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The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in coronavirus disease 2019 (COVID-19). The present study was designed to conduct a systematic investigation of the interaction of human humoral fluid-phase pattern recognition molecules (PRMs) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of 12 PRMs tested, the long pentraxin 3 (PTX3) and mannose-binding lectin (MBL) bound the viral nucleocapsid and spike proteins, respectively. MBL bound trimeric spike protein, including that of variants of concern (VoC), in a glycan-dependent manner and inhibited SARS-CoV-2 in three in vitro models. Moreover, after binding to spike protein, MBL activated the lectin pathway of complement activation. Based on retention of glycosylation sites and modeling, MBL was predicted to recognize the Omicron VoC. Genetic polymorphisms at the MBL2 locus were associated with disease severity. These results suggest that selected humoral fluid-phase PRMs can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.

Adeno-associated Viral vectors (AAVs) have been successful exploited in gene therapy applications for the treatment of several genetic disorders. AAV is considered an episomal vector but it has been shown to integrate within the host-cell genome following generation of double-strand DNA breaks or nicks. While AAV integration raises some safety concerns, it can also provide therapeutic benefit; the direct intrathymic injection of an AAV harboring a therapeutic transgene results in integration in T cell progenitors and long-term T cell immunity. To assess the mechanisms of AAV integration, we retrieved and analyzed hundreds of AAV integration sites from lymph node-derived mature T-cells as compared to liver and brain tissue from treated mice. Notably, we found that while AAV integrations in the liver and brain were distributed across the entire mouse genome, >90% of the integrations in T-cells were clustered within the T cell receptor α, β and γ genes. More precisely, the insertion mapped to DNA breaks created by the enzymatic activity of recombination activating genes (RAG) during V(D)J recombination. Our data indicate that RAG activity during T-cell receptor maturation induces a site-specific integration of AAV genomes and opens new therapeutic avenues for achieving long-term AAV-mediated gene transfer in dividing cells.

Longitudinal clonal tracking studies based on high-throughput sequencing technologies supported safety and long-term efficacy and unraveled hematopoietic reconstitution in many gene therapy applications with unprecedented resolution. However, monitoring patients over a decade-long follow-up entails a constant increase of large data volume with the emergence of critical computational challenges, unfortunately not addressed by currently available tools. Here we present ISAnalytics, a new R package for comprehensive and high-throughput clonal tracking studies using vector integration sites as markers of cellular identity. Once identified the clones externally from ISAnalytics and imported in the package, a wide range of implemented functionalities are available to users for assessing the safety and long-term efficacy of the treatment, here described in a clinical trial use case for Hurler disease, and for supporting hematopoietic stem cell biology in vivo with longitudinal analysis of clones over time, proliferation and differentiation. ISAnalytics is conceived to be metadata-driven, enabling users to focus on biological questions and hypotheses rather than on computational aspects. ISAnalytics can be fully integrated within laboratory workflows and standard procedures. Moreover, ISAnalytics is designed with efficient and scalable data structures, benchmarked with previous methods, and grants reproducibility and full analytical control through interactive web-reports and a module with Shiny interface. The implemented functionalities are flexible for all viral vector-based clonal tracking applications as well as genetic barcoding or cancer immunotherapies.

Background Allogeneic hematopoietic stem-cell transplantation is the standard of care for Hurler syndrome (mucopolysaccharidosis type I, Hurler variant [MPSIH]). However, this treatment is only partially curative and is associated with complications. Methods We are conducting an ongoing study involving eight children with MPSIH. At enrollment, the children lacked a suitable allogeneic donor and had a Developmental Quotient or Intelligence Quotient score above 70 (i.e., none had moderate or severe cognitive impairment). The children received autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with an alpha-L-iduronidase (IDUA)-encoding lentiviral vector after myeloablative conditioning. Safety and correction of blood IDUA activity up to supraphysiologic levels were the primary end points. Clearance of lysosomal storage material as well as skeletal and neurophysiological development were assessed as secondary and exploratory end points. The planned duration of the study is 5 years. Results We now report interim results. The children's mean (+/- SD) age at the time of HSPC gene therapy was 1.9 +/- 0.5 years. At a median follow-up of 2.10 years, the procedure had a safety profile similar to that known for autologous hematopoietic stem-cell transplantation. All the patients showed prompt and sustained engraftment of gene-corrected cells and had supraphysiologic blood IDUA activity within a month, which was maintained up to the latest follow-up. Urinary glycosaminoglycan (GAG) excretion decreased steeply, reaching normal levels at 12 months in four of five patients who could be evaluated. Previously undetectable levels of IDUA activity in the cerebrospinal fluid became detectable after gene therapy and were associated with local clearance of GAGs. Patients showed stable cognitive performance, stable motor skills corresponding to continued motor development, improved or stable findings on magnetic resonance imaging of the brain and spine, reduced joint stiffness, and normal growth in line with World Health Organization growth charts. Conclusions The delivery of HSPC gene therapy in patients with MPSIH resulted in extensive metabolic correction in peripheral tissues and the central nervous system. (Funded by Fondazione Telethon and others; ClinicalTrials.gov number, ; EudraCT number, .)Hematopoietic Gene Therapy for Hurler Syndrome Eight patients with Hurler syndrome who lacked suitable allogeneic donors received autologous hematopoietic stem and progenitor cells transduced ex vivo with an alpha-L-iduronidase-encoding lentiviral vector. This therapy resulted in extensive metabolic correction in peripheral tissues and the central nervous system.

