Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 107 TU mL-1 in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 107 TU mL-1 in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL-1 day-1 for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

High yield sterile filtration process for highly concentrated lentiviral vectors.

The development and manufacture of biopharmaceuticals are subject to strict regulations that specify the required minimum quality of the products. A key measure to meet these quality requirements is the integration of a sterile filtration step into the commercial manufacturing process. Whereas common procedures for most biologics exist, this is challenging for lentiviral vector (LVV) production for ex vivo gene therapy. LVVs nominal size is more than half the pore size (0.2 µm) of filters used for sterile filtration. Hence, highly concentrated virus solutions are prone to filter clogging if aggregation of viruses occurs or impurities attach to the viruses. Several filters were screened aiming to identify those which allow filtering highly concentrated stocks of LVVs of up to 1E + 9 transducing units mL-1 , which corresponds to 4.5E + 12 particles mL-1 . In addition, the effect of endonuclease treatment upstream of the purification process on filter performance was studied. In summary, three suitable filters were identified in a small-scale study (<15 mL) with virus yields >80% and the process was successfully scaled-up to a final scale of 100 mL LVV stock solution.

Development of a perfusion process for continuous lentivirus production using stable suspension producer cell lines.

The large-scale production of clinical-grade lentiviral vectors (LVs) for gene therapy applications is a remaining challenge. The use of adherent cell lines and methods like transient transfection are cost-intensive and hamper process scalability as well as reproducibility. This study describes the use of two suspension-adapted stable packaging cell lines, called GPRGs and GPRTGs, for the development of a scalable and serum-free LV production process. Both stable packaging cell lines are based on an inducible Tet-off system, thus requiring doxycycline removal for initiation of the virus production. Therefore, we compared different methods for doxycycline removal and inoculated three independent 5 L bioreactors using a scalable induction method by dilution, an acoustic cell washer and manual centrifugation. The bioreactors were inoculated with a stable producer cell line encoding for a LV carrying a clinically relevant gene. LV production was performed in perfusion mode using a cell retention device based on acoustic wave separation. Comparable cell-specific productivities were obtained with all three methods and cumulative functional yields up to 6.36 × 1011 transducing units per bioreactor were generated in a 234-h long process, demonstrating the usability of stable Tet-off cell lines for an easily scalable suspension process. Remarkably, cell viabilities >90% were maintained at high cell densities without compromising productivity throughout the whole process, allowing to further extend the process time. Given its low effects of toxicity during virus production, the presented cell lines are excellent candidates to develop a fully continuous LV production process to overcome the existing bottlenecks in LV manufacturing.

The process of somatic hypermutation increases polyreactivity for central nervous system antigens in primary central nervous system lymphoma.

The immunoglobulin (Ig) heavy and light chain variable gene mutational pattern of the B cell receptor (BCR) in primary central nervous system (CNS) lymphoma (PCNSL) cells suggests antigenic selection to drive pathogenesis and confinement to the CNS. This hypothesis is supported by the observation that the tumor B cell receptor (tBCR) of PCNSL is polyreactive and may be stimulated by CNS proteins. To obtain further insight into the role of the germinal center (GC) reaction on BCR reactivity, we constructed recombinant antibodies (recAb) with Ig heavy and light chain sequences of the corresponding naive BCR (nBCR) by reverting tBCR somatic mutations in 10 PCNSL. Analysis of nBCR-derived recAb reactivity by a protein microarray and immunoprecipitation demonstrated auto- and polyreactivity in all cases. Self-/polyreactivity was not lost during the GC reaction; surprisingly, tBCR significantly increased self-/polyreactivity. In addition to proteins recognized by both the nBCR and tBCR, tBCR gained self-/polyreactivity particularly for proteins expressed in the CNS including proteins of oligodendrocytes/myelin, the S100 protein family, and splicing factors. Thus, in PCNSL pathogenesis, a faulty GC reaction may increase self-/polyreactivity, hereby facilitating BCR signaling via multiple CNS antigens, and may ultimately foster tumor cell survival in the CNS.

