Common clonal origin of conventional T cells and induced regulatory T cells in breast cancer patients.

Regulatory CD4+ T cells (Treg) prevent tumor clearance by conventional T cells (Tconv) comprising a major obstacle of cancer immune-surveillance. Hitherto, the mechanisms of Treg repertoire formation in human cancers remain largely unclear. Here, we analyze Treg clonal origin in breast cancer patients using T-Cell Receptor and single-cell transcriptome sequencing. While Treg in peripheral blood and breast tumors are clonally distinct, Tconv clones, including tumor-antigen reactive effectors (Teff), are detected in both compartments. Tumor-infiltrating CD4+ cells accumulate into distinct transcriptome clusters, including early activated Tconv, uncommitted Teff, Th1 Teff, suppressive Treg and pro-tumorigenic Treg. Trajectory analysis suggests early activated Tconv differentiation either into Th1 Teff or into suppressive and pro-tumorigenic Treg. Importantly, Tconv, activated Tconv and Treg share highly-expanded clones contributing up to 65% of intratumoral Treg. Here we show that Treg in human breast cancer may considerably stem from antigen-experienced Tconv converting into secondary induced Treg through intratumoral activation.

Mapping Active Gene-Regulatory Regions in Human Repopulating Long-Term HSCs.

Genes that regulate hematopoietic stem cell (HSC) self-renewal, proliferation, and differentiation are tightly controlled by regulatory regions. However, mapping such regions relies on surface markers and immunophenotypic definition of HSCs. Here, we use γ-retroviral integration sites (γRV ISs) from a gene therapy trial for 10 patients with Wiskott-Aldrich syndrome to mark active enhancers and promoters in functionally defined long-term repopulating HSCs. Integration site clusters showed the highest ATAC-seq signals at HSC-specific peaks and strongly correlated with hematopoietic risk variants. Tagged genes were significantly enriched for HSC gene sets. We were able to map over 3,000 HSC regulatory regions in late-contributing HSCs, and we used these data to identify miR-10a and miR-335 as two miRNAs regulating early hematopoiesis. In this study, we show that viral insertion sites can be used as molecular tags to assess chromatin conformation on functionally defined cell populations, thereby providing a genome-wide resource for regulatory regions in human repopulating long-term HSCs.

AAVvector-mediated in vivo reprogramming into pluripotency.

In vivo reprogramming of somatic cells into induced pluripotent stem cells (iPSC) holds vast potential for basic research and regenerative medicine. However, it remains hampered by a need for vectors to express reprogramming factors (Oct-3/4, Klf4, Sox2, c-Myc; OKSM) in selected organs. Here, we report OKSM delivery vectors based on pseudotyped Adeno-associated virus (AAV). Using the AAV-DJ capsid, we could robustly reprogram mouse embryonic fibroblasts with low vector doses. Swapping to AAV8 permitted to efficiently reprogram somatic cells in adult mice by intravenous vector delivery, evidenced by hepatic or extra-hepatic teratomas and iPSC in the blood. Notably, we accomplished full in vivo reprogramming without c-Myc. Most iPSC generated in vitro or in vivo showed transcriptionally silent, intronic or intergenic vector integration, likely reflecting the increased host genome accessibility during reprogramming. Our approach crucially advances in vivo reprogramming technology, and concurrently facilitates investigations into the mechanisms and consequences of AAV persistence.

Lentiviral Vector Promoter is Decisive for Aberrant Transcript Formation.