Gene therapy (GT) has rapidly attracted renewed interest as a treatment for otherwise incurable diseases, with several GT products already on the market and many more entering clinical testing for selected indications. Clonal tracking techniques based on vector integration enable monitoring of the fate of engineered cells in the blood of patients receiving GT and allow assessment of the safety and efficacy of these procedures. However, owing to the limited number of cells that can be tested and the impracticality of studying cells residing in peripheral organs without performing invasive biopsies, this approach provides only a partial snapshot of the clonal repertoire and dynamics of genetically modified cells and reduces the predictive power as a safety readout. In this study, we developed liquid biopsy integration site sequencing, or LiBIS-seq, a polymerase chain reaction technique optimized to quantitatively retrieve vector integration sites from cell-free DNA released into the bloodstream by dying cells residing in several tissues. This approach enabled longitudinal monitoring of in vivo liver-directed GT and clonal tracking in patients receiving hematopoietic stem cell GT, improving our understanding of the clonal composition and turnover of genetically modified cells in solid tissues and, in contrast to conventional analyses based only on circulating blood cells, enabling earlier detection of vector-marked clones that are aberrantly expanding in peripheral tissues.

Long-range gene editing by homology-directed repair (HDR) in hematopoietic stem/progenitor cells (HSPCs) often relies on viral transduction with recombinant adeno-associated viral vector (AAV) for template delivery. Here, we uncover unexpected load and prolonged persistence of AAV genomes and their fragments, which trigger sustained p53-mediated DNA damage response (DDR) upon recruiting the MRE11-RAD50-NBS1 (MRN) complex on the AAV inverted terminal repeats (ITRs). Accrual of viral DNA in cell-cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITRs, at nuclease on- and off-target sites. Optimized delivery of integrase-defective lentiviral vector (IDLV) induced lower DNA load and less persistent DDR, improving clonogenic capacity and editing efficiency in long-term repopulating HSPCs. Because insertions of viral DNA fragments are less frequent with IDLV, its choice for template delivery mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate its clinical translation in HSPC gene therapy.

High transduction rates of viral vectors in gene therapies (GT) and experimental hematopoiesis ensure a high frequency of gene delivery, although multiple integration events can occur in the same cell. Therefore, tracing of integration sites (IS) leads to misquantification of the true clonal spectrum and limits safety considerations in GT.Hence, we use correlations between repeated measurements of IS abundances to estimate their mutual similarity and identify clusters of co-occurring IS, for which we assume a clonal origin. We evaluate the performance, robustness and specificity of our methodology using clonal simulations. The reconstruction methods, implemented and provided as an R-package, are further applied to experimental clonal mixes and to a preclinical model of hematopoietic GT.Our results demonstrate that clonal reconstruction from IS data allows to overcome systematic biases in the clonal quantification as an essential prerequisite for the assessment of safety and long-term efficacy of GT involving integrative vectors.

SummaryThe humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in COVID-19. The present study was designed to conduct a systematic investigation of the interaction of humoral fluid phase pattern recognition molecules (PRM) with SARS-CoV-2. Out of 10 PRM tested, the long pentraxin PTX3 and Mannose Binding Lectin (MBL) bound the viral Nucleoprotein and Spike, respectively. MBL bound trimeric Spike, including that of variants of concern, in a glycan- dependent way and inhibited SARS-CoV-2 in threein vitromodels. Moreover, upon binding to Spike, MBL activated the lectin pathway of complement activation. Genetic polymorphisms at the MBL locus were associated with disease severity. These results suggest that selected humoral fluid phase PRM can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.