The RNA-associated proteins MKT1 and MKT1L form alternative PBP1-containing complexes in Trypanosoma brucei.

Control of gene expression in kinetoplastids such as trypanosomes depends heavily on RNA-binding proteins that influence mRNA decay and translation. We previously showed that the trypanosome protein MKT1 forms a multicomponent protein complex: MKT1 interacts with PBP1, which in turn recruits LSM12 and poly(A)-binding protein. MKT1 is recruited to mRNAs by sequence-specific RNA-binding proteins, resulting in stabilization of the bound mRNA. We here show that PBP1, LSM12, and a 117-residue protein, XAC1 (Tb927.7.2780), are present in complexes that contain either MKT1 or an MKT1-like protein, MKT1L (Tb927.10.1490). All five proteins are present predominantly in the complexes, and we found evidence for a minor subset of complexes containing both MKT1 and MKT1L. XAC1-containing complexes reproducibly contained RNA-binding proteins that were previously found associated with MKT1. Moreover, XAC1- or MKT1-containing complexes specifically recruited one of the two poly(A)-binding proteins, PABP2, and one of the six cap-binding translation initiation complexes, EIF4E6-EIF4G5. Yeast two-hybrid assay results indicated that MKT1 directly interacts with EIF4G5. MKT1-PBP1 complexes can therefore interact with mRNAs via their poly(A) tails and caps, as well as through sequence-specific RNA-binding proteins. Correspondingly, MKT1 is associated with many mRNAs, although not with those encoding ribosomal proteins. Meanwhile, MKT1L resembles MKT1 at the C terminus but additionally features an N-terminal extension with low-complexity regions. Although MKT1L depletion inhibited cell proliferation, we found no evidence that it specifically interacts with RNA-binding proteins or mRNA. We speculate that MKT1L may compete with MKT1 for PBP1 binding and thereby modulate the function of MKT1-containing complexes.

Insights on a putative aminoacyl-tRNA-protein transferase of Leishmania major.

The N-end rule pathway leads to regulated proteolysis as an adaptive response to external stress and is ubiquitous from bacteria to mammals. In this study, we investigated a gene coding for a putative core enzyme of this post-translational regulatory pathway in Leishmania major, which may be crucial during cytodifferentiation and the environment adaptive responses of the parasite. Leucyl, phenylalanyl-tRNA protein transferase and arginyl-tRNA protein transferase are key components of this pathway in E. coli and eukaryotes, respectively. They catalyze the specific conjugation of leucine, phenylalanine or arginine to proteins containing exposed N-terminal amino acid residues, which are recognized by the machinery for the targeted proteolysis. Here, we characterized a conserved hypothetical protein coded by the LmjF.21.0725 gene in L. major. In silico analysis suggests that the LmjF.21.0725 protein is highly conserved among species of Leishmania and might belong to the Acyl CoA-N-acyltransferases (NAT) superfamily of proteins. Immunofluorescence cell imaging indicates that the cytosolic localization of the studied protein and the endogenous levels of the protein in promastigotes are barely detectable by western blotting assay. The knockout of the two alleles of LmjF.21.0725 by homologous recombination was only possible in the heterozygous transfectant expressing LmjF.21.0725 as a transgene from a plasmid. Moreover, the kinetics of loss of the plasmid in the absence of drug pressure suggests that maintenance of the gene is essential for promastigote survival. Here, evidence is provided that this putative aminoacyl tRNA-protein transferase is essential for parasite survival. The enzyme activity and corresponding post-translational regulatory pathway are yet to be investigated.

The suppressive cap-binding complex factor 4EIP is required for normal differentiation.