Lentiviral vectors hold great promise for the genetic correction of various inherited diseases. However, lentiviral vector biology is still not completely understood and warrants the precise decoding of molecular mechanisms underlying integration and post-translational modification. This study investigated a series of self-inactivating (SIN) and full long terminal repeat (LTR) lentiviral vectors that contained different types of promoters with or without a transgene to gain deeper insights in lentiviral target site selection and potential perturbation of cellular gene expression. Using an optimized nonrestrictive linear amplification-mediated polymerase chain reaction (nrLAM-PCR) protocol, vector structure-dependent integration site profiles were observed upon transduction of mouse lin- hematopoietic progenitors in vitro. Initial target site selection mainly depended on the presence of the promoter while being independent of its nature. Despite the increased propensity for read-through transcription of SIN lentiviral vectors, the incidence of viral-cellular fusion transcript formation involving the canonical viral splice donor or cryptic splice sites was reduced in both unselected primary lin- cells and transformed 32D cells. Moreover, the strength of the internal promoter in vectors with SIN LTRs is decisive for in vitro selection and for the abundance of chimeric transcripts, which are decreased by moderately active promoters. These results will help to better understand vector biology and to optimize therapeutic vectors for future gene therapy applications.

Genetic subclone architecture of tumor clone-initiating cells in colorectal cancer.

A hierarchically organized cell compartment drives colorectal cancer (CRC) progression. Genetic barcoding allows monitoring of the clonal output of tumorigenic cells without prospective isolation. In this study, we asked whether tumor clone-initiating cells (TcICs) were genetically heterogeneous and whether differences in self-renewal and activation reflected differential kinetics among individual subclones or functional hierarchies within subclones. Monitoring genomic subclone kinetics in three patient tumors and corresponding serial xenografts and spheroids by high-coverage whole-genome sequencing, clustering of genetic aberrations, subclone combinatorics, and mutational signature analysis revealed at least two to four genetic subclones per sample. Long-term growth in serial xenografts and spheroids was driven by multiple genomic subclones with profoundly differing growth dynamics and hence different quantitative contributions over time. Strikingly, genetic barcoding demonstrated stable functional heterogeneity of CRC TcICs during serial xenografting despite near-complete changes in genomic subclone contribution. This demonstrates that functional heterogeneity is, at least frequently, present within genomic subclones and independent of mutational subclone differences.

GENE-IS: Time-Efficient and Accurate Analysis of Viral Integration Events in Large-Scale Gene Therapy Data.

Integration site profiling and clonality analysis of viral vector distribution in gene therapy is a key factor to monitor the fate of gene-corrected cells, assess the risk of malignant transformation, and establish vector biosafety. We developed the Genome Integration Site Analysis Pipeline (GENE-IS) for highly time-efficient and accurate detection of next-generation sequencing (NGS)-based viral vector integration sites (ISs) in gene therapy data. It is the first available tool with dual analysis mode that allows IS analysis both in data generated by PCR-based methods, such as linear amplification method PCR (LAM-PCR), and by rapidly evolving targeted sequencing (e.g., Agilent SureSelect) technologies. GENE-IS makes use of trimming strategies, customized reference genome, and soft-clipped information with sequential filtering steps to provide annotated IS with clonality information. It is a scalable, robust, precise, and reliable tool for large-scale pre-clinical and clinical data analysis that provides users complete flexibility and control over analysis with a broad range of configurable parameters. GENE-IS is available at https://github.com/G100DKFZ/gene-is.

A Graph Based Framework to Model Virus Integration Sites.

With next generation sequencing thousands of virus and viral vector integration genome targets are now under investigation to uncover specific integration preferences and to define clusters of integration, termed common integration sites (CIS), that may allow to assess gene therapy safety or to detect disease related genomic features such as oncogenes. Here, we addressed the challenge to: 1) define the notion of CIS on graph models, 2) demonstrate that the structure of CIS enters in the category of scale-free networks and 3) show that our network approach analyzes CIS dynamically in an integrated systems biology framework using the Retroviral Transposon Tagged Cancer Gene Database (RTCGD) as a testing dataset.

Recombinant AAV Integration Is Not Associated With Hepatic Genotoxicity in Nonhuman Primates and Patients.