Activating mutations in the BRAF-MAPK pathway have been reported in histiocytoses, hematological inflammatory neoplasms characterized by multi-organ dissemination of pro-inflammatory myeloid cells. Here, we generate a humanized mouse model of transplantation of human hematopoietic stem and progenitor cells (HSPCs) expressing the activated form of BRAF (BRAFV600E). All mice transplanted with BRAFV600E-expressing HSPCs succumb to bone marrow failure, displaying myeloid-restricted hematopoiesis and multi-organ dissemination of aberrant mononuclear phagocytes. At the basis of this aggressive phenotype, we uncover the engagement of a senescence program, characterized by DNA damage response activation and a senescence-associated secretory phenotype, which affects also non-mutated bystander cells. Mechanistically, we identify TNFα as a key determinant of paracrine senescence and myeloid-restricted hematopoiesis and show that its inhibition dampens inflammation, delays disease onset and rescues hematopoietic defects in bystander cells. Our work establishes that senescence in the human hematopoietic system links oncogene-activation to the systemic inflammation observed in histiocytic neoplasms., BRAF-MAPK activating mutations are reported in histiocytoses—hematological neoplasms with widespread pro-inflammatory myeloid cells. Here, the authors show that an activating mutant BRAF in haematopoietic stem and progenitor cells causes an oncogene-induced senescence response leading to myeloid restricted haematopoiesis, inflammation and histiocytosis.

Trained immunity (TI) is a proinflammatory program induced in monocyte/macrophages upon sensing of specific pathogens and is characterized by immunometabolic and epigenetic changes that enhance cytokine production. Maladaptive activation of TI (ie, in the absence of infection) may result in detrimental inflammation and development of disease; however, the exact role and extent of inappropriate activation of TI in the pathogenesis of human diseases is undetermined. In this study, we uncovered the oncogene-induced, maladaptive induction of TI in the pathogenesis of a human inflammatory myeloid neoplasm (Erdheim-Chester disease, [ECD]), characterized by the BRAFV600E oncogenic mutation in monocyte/macrophages and excess cytokine production. Mechanistically, myeloid cells expressing BRAFV600E exhibit all molecular features of TI: activation of the AKT/mammalian target of rapamycin signaling axis; increased glycolysis, glutaminolysis, and cholesterol synthesis; epigenetic changes on promoters of genes encoding cytokines; and enhanced cytokine production leading to hyperinflammatory responses. In patients with ECD, effective therapeutic strategies combat this maladaptive TI phenotype; in addition, pharmacologic inhibition of immunometabolic changes underlying TI (ie, glycolysis) effectively dampens cytokine production by myeloid cells. This study revealed the deleterious potential of inappropriate activation of TI in the pathogenesis of human inflammatory myeloid neoplasms and the opportunity for inhibition of TI in conditions characterized by maladaptive myeloid-driven inflammation.

Referred to by : Gregori Silvia, Aiuti Alessandro - Intrathymic delivery a new route for adenoviral-associated vector gene therapy - Journal of Allergy and Clinical Immunology, Volume 145, Issue 2, February 2020, Pages 499-501; International audience; BACKGROUND: Patients with T-cell immunodeficiencies are generally treated with allogeneic hematopoietic stem cell transplantation, but alternatives are needed for patients without matched donors. An innovative intrathymic gene therapy approach that directly targets the thymus might improve outcomes.OBJECTIVE: We sought to determine the efficacy of intrathymic adeno-associated virus (AAV) serotypes to transduce thymocyte subsets and correct the T-cell immunodeficiency in a zeta-associated protein of 70 kDa (ZAP-70)-deficient murine model.METHODS: AAV serotypes were injected intrathymically into wild-type mice, and gene transfer efficiency was monitored. ZAP-70-/- mice were intrathymically injected with an AAV8 vector harboring the ZAP70 gene. Thymus structure, immunophenotyping, T-cell receptor clonotypes, T-cell function, immune responses to transgenes and autoantibodies, vector copy number, and integration were evaluated.RESULTS: AAV8, AAV9, and AAV10 serotypes all transduced thymocyte subsets after in situ gene transfer, with transduction of up to 5% of cells. Intrathymic injection of an AAV8-ZAP-70 vector into ZAP-70-/- mice resulted in a rapid thymocyte differentiation associated with the development of a thymic medulla. Strikingly, medullary thymic epithelial cells expressing the autoimmune regulator were detected within 10 days of gene transfer, correlating with the presence of functional effector and regulatory T-cell subsets with diverse T-cell receptor clonotypes in the periphery. Although thymocyte reconstