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Several viral and non-viral vectors have been developed for exogenous protein expression in specific cells. Conventionally, this purpose is achieved through the use of recombinant DNA. But mainly due to the risks associated with permanent genetic alteration of cells, safety and ethical concerns have been raised for the use of DNA-based vectors in human clinical therapy. In the last years, synthetic messenger RNA has emerged as powerful tool to deliver genetic information. RNA vectors exhibit several advantages compared to DNA and are particularly interesting for applications that require transient gene expression. RNA stability and translation efficiency can be increased by cis-acting structural elements in the RNA such as the 5'-cap, the poly(A)-tail, untranslated regions and the sequence of the coding region. Here we review recent developments in the optimization of messenger RNA as vector for modulation of protein expression emphasizing on stability, transfection and immunogenicity. In addition, we summarize current pre-clinical and clinical studies using RNA-based vectors for immunotherapy, T cell, stem cell as well as gene therapy.

In an attempt to develop a novel strategy for the identification of new members of protein families by in silico approaches, we have developed a semi-automated procedure of consecutive PSI-BLAST (Position-Specific-Iterated Basic Local Alignment Search Tool) searches incorporating identificiation as well as subsequent validation of putative candidates. For a proof of concept study we chose the search for novel members of the claudin family. The initial step was an iterated PSI-BLAST search starting with the PMP22_Claudin domain of each known member of the claudin family against the human part of the RefSeq Database. Putative new claudin domains derived from the converged list were evaluated by a validating PSI-BLAST in which each sequence was assessed for finding back the starting set of known claudin domains. The local PSI-BLAST searches and validation were automated by a set of PERL scripts. With this strategy a total of three additional putative claudin domains in three different proteins were identified. One of them was subjected to further characterization and was shown to exhibit claudin-like features in terms of protein structure and expression pattern. The strategy we present is an efficient and versatile tool to identify novel members of domain-sharing protein families. Low rates of false positives achieved by inclusion of a validation step into the in silico procedure make this strategy particularly attractive to select candidates for subsequent labor-intensive wet bench characterization., ((c) 2006 Wiley-Liss, Inc.)

Little is known about the distribution in normal cells of CLIP-170, a linkage mediator between endocytic vesicles and microtubules, and restin, a splice variant encoded by the same gene and marker for Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin disease. Although only trace amounts of CLIP-170/restin are present in peripheral blood mononuclear cell subpopulations, monocyte-derived dendritic cells (DCs) and interleukin-4 (IL-4) + CD40L-activated B cells express high levels of CLIP-170/restin. CLIP-170/restin colocalizes preferentially with membranes of intermediate macropinocytic vesicles, suggesting a new function of CLIP-170/restin in the trafficking of macropinosomes to the cytoskeleton, which is a crucial step in antigen presentation. The strong expression of CLIP-170/restin in HRS cells, DCs, and activated B cells underscores their functional similarities supporting a function-based concept of HRS cells as professional antigen-presenting cells.

Cancer/testis-antigens (CTA), a novel and expanding family of immunogenic proteins detected by serological screening of recombinant cDNA expression libraries, encompass promising candidate targets for T-cell based immunotherapy. We screened kryo-preserved tissue of cutaneous T cell lymphoma (CTCL, n=36) such as mycosis fungoides (MF, n=17), pleomorphic cutaneous T-cell lymphoma (n=8) and Sezary's syndrome (SS, n= 11) as well as a non-malignant entity (small plaques parapsoriasis, SPP, n=5), for the expression of CTA by RT-PCR and Northern blot hybridization. From a panel of eleven CTA (MAGE-1, MAGE-C1, MAGE-3, BAGE, GAGE, SSX-1, SSX-2, SSX4, SCP-1, NY-ESO-1 and TS85) (HOM-Tes-85), mRNA expression could be detected for SCP-1 in 8/17 MF and 6/8 pleomorphic CTCL patients but was completely absent in small plaques parapsoriasis. SS patients had a more heterogeneous antigen expression pattern: Gage (1/11), MAGE-1 (3/11), MAGE-3 (6/11), MAGE-C1 (5/11), NY-ESO-1 (7/11) and TS85 (5/11), with expression of MAGE-3 confirmed by immunohistochemistry. CTA could provide defined targets for antigen-based vaccination in a high percentage of cases with CTCL. SCP-1 might serve as an additional diagnostic indicator in early and clinically indistinct lesions suspicious for cutaneous T-cell lymphoma., (Copyright 2001 Wiley-Liss, Inc.)

With an estimated annual incidence of about one million cases, hepatocellular carcinoma (HCC) is one of the most common neoplasms worldwide. Of all malignant diseases, it is the major cause of death in some regions of Africa and Asia. The pathogenic mechanisms responsible for HCC are not well defined, and therapeutic means, especially in inoperable HCCs, are still unsatisfactory and await improvement. In the quest for tumor antigens exploitable for gene therapy, we studied immune responses in the context of HCC. A cDNA library derived from a human HCC sample was screened using the SEREX approach. Nineteen distinct antigens reactive with autologous IgG were identified. Sequence analysis revealed three of the cDNA clones to code for hitherto unknown proteins and 16 known genes products. Proteins as diverse in function as LDH, albumin, and kinectin were found. Furthermore, proteins involved in the transcription/translation machinery had elicited an immune response in the autologous host. A panel of allogenic sera including sera from patients with hepatitis, liver cirrhosis, HCC, and other tumor entities, as well as sera from normal individuals, was used for frequency analysis of antibody responses. Whereas allogenic sera of HCC patients detected most antigens at a high percentage, control sera were rarely antibody-positive. The nature of the major fraction of antigens described here are linked to liver. Thus, our findings demonstrate not only the complexity of the humoral immune response against HCC, but may also offer new insight into mechanisms underlying transformation of the liver cell.