Recombinant adeno-associated viral vectors (rAAV) currently constitute a real therapeutic strategy for the sustained correction of diverse genetic conditions. Though a wealth of preclinical and clinical studies have been conducted with rAAV, the oncogenic potential of these vectors is still controversial, particularly when considering liver-directed gene therapy. Few preclinical studies and the recent discovery of incomplete wild-type AAV2 genomes integrated in human hepatocellular carcinoma biopsies have raised concerns on rAAV safety. In the present study, we have characterized the integration of both complete and partial rAAV2/5 genomes in nonhuman primate tissues and clinical liver biopsies from a trial aimed to treat acute intermittent porphyria. We applied a new multiplex linear amplification-mediated polymerase chain reaction (PCR) assay capable of detecting integration events that are originated throughout the rAAV genome. The integration rate was low both in nonhuman primates and patient's samples. Importantly, no integration clusters or events were found in genes previously reported to link rAAV integration with hepatocellular carcinoma development, thus showing the absence of genotoxicity of a systemically administered rAAV2/5 in a large animal model and in the clinical context.

Tracking genetically engineered lymphocytes long-term reveals the dynamics of T cell immunological memory.

Long-lasting immune protection from pathogens and cancer requires the generation of memory T cells able to survive long-term. To unravel the immunological requirements for long-term persistence of human memory T cells, we characterized and traced, over several years, T lymphocytes genetically modified to express the thymidine kinase (TK) suicide gene that were infused in 10 patients after haploidentical hematopoietic stem cell transplantation (HSCT). At 2 to 14 years after infusion and in the presence of a broad and resting immune system, we could still detect effectors/effector memory (TEM/EFF), central memory (TCM), and stem memory (TSCM) TK(+) cells, circulating at low but stable levels in all patients. Longitudinal analysis of cytomegalovirus (CMV)- and Flu-specific TK(+) cells indicated that antigen recognition was dominant in driving in vivo expansion and persistence at detectable levels. The amount of infused TSCM cells positively correlated with early expansion and with the absolute counts of long-term persisting gene-marked cells. By combining T cell sorting with sequencing of integration (IS), TCRα and TCRß clonal markers, we showed that T cells retrieved long-term were enriched in clones originally shared in different memory T cell subsets, whereas dominant long-term clonotypes appeared to preferentially originate from infused TSCM and TCM clones. Together, these results indicate that long-term persistence of gene-modified memory T cells after haploidentical HSCT is influenced by antigen exposure and by the original phenotype of infused cells. Cancer adoptive immunotherapy might thus benefit from cellular products enriched in lymphocytes with an early-differentiated phenotype.

High-resolution analysis of the human T-cell receptor repertoire.

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 ß-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.

Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against cytomegalovirus after stem cell transplantation.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.

Presence of a trs-Like Motif Promotes Rep-Mediated Wild-Type Adeno-Associated Virus Type 2 Integration.

High-throughput integration site (IS) analysis of wild-type adeno-associated virus type 2 (wtAAV2) in human dermal fibroblasts (HDFs) and HeLa cells revealed that juxtaposition of a Rep binding site (RBS) and terminal resolution site (trs)-like motif leads to a 4-fold-increased probability of wtAAV integration. Electrophoretic mobility shift assays (EMSAs) confirmed binding of Rep to off-target RBSs. For the first time, we show Rep protein off-target nicking activity, highlighting the importance of the nicking substrate for Rep-mediated integration.

Preclinical evaluation of efficacy and safety of an improved lentiviral vector for the treatment of ß-thalassemia and sickle cell disease.

A previously published clinical trial demonstrated the benefit of autologous CD34(+) cells transduced with a selfinactivating lentiviral vector (HPV569) containing an engineered ß-globin gene (ß(A-T87Q)-globin) in a subject with ß 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 ß-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 ß-thalassemia major and sickle cell disease.

Comparative next-generation sequencing of adeno-associated virus inverted terminal repeats.

The inverted terminal repeats (ITRs) of adeno-associated virus (AAV) are notoriously difficult to sequence owing to their high GC-content (70%) and palindromic sequences that result in the formation of a very stable, 125 bp long, T-shaped hairpin structure. Here we evaluate the performance of two widely used next-generation sequencing platforms, 454 GS FLX (Roche) and MiSeq Benchtop Sequencer (Illumina), in analyzing ITRs in comparatively sequencing linear amplification-meditated PCR (LAM-PCR) amplicons derived from AAV-concatemeric structures. While our data indicate that both platforms can sequence complete ITRs, efficiencies (MiSeq: 0.11% of sequence reads; 454: 0.02% of reads), frequencies (MiSeq: 171 full ITRs, 454: 3 full ITRs), and rates of deviation from the derived ITR consensus sequence (MiSeq: 0.8%-1.3%; 454: 0.5%) did differ. These results suggest that next-generation sequencing platforms can be used to specifically detect ITR mutations and sequence complete ITRs.

Gene therapy for Wiskott-Aldrich syndrome--long-term efficacy and genotoxicity.

Wiskott-Aldrich syndrome (WAS) is characterized by microthrombocytopenia, immunodeficiency, autoimmunity, and susceptibility to malignancies. In our hematopoietic stem cell gene therapy (GT) trial using a γ-retroviral vector, 9 of 10 patients showed sustained engraftment and correction of WAS protein (WASP) expression in lymphoid and myeloid cells and platelets. GT resulted in partial or complete resolution of immunodeficiency, autoimmunity, and bleeding diathesis. Analysis of retroviral insertion sites revealed >140,000 unambiguous integration sites and a polyclonal pattern of hematopoiesis in all patients early after GT. Seven patients developed acute leukemia [one acute myeloid leukemia (AML), four T cell acute lymphoblastic leukemia (T-ALL), and two primary T-ALL with secondary AML associated with a dominant clone with vector integration at the LMO2 (six T-ALL), MDS1 (two AML), or MN1 (one AML) locus]. Cytogenetic analysis revealed additional genetic alterations such as chromosomal translocations. This study shows that hematopoietic stem cell GT for WAS is feasible and effective, but the use of γ-retroviral vectors is associated with a substantial risk of leukemogenesis.

From bench to bedside: preclinical evaluation of a self-inactivating gammaretroviral vector for the gene therapy of X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by impaired antimicrobial activity in phagocytic cells. As a monogenic disease affecting the hematopoietic system, CGD is amenable to gene therapy. Indeed in a phase I/II clinical trial, we demonstrated a transient resolution of bacterial and fungal infections. However, the therapeutic benefit was compromised by the occurrence of clonal dominance and malignant transformation demanding alternative vectors with equal efficacy but safety-improved features. In this work we have developed and tested a self-inactivating (SIN) gammaretroviral vector (SINfes.gp91s) containing a codon-optimized transgene (gp91(phox)) under the transcriptional control of a myeloid promoter for the gene therapy of the X-linked form of CGD (X-CGD). Gene-corrected cells protected X-CGD mice from Aspergillus fumigatus challenge at low vector copy numbers. Moreover, the SINfes.gp91s vector generates substantial amounts of superoxide in human cells transplanted into immunodeficient mice. In vitro genotoxicity assays and longitudinal high-throughput integration site analysis in transplanted mice comprising primary and secondary animals for 11 months revealed a safe integration site profile with no signs of clonal dominance.

A largely random AAV integration profile after LPLD gene therapy.

The clinical application of adeno-associated virus vectors (AAVs) is limited because of concerns about AAV integration-mediated tumorigenicity. We performed integration-site analysis after AAV1-LPL(S447X) intramuscular injection in five lipoprotein lipase-deficient subjects, revealing random nuclear integration and hotspots in mitochondria. We conclude that AAV integration is potentially safe and that vector breakage and integration may occur from each position of the vector genome. Future viral integration-site analyses should include the mitochondrial genome.

Integrase-deficient lentiviral vectors mediate efficient gene transfer to human vascular smooth muscle cells with minimal genotoxic risk.

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.

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.