Trypanosoma brucei live in mammals as bloodstream forms and in the Tsetse midgut as procyclic forms. Differentiation from one form to the other proceeds via a growth-arrested stumpy form with low messenger RNA (mRNA) content and translation. The parasites have six eIF4Es and five eIF4Gs. EIF4E1 pairs with the mRNA-binding protein 4EIP but not with any EIF4G. EIF4E1 and 4EIP each inhibit expression when tethered to a reporter mRNA, but while tethered EIF4E1 suppresses only when 4EIP is present, suppression by tethered 4EIP does not require the interaction with EIF4E1. In growing bloodstream forms, 4EIP is preferentially associated with unstable mRNAs. Bloodstream- or procyclic-form trypanosomes lacking 4EIP have only a marginal growth disadvantage. Bloodstream forms without 4EIP are, however, defective in translation suppression during stumpy-form differentiation and cannot subsequently convert to growing procyclic forms. Intriguingly, the differentiation defect can be complemented by a truncated 4EIP that does not interact with EIF4E1. In contrast, bloodstream forms lacking EIF4E1 have a growth defect, stumpy formation seems normal, but they appear unable to grow as procyclic forms. We suggest that 4EIP and EIF4E1 fine-tune mRNA levels in growing cells, and that 4EIP contributes to translation suppression during differentiation to the stumpy form.

Disclosing 3' UTR cis-elements and putative partners involved in gene expression regulation in Leishmania spp.

To identify putative cis-elements involved in gene expression regulation in Leishmania, we previously conducted an in silico investigation to find conserved intercoding sequences (CICS) in the genomes of L. major, L. infantum, and L. braziliensis. Here, the CICS databank was explored to search for sequences that were present in the untranslated regions (UTRs) of groups of genes showing similar expression profiles during in vitro differentiation. Using a selectable marker as a reporter gene, flanked by either an intact 3' UTR or a UTR lacking the conserved element, the regulatory role of a CICS was confirmed. We observed that the pattern of modulation of the mRNA levels was altered in the absence of the CICS. We also identified putative CICS RNA-binding proteins. This study suggests that the publicly available CICS database is a useful tool for identifying regulatory cis-elements for Leishmania genes and suggests the existence of post-transcriptional regulons in Leishmania.

The role of the zinc finger protein ZC3H32 in bloodstream-form Trypanosoma brucei.

Kinetoplastids rely heavily on post-transcriptional mechanisms for control of gene expression, with regulation of mRNA processing, translation and degradation by RNA-binding proteins. ZC3H32 is a cytosolic mRNA-binding protein with three non-canonical CCCH zinc finger domains. It is much more abundant in bloodstream-form Trypanosoma brucei than in procyclic forms. Tethering of ZC3H32 to a reporter mRNA suppressed translation and resulted in mRNA degradation, and deletion analysis suggested that this activity was present in both the N- and C-terminal domains, but not the central zinc finger-containing domain. Tandem affinity purification, however, revealed no interaction partners that might account for this activity. RNASeq analyses did not yield any evidence for sequence-specific binding or regulation of specific mRNAs. The presence of ZC3H32 homologues in monogenetic and free-living Euglenids also argues against a role in developmental regulation, although its function may have diverged in evolution. T. brucei ZC3H32 might be implicated in basal mRNA metabolism, with this role perhaps being taken over by another protein in procyclic forms.

Translation Regulation and RNA Granule Formation after Heat Shock of Procyclic Form Trypanosoma brucei: Many Heat-Induced mRNAs Are also Increased during Differentiation to Mammalian-Infective Forms.

African trypanosome procyclic forms multiply in the midgut of tsetse flies, and are routinely cultured at 27°C. Heat shocks of 37°C and above result in general inhibition of translation, and severe heat shock (41°C) results in sequestration of mRNA in granules. The mRNAs that are bound by the zinc-finger protein ZC3H11, including those encoding refolding chaperones, escape heat-induced translation inhibition. At 27°C, ZC3H11 mRNA is predominantly present as an untranslated cytosolic messenger ribonucleoprotein particle, but after heat shocks of 37°C-41°C, the ZC3H11 mRNA moves into the polysomal fraction. To investigate the scope and specificities of heat-shock translational regulation and granule formation, we analysed the distributions of mRNAs on polysomes at 27°C and after 1 hour at 39°C, and the mRNA content of 41°C heat shock granules. We found that mRNAs that bind to ZC3H11 remained in polysomes at 39°C and were protected from sequestration in granules at 41°C. As previously seen for starvation stress granules, the mRNAs that encode ribosomal proteins were excluded from heat-shock granules. 70 mRNAs moved towards the polysomal fraction after the 39°C heat shock, and 260 increased in relative abundance. Surprisingly, many of these mRNAs are also increased when trypanosomes migrate to the tsetse salivary glands. It therefore seems possible that in the wild, temperature changes due to diurnal variations and periodic intake of warm blood might influence the efficiency with which procyclic forms develop into mammalian-infective forms.

