Flemish network on rare connective tissue diseases (CTD): patient pathways in systemic sclerosis. First steps taken.

Despite the low prevalence of each rare disease, the total burden is high. Patients with rare diseases encounter numerous barriers, including delayed diagnosis and limited access to high-quality treatments. In order to tackle these challenges, the European Commission launched the European Reference Networks (ERNs), cross-border networks of healthcare providers and patients representatives. In parallel, the aims and structure of these ERNs were translated at the federal and regional levels, resulting in the creation of the Flemish Network of Rare Diseases. In line with the mission of the ERNs and to ensure equal access to care, we describe as first patient pathways for systemic sclerosis (SSc), as a pilot model for other rare connective and musculoskeletal diseases. Consensus was reached on following key messages: 1. Patients with SSc should have multidisciplinary clinical and investigational evaluations in a tertiary reference expert centre at baseline, and subsequently every three to 5 years. Intermediately, a yearly clinical evaluation should be provided in the reference centre, whilst SSc technical evaluations are permissionably executed in a centre that follows SSc-specific clinical practice guidelines. In between, monitoring can take place in secondary care units, under the condition that qualitative examinations and care including interactive multidisciplinary consultations can be provided. 2. Patients with early diffuse cutaneous SSc, (progressive) interstitial lung disease and/or pulmonary arterial hypertension should undergo regular evaluations in specialised tertiary care reference institutions. 3. Monitoring of patients with progressive interstitial lung disease and/or pulmonary (arterial) hypertension will be done in agreement with experts of ERN LUNG.

Single-copy T-DNAs integrated at different positions in the Arabidopsis genome display uniform and comparable beta-glucuronidase accumulation levels.

This study aimed at determining whether transgene expression variability is observed in single-copy T-DNA plants and whether it can be correlated with the T-DNA integration position. Among a population of 135 Arabidopsis thaliana transformants, selected on the basis of antibiotic resistance marker expression, 21 single-copy T-DNA transformants were identified and characterized. In 19 of these 21 lines, 35S-beta-glucuronidase transgene expression, measured in two subsequent generations, was similar. This observation means that the intra-transformant variability was as high as the inter-transformant variability. Integration into an intergenic or genic region, into an exon or intron, in sense or antisense orientation, did not result in differential transgene expression. Remarkably, single-copy transformants were not always the highest expressers, implying that low transgene expression is not always induced by multicopy transformants. In only 2 of the 21 single-copy plants was the transgene expression more than 20-fold lower. However, characteristics of the insertion position in one of these lines did not differ significantly when compared to high-expressing lines. In the remaining line, methylation of the transgene was clearly demonstrated. In conclusion, screening for single-copy T-DNA transformants greatly enriches for stable and high transgene expression, because the integration position is not a major determinant of transgene expression variability in Arabidopsis.

Highly efficient targeting and accumulation of a F(ab) fragment within the secretory pathway and apoplast of Arabidopsis thaliana.

To further improve antibody production in plants, constructs were designed to minimize transgene silencing and to retain a F(ab) fragment within the secretory pathway of transgenic Arabidopsis thaliana plants. The levels of antibody accumulation suggest that placing the sequences that encode Fd and light chain under the control of nonidentical 3' regions reduces susceptibility to post-transcriptional gene silencing compared with when the individual polypeptide-encoding sequences are placed under the control of identical 3' regions. High levels of accumulation (up to 6% of total soluble protein) were found for both secreted and intracellularly targeted antibody fragments. Immunofluorescence microscopic analysis showed that F(ab) fragments devoid of any additional C-terminal sequence were efficiently secreted, whereas retention of F(ab) fragments within the endomembrane system of the secretory pathway was achieved by C-terminal fusion of the DIKDEL sequence to the antibody light chain. Furthermore, analysis by immunoprecipitation and ELISA showed that intracellular retention of antibody fragments did not affect antigen-binding activity, and more than 80% of the isolated antibody fragments were found to bind antigen. Taken together, our results provide improvements to the technology of recombinant antibody production in transgenic plants.

