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1.  High-resolution analysis of the human T-cell receptor repertoire 
Nature Communications  2015;6:8081.
Unbiased dissection of T-cell receptor (TCR) repertoire diversity at the nucleotide level could provide important insights into human immunity. Here we show that TCR ligation-anchored-magnetically captured PCR (TCR-LA-MC PCR) identifies TCR α- and β-chain diversity without sequence-associated or quantitative restrictions in healthy and diseased conditions. TCR-LA-MC PCR identifies convergent recombination events, classifies different stages of cutaneous T-cell lymphoma in vivo and demonstrates TCR reactivation after in vitro cytomegalovirus stimulation. TCR-LA-MC PCR allows ultra-deep data access to both physiological TCR diversity and mechanisms influencing clonality in all clinical settings with restricted or distorted TCR repertoires.
Immune system diversity is generated by V(D)J recombination, leading to clonal T-cell lineages. Here the authors investigate the events leading to T-cell diversity through the use of a modified PCR technique combined with deep sequencing.
PMCID: PMC4569693  PMID: 26324409
2.  Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against cytomegalovirus after stem cell transplantation 
Reactivation of latent viruses such as human cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naïve T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs (“SmyleDCpp65”) that accelerated antigen-specific T cell development.
Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the cytomegalovirus antigen pp65) that were cryopreserved and thawed.
Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCpp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCpp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCpp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCpp65 was validated.
These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-015-0599-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4511080  PMID: 26198406
Monocyte; Dendritic cell; Lentiviral vector; Stem cell transplantation; Cytomegalovirus
3.  Preclinical Evaluation of Efficacy and Safety of an Improved Lentiviral Vector for the Treatment of β-Thalassemia and Sickle Cell Disease 
Current Gene Therapy  2015;15(1):64-81.
A previously published clinical trial demonstrated the benefit of autologous CD34+ cells transduced with a self-inactivating lentiviral vector (HPV569) containing an engineered β-globin gene (βA-T87Q globin) in a subject with β-thalassemia major. This vector has been modified to increase transduction efficacy without compromising safety. In vitro analyses indicated that the changes resulted in both increased vector titers (3 to 4 fold) and increased transduction efficacy (2 to 3 fold). An in vivo study in which 58 β-thalassemic mice were transplanted with vector- or mock-transduced syngenic bone marrow cells indicated sustained therapeutic efficacy. Secondary transplantations involving 108 recipients were performed to evaluate long-term safety. The six month study showed no hematological or biochemical toxicity. Integration site (IS) profile revealed an oligo/polyclonal hematopoietic reconstitution in the primary transplants and reduced clonality in secondary transplants. Tumor cells were detected in the secondary transplant mice in all treatment groups (including the control group), without statistical differences in the tumor incidence. Immunohistochemistry and quantitative PCR demonstrated that tumor cells were not derived from transduced donor cells. This comprehensive efficacy and safety data provided the basis for initiating two clinical trials with this second generation vector (BB305) in Europe and in the USA in patients with β-thalassemia major and sickle cell disease.
PMCID: PMC4440358  PMID: 25429463
β-hemoglobinopathy; β-thalassemia; gene therapy; lentiviral vector; mouse model
4.  Integrase-Deficient Lentiviral Vectors Mediate Efficient Gene Transfer to Human Vascular Smooth Muscle Cells with Minimal Genotoxic Risk 
Human Gene Therapy  2012;23(12):1247-1257.
We have previously shown that injury-induced neointima formation was rescued by adenoviral-Nogo-B gene delivery. Integrase-competent lentiviral vectors (ICLV) are efficient at gene delivery to vascular cells but present a risk of insertional mutagenesis. Conversely, integrase-deficient lentiviral vectors (IDLV) offer additional benefits through reduced mutagenesis risk, but this has not been evaluated in the context of vascular gene transfer. Here, we have investigated the performance and genetic safety of both counterparts in primary human vascular smooth muscle cells (VSMC) and compared gene transfer efficiency and assessed the genotoxic potential of ICLVs and IDLVs based on their integration frequency and insertional profile in the human genome. Expression of enhanced green fluorescent protein (eGFP) mediated by IDLVs (IDLV-eGFP) demonstrated efficient transgene expression in VSMCs. IDLV gene transfer of Nogo-B mediated efficient overexpression of Nogo-B in VSMCs, leading to phenotypic effects on VSMC migration and proliferation, similar to its ICLV version and unlike its eGFP control and uninfected VSMCs. Large-scale integration site analyses in VSMCs indicated that IDLV-mediated gene transfer gave rise to a very low frequency of genomic integration compared to ICLVs, revealing a close-to-random genomic distribution in VSMCs. This study demonstrates for the first time the potential of IDLVs for safe and efficient vascular gene transfer.
Chick and colleagues demonstrate that integrase-deficient lentiviral (IDLV) vector gene transfer of Nogo-B in human vascular smooth muscle cells (VSMCs) leads to significant phenotypic effects on VSMC migration and proliferation. Large-scale integration analysis in VSMCs revealed that the IDLV vector gave rise to a low frequency of genome integration compared with integrase-competent lentiviral vectors.
PMCID: PMC3523254  PMID: 22931362
5.  Brain cancer prognosis: independent validation of a clinical bioinformatics approach 
Translational and evidence based medicine can take advantage of biotechnology advances that offer a fast growing variety of high-throughput data for screening molecular activities of genomic, transcriptional, post-transcriptional and translational observations. The clinical information hidden in these data can be clarified with clinical bioinformatics approaches. We have recently proposed a method to analyze different layers of high-throughput (omic) data to preserve the emergent properties that appear in the cellular system when all molecular levels are interacting. We show here that this method applied to brain cancer data can uncover properties (i.e. molecules related to protective versus risky features in different types of brain cancers) that have been independently validated as survival markers, with potential important application in clinical practice.
PMCID: PMC3296594  PMID: 22297051
glioblastoma; survival; system; emergent property; high-throughput biology
6.  Joint analysis of transcriptional and post- transcriptional brain tumor data: searching for emergent properties of cellular systems 
BMC Bioinformatics  2011;12:86.
Advances in biotechnology offer a fast growing variety of high-throughput data for screening molecular activities of genomic, transcriptional, post-transcriptional and translational observations. However, to date, most computational and algorithmic efforts have been directed at mining data from each of these molecular levels (genomic, transcriptional, etc.) separately. In view of the rapid advances in technology (new generation sequencing, high-throughput proteomics) it is important to address the problem of analyzing these data as a whole, i.e. preserving the emergent properties that appear in the cellular system when all molecular levels are interacting. We analyzed one of the (currently) few datasets that provide both transcriptional and post-transcriptional data of the same samples to investigate the possibility to extract more information, using a joint analysis approach.
We use Factor Analysis coupled with pre-established knowledge as a theoretical base to achieve this goal. Our intention is to identify structures that contain information from both mRNAs and miRNAs, and that can explain the complexity of the data. Despite the small sample available, we can show that this approach permits identification of meaningful structures, in particular two polycistronic miRNA genes related to transcriptional activity and likely to be relevant in the discrimination between gliosarcomas and other brain tumors.
This suggests the need to develop methodologies to simultaneously mine information from different levels of biological organization, rather than linking separate analyses performed in parallel.
PMCID: PMC3078861  PMID: 21450054

Results 1-6 (6)