Integrative analysis of the Trypanosoma brucei gene expression cascade predicts differential regulation of mRNA processing and unusual control of ribosomal protein expression.

BACKGROUND: Trypanosoma brucei is a unicellular parasite which multiplies in mammals (bloodstream form) and Tsetse flies (procyclic form). Trypanosome RNA polymerase II transcription is polycistronic, individual mRNAs being excised by trans splicing and polyadenylation. We previously made detailed measurements of mRNA half-lives in bloodstream and procyclic forms, and developed a mathematical model of gene expression for bloodstream forms. At the whole transcriptome level, many bloodstream-form mRNAs were less abundant than was predicted by the model. RESULTS: We refined the published mathematical model and extended it to the procyclic form. We used the model, together with known mRNA half-lives, to predict the abundances of individual mRNAs, assuming rapid, unregulated mRNA processing; then we compared the results with measured mRNA abundances. Remarkably, the abundances of most mRNAs in procyclic forms are predicted quite well by the model, being largely explained by variations in mRNA decay rates and length. In bloodstream forms substantially more mRNAs are less abundant than predicted. We list mRNAs that are likely to show particularly slow or inefficient processing, either in both forms or with developmental regulation. We also measured ribosome occupancies of all mRNAs in trypanosomes grown in the same conditions as were used to measure mRNA turnover. In procyclic forms there was a weak positive correlation between ribosome density and mRNA half-life, suggesting cross-talk between translation and mRNA decay; ribosome density was related to the proportion of the mRNA on polysomes, indicating control of translation initiation. Ribosomal protein mRNAs in procyclics appeared to be exceptionally rapidly processed but poorly translated. CONCLUSIONS: Levels of mRNAs in procyclic form trypanosomes are determined mainly by length and mRNA decay, with some control of precursor processing. In bloodstream forms variations in nuclear events play a larger role in transcriptome regulation, suggesting aquisition of new control mechanisms during adaptation to mammalian parasitism.

Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins.

We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites.

RNA-seq approaches for determining mRNA abundance in Leishmania.

High-throughput sequencing of cDNA copies of mRNA (RNA-seq) provides a digital read-out of mRNA levels over several orders of magnitude, as well as mapping the transcripts to the nucleotide level. Here we describe an RNA-seq approach that exploits the 39-nucleotide mini-exon or spliced leader (SL) sequence found at the 5' end of all Leishmania (and other trypanosomatid) mRNAs.

Current treatment and drug discovery against Leishmania spp. and Plasmodium spp.: a review.

Malaria and leishmaniasis are the most prevalent tropical diseases caused by protozoan parasites. Half of world's population is at risk of malaria and more than 2 million of new cases of leishmaniasis occur annually. There are no vaccines available for these diseases and current treatments suffer from several limitations. Therefore, novel drugs for malaria and leishmaniasis are much-needed. This article reviews the agents currently in use for treatment of these diseases, their known mechanisms of action and weaknesses. We present an overview of the main strategies for drug discovery and the relevance of these parasites genomics/proteomics data for a rational search of molecular targets and matching leads. In this direction, we emphasize the importance of the highly integrated partnerships and networks between scientists in academic institutions and industry involving several countries that promise to increase the chances of success and enhance cost-effectiveness in drug discovery against these parasitic diseases. In addition, we approach the available assays for testing lead compounds in large scale and their limitations for they represent one of the bottlenecks in the pipeline for novel drug discovery. We conclude the article presenting a recent coordinated initiative (TDR Transfection Network) established to overcome some of these limitations by the generation of Plasmodium and Leishmania transgenic parasites better suited for HTS platforms.