Transgene silencing of invertedly repeated transgenes is released upon deletion of one of the transgenes involved.

To analyse experimentally the correlation between transgene silencing and the presence of an inverted repeat in transgenic Arabidopsis thaliana plants, expression of the beta-glucuronidase (gus) gene was studied when present as a convergently transcribed inverted repeat or as a single copy in otherwise isogenic lines. In transformants containing two invertedly repeated gus genes separated by a 732 bp palindromic sequence, gus expression was low, as exemplified by the expression levels in the parental line KH15. The parental KH15 locus could induce efficiently in trans silencing of gus copies at allelic and non-allelic positions. In transformants containing two invertedly repeated gus genes separated by a 826 bp non-repetitive spacer region, gus expression was high or intermediate, especially in hemizygous state and at late developmental stages, as demonstrated in detail for line KHsb67. Removal of one of the gus copies by Cre recombinase resulted in all cases in constitutively high gus expression in hemizygous as well as in homozygous state. The derived deletion lines could no longer induce in trans silencing of homologous gus copies. The results show that convergent transcription of transgenes in an inverted repeat is an important parameter to trigger their silencing and that co-transformation of two T-DNAs with identical transgenes can be used to obtain inverted repeats and targeted co-suppression of the homologous endogenes. Moreover, the data suggest that the spacer region in between the inverted genes plays a role in the efficiency of initiating and maintaining silencing.

Disruption of their palindromic arrangement leads to selective loss of DNA methylation in inversely repeated gus transgenes in Arabidopsis.

The transgene locus KH15, which is highly susceptible to silencing in Arabidopsis thaliana, contains two inversely repeated beta-glucuronidase (gus) genes separated by a palindromic sequence and has a low GUS activity, was found to be heavily methylated in the gus coding sequence and in the center of the inverted repeat. The locus KHsb67, which is less prone to silencing, was found to be less densely methylated in the non-repetitive region that separates the inversely repeated gus genes. After the removal of one of the gus genes by Cre-mediated recombination, methylation in both loci decreased or was totally lost. Despite the presence of a 732-bp palindromic sequence in the deletion line derived from KH15, this sequence was not methylated. Whereas the KH15 locus triggers methylation of homologous gus genes when placed in trans to them, the deletion derivative did not, suggesting that the capacity for cross-talk was severely affected by disruption of the palindromic arrangement. This result suggests that the transcribed palindromic sequences are required to maintain the methylation of both symmetrically and non-symmetrically arranged cytosines.

Silencing of antibody genes in plants with single-copy transgene inserts as a result of gene dosage effects.

The stability of Fab antibody fragment expression during plant development was studied using two homozygous Arabidopsis thaliana lines that contain single copies of the transgenes. These lines exhibited expression characteristics that are typical for homology-based post-transcriptional gene silencing. Their developmental silencing profiles differed markedly, presumably due to the influence of the genomic context on the T-DNAs. In both lines, a clear gene dosage effect could be observed: in contrast to the homozygous lines, derived hemizygous plants accumulated high levels of Fab fragments throughout development. Interestingly, silencing also occurred in double-hemizygous plants, which resulted from a cross between the two homozygous lines and had two copies of each T-DNA at non-allelic positions in their genome. In all cases, down-regulation of the Fab levels was strictly correlated with methylation of cytosine residues in the transcribed regions of the transgenes. Remarkably, this methylation was also found in regions in which the transgenes were non-homologous regions. Finally, the time point of down-regulation depended on the culture conditions and differed for leaves and roots of the same transgenic plant.

Production of antibodies and antibody fragments in plants.

Our current knowledge allows the generation of transgenic plants that efficiently produce heterologous proteins from plant, bacterial, fungal or animal origin. Among all types of recombinant proteins, antibodies are particularly attractive because of their ability to specifically recognize and bind virtually any type of antigen. Plants show several advantages as a large-scale antibody production system: they can be grown easily and inexpensively in large quantities that can be harvested, stored and processed by using existing infrastructures. Isolation and purification of plant-made antibodies, if necessary, allow fundamental, industrial, and therapeutical applications. In the past, we and others have successfully generated antibody-producing plants. The maximal accumulation levels of antibodies and antibody fragments that we observed are 1-5% of the extracted proteins. Currently, several biotechnological companies grow field crops to produce antibodies for ex planta applications on an industrial scale.

