% PS), general physical status (51% RA, 51% SA, very positive 43% PS), mental well-being (46% RA, 47% SA, 48% JIA, very positive 43% PS), sexual life (39% SA, 38% PS, 31% RA), sleep (39% RA, 41% SA, 36% PS, 40% JIA), in smaller part of the patients it had effect on working and financial situation. In general, satisfaction of the treatment was evaluated as positive or very positive in 84% patients with RA, 72% with SA, 87% with PS and 81% with JIA. In almost half of the patients, transportation to the medical center was a disadvantage of the treatment. 79% did not consider blood sampling as a problem for them. About one-third of the patients considered laboratory and imagine tests to be done before initiation of the medication as a difficulty, and for 27-47% waiting time for qualification for the medication was a significant disadvantage. The route of drug administration was without importance for 4/5 of the patients. Conclusions Summing up, the results were similar in the patients suffering from various diseases although those with PS felt the highest satisfaction (possible due to positive aesthetic effect), and those with SA had significant improvement in sexual life (probably due to younger age). Relatively low satisfaction was found in patients with JIA. There was low influence of medication on financial status of the patients however some of them returned to work. Application of biologics has low disadvantages and most of them are associated with organization of health services (waiting time for the tests, transportation to the medical centers). Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3923

Apoptosis has an essential role in controlling T cell homeostasis, especially during the contraction phase of an immune response. However, its contribution to the balance between effector and regulatory populations remains unclear. We found that Rag1(-/-) hosts repopulated with Bim(-/-) conventional CD4(+) T cells (Tconv) resulted in a larger induced regulatory T cell (iTreg) population than mice given wild-type (WT) Tconv. This appears to be due to an increased survival advantage of iTregs compared with activated Tconv in the absence of Bim. Downregulation of Bcl-2 expression and upregulation of Bim expression were more dramatic in WT iTregs than activated Tconv in the absence of IL-2 in vitro. The iTregs generated following Tconv reconstitution of Rag1(-/-) hosts exhibited lower Bcl-2 expression and higher Bim/Bcl-2 ratio than Tconv, which indicates that iTregs were in an apoptosis-prone state in vivo. A significant proportion of the peripheral iTreg pool exhibits low Bcl-2 expression indicating increased sensitivity to apoptosis, which may be a general characteristic of certain Treg subpopulations. In summary, our data suggest that iTregs and Tconv differ in their sensitivity to apoptotic stimuli due to their altered ratio of Bim/Bcl-2 expression. Modulating the apoptosis pathway may provide novel therapeutic approaches to alter the balance between effector T cells and Tregs.

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. This article describes the design and construction of 2A peptide-linked multicistronic vectors, which can be used to express multiple proteins from a single open reading frame (ORF). The small 2A peptide sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel “cleavage” event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18–22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector.

Engagement of peptide-MHC by the TCR induces a conformational change in CD3ε that exposes a proline-rich sequence (PRS) and recruits the cytoskeletal adaptor Nck. This event, which precedes phosphorylation of the CD3ε ITAM, has been implicated in synapse formation and T cell function. However, there is compelling evidence that responsiveness to TCR ligation is CD3ε PRS independent. In this study, we show that the CD3ε PRS is necessary for peptide-MHC-induced phosphorylation of CD3ε and for recruitment of protein kinase Cθ to the immune synapse in differentiated CD8+ T lymphocytes. However, whereas these two events are dispensable for functional T cell responsiveness to high-avidity ligands, they are required for responsiveness to low-avidity ones. Thus, in at least certain T cell clonotypes, the CD3ε PRS amplifies weak TCR signals by promoting synapse formation and CD3ε phosphorylation.