Silencing of invertedly repeated transgenes in Arabidopsis thaliana.

Locus KH15, containing two invertedly repeated beta-glucuronidase (gus) genes separated by a palindromic sequence showed low gus expression in both hemizygous and homozygous seedlings, in all plant tissues tested and throughout development assayed up to 70 days after sowing. Removal of one of the gus genes by Cre-mediated recombination resulted in a 10- to 100-fold increase of gus expression, confirming that inverted repeats and transgene silencing are strictly correlated. Crosses between the locus KH15 and highly expressing gus transformants further revealed that invertedly repeated gus genes could induce in trans silencing of gus copies at allelic and nonallelic positions. Double-stranded RNA produced by this KH15 may be responsible for this in trans inactivation.

Isolation and characterization of recombinant antibody fragments against CDC2a from Arabidopsis thaliana.

In order to obtain recombinant antibody fragments that bind the cell-cycle protein CDC2a from Arabidopsis thaliana (CDC2aAt), two phage display libraries of single-chain variable (scFv) fragments were constructed. One library was derived from mice immunized with recombinant CDC2aAt N-terminally fused to a His6-tag (His-CDC2aAt) and the other was made out of an anti-PSTAIRE hybridoma cell line. Six specific His-CDC2aAt-binding phage clones (3D1, 3D2, 3D10, 3D25, 4D21 and 4D47) were isolated by panning. The isolated monoclonal phage clones, as well as the soluble scFv fragments produced in the periplasm of Escherichia coli, bind His-CDC2aAt in ELISA and on Western blots. Moreover, four clones (3D1, 3D2, 3D10 and 4D21) detect specifically CDC2aAt from Arabidopsis cell suspensions on Western blots. Clone 4D21 binds the PSTAIRE epitope, whereas the 3D1, 3D2 and 3D10 clones bind, as yet unidentified, epitopes of CDC2aAt. Furthermore, the accumulation and antigen-binding activity of these scFv fragments in a reducing environment were assessed. No interaction could be shown between the scFv fragments and CDC2aAt in a yeast two-hybrid assay. However, after transient expression of the scFv fragments in the cytosol of tobacco leaves, three of six scFv fragments (3D1, 3D2 and 3D10) accumulated in the plant cytosol and ELISA results indicate that these scFv fragments retained antigen-binding activity.

The plantibody approach: expression of antibody genes in plants to modulate plant metabolism or to obtain pathogen resistance.

Immunomodulation is a molecular technique that allows the interference with cellular metabolism or pathogen infectivity by the ectopic expression of genes encoding antibodies or antibody fragments. In recent years, several reports have proven the value of this tool in plant research for modulation of phytohormone activity and for blocking plant-pathogen infection. Efficient application of the plantibody approach requires different levels of investigation. First of all, methods have to be available to clone efficiently the genes coding for antibodies or antibody fragments that bind the target antigen. Secondly, conditions to obtain high accumulation of antigen-binding antibodies and antibody fragments in plants are being investigated and optimized. Thirdly, different strategies are being evaluated to interfere with the function of the target molecule, thus enabling immunomodulation of metabolism or pathogen infectivity. In the near future, optimized antibody gene isolation and expression, especially in reducing subcellular environments, such as the cytosol and nucleus, should turn immunomodulation into a powerful and attractive tool for gene inactivation, complementary to the classical antisense and co-suppression approaches.

Plants as bioreactors for protein production: avoiding the problem of transgene silencing.

Plants are particularly attractive as large-scale production systems for proteins intended for therapeutical or industrial applications: they can be grown easily and inexpensively in large quantities that can be harvested and processed with the available agronomic infrastructures. The effective use of plants as bioreactors depends on the possibility of obtaining high protein accumulation levels that are stable during the life cycle of the transgenic plant and in subsequent generations. Silencing of the introduced transgenes has frequently been observed in plants, constituting a major commercial risk and hampering the general economic exploitation of plants as protein factories. Until now, the most efficient strategy to avoid transgene silencing involves careful design of the transgene construct and thorough analysis of transformants at the molecular level. Here, we focus on different aspects of the generation of transgenic plants intended for protein production and on their influence on the stability of heterologous gene expression.

Both sense and antisense RNAs are targets for the sense transgene-induced posttranscriptional silencing mechanism.

Two stable transgenic tobacco lines were obtained as segregants from a primary transformant. Plants homozygous for a T-DNA inverted repeat locus (HOlo1) showed posttranscriptional gene silencing (PTGS) of the neomycin phosphotransferase II (nptII) transgenes, whereas HOlo2 plants, homozygous for a single T-DNA insert, expressed the nptII genes normally. Transient expression of nptII genes newly introduced into leaves of both the HOlo2 and nptII-silenced HOlo1 plants was downregulated only in the silenced background. Different chimeric beta-glucuronidase (gus) genes with parts of the nptII transgene inserted in sense or antisense orientation into the 3'-untranslated region, which encoded transcripts that had homology or complementarity to nptII transcripts. showed reduced transient expression specifically in nptII-silenced tissue. Therefore, we conclude that RNAs of both polarities are targets for PTGS-induced RNA degradation, which supports the notion that double-stranded RNA acts as an inducing signal for silencing.

Determination of the T-DNA transfer and the T-DNA integration frequencies upon cocultivation of Arabidopsis thaliana root explants.

Using the Cre/lox recombination system, we analyzed the extent to which T-DNA transfer to the plant cell and T-DNA integration into the plant genome determine the transformation and cotransformation frequencies of Arabidopsis root cells. Without selection for transformation competence, the stable transformation frequency of shoots obtained after cocultivation and regeneration on nonselective medium is below 0.5%. T-DNA transfer and expression occur in 5% of the shoots, indicating that the T-DNA integrates in less than 10% of the transiently expressing plant cells. A limited fraction of root cells, predominantly located at the wounded sites and in the pericycle, are competent for interaction with agrobacteria and the uptake of a T-DNA, as demonstrated by histochemical GUS staining. When selection for transformation competence is applied, the picture is completely different. Then, approximately 50% of the transformants show transient expression of a second, nonselected T-DNA and almost 50% of these cotransferred T-DNAs are integrated into the plant genome. Our results indicate that both T-DNA transfer and T-DNA integration limit the transformation and cotransformation frequencies and that plant cell competence for transformation is based on these two factors.

Analysis of the interaction between single-chain variable fragments and their antigen in a reducing intracellular environment using the two-hybrid system.

The coding sequences of three single-chain variable (scFv) fragments (A4, G4 and H3), which bind to dihydroflavonol-4-reductase (DFR) of Petunia hybrida, and the DFR-encoding sequence were cloned in two-hybrid vectors. The vectors were transformed in the yeast strain HF7c (his3-200, trp1-901, leu2-3) and the scFv-DFR interaction was analyzed by measuring yeast growth on medium without histidine. ScFv-G4 and, to a lesser extent, scFv-A4 could interact with DFR in the yeast nucleus. On the contrary, scFv-H3 showed no interaction with its antigen in yeast. The results of a previous expression analysis of the same scFv fragments in the plant cytosol correlate with those of the two-hybrid test. This suggests that it is possible to evaluate the antigen-scFv interaction in a reducing subcellular environment with the two-hybrid test. Therefore, the yeast two-hybrid system can be useful to identify candidate scFv fragments for intracellular antibody applications.

Cross-talk between posttranscriptionally silenced neomycin phosphotransferase II transgenes.

Tobacco plants containing a transgene locus with two chimeric neomycin phosphotransferase II (nptII) genes in tail-to-tail orientation (locus 1) show posttranscriptional gene silencing. The silenced nptII transgenes of locus 1 can downregulate the expression of homologous nptII transgenes in hybrid plants. The 3' region of the silenced nptII genes located in the center of the inverted repeat locus 1 is extensively methylated. Moreover, 3' segments of in trans-inactivated transgenes also become methylated, suggesting cross-talk between homologous posttranscriptionally silenced genes. Our results are in accordance with the hypothesis that this cross-talk can be mediated by specially featured RNAs.

Drug-induced hypomethylation of a posttranscriptionally silenced transgene locus of tobacco leads to partial release of silencing.

The effect of DNA methylation upon posttranscriptional gene silencing (PTGS) has been investigated in transgenic tobacco lines showing PTGS and methylation of the neomycin phosphotransferase II (nptII) reporter genes. Application of the hypomethylation drugs dihydroxypropyladenine or 5-azacytidine resulted in approximately 30% reduced methylation of cytosines located in a non-symmetrical context in the 3' untranslated region of the nptII transgenes. The hypomethylation was accompanied by up to 12-fold increase in NPTII protein levels, suggesting that methylation of non-symmetrical motifs may account for an increased degree of PTGS. Models for the possible role of DNA methylation in PTGS are discussed.

The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a recombination process resembling T-DNA integration.

After Agrobacterium-mediated plant transformation, multiple T-DNAs frequently integrate at the same position in the plant genome, resulting in the formation of inverted and direct repeats. Because these inverted repeats cannot be amplified and analyzed by PCR, Arabidopsis root cells were co-transformed with two different T-DNAs with distinct sequences adjacent to the T-DNA borders. Nine direct or inverted T-DNA border junctions were analyzed at the sequence level. Precise end-to-end fusions were found between two right border ends, whereas imprecise fusions and filler DNA were present in T-DNA linkages containing a left border end. The results suggest that end-to-end ligation of double-stranded T-DNAs occurs especially between right T-DNA ends and that illegitimate recombination on the basis of microhomology, deletions, repair activities and insertions of filler DNA is involved in the formation of left border T-DNA junctions. Therefore, a similar illegitimate recombination mechanism is proposed that is involved in the formation of complex T-DNA inserts as well as in the integration of the T-DNA in the plant genome.

Gene silencing results in instability of antibody production in transgenic plants.

The stability of antibody and Fab expression was assessed in five different homozygous transgenic Arabidopsis lines. Each of these lines showed silencing of the transgenes that encode the antibody polypeptides, leading to instability of antibody production. However, each line had a different and specific instability profile. The characteristic variation in the level of antibody accumulation in each line as a function of developmental stage indicated that the T-DNA integration pattern played a role in triggering silencing, and also that the history and the integration position of simple transgene loci can influence the susceptibility to epigenetic silencing. In different lines with low antibody accumulation levels, methylation was found either in the promoter alone, in both the promoter and the transcribed region, in the transcribed region only, or in the transcribed region and downstream sequences. In conclusion, our data suggest that epigenetic effects result in different transgene expression profiles in each of the five Arabidopsis lines analyzed.

High level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida.

The accumulation of five murine single-chain variable fragments, binding to dihydroflavonol 4-reductase, was analyzed in transgenic Petunia hybrida plants. The five scFv-encoding sequences were cloned in an optimized plant transformation vector for expression in the cytosol under control of the 35S promoter. In a transient expression assay we found that the scFv expression levels were reproducible and correlated with those in stably transformed petunia. Our results show that accumulation in the cytosol strongly depends on the intrinsic properties of the scFv fragment. Three of the five scFv fragments accumulated to unexpectedly high levels in the cytosol of the primary transformants, but no phenotypic effect could be detected. Experimental results indicate that one of the scFv fragments accumulated in the cytosol to 1% of the total soluble protein as a functional antigen-binding protein in the absence of disulphide bonds. This observation supports the idea that certain antibody fragments do not need disulphide bonds to be stable and functional. Such scFv scaffolds provide new opportunities to design scFv fragments for immunomodulation in the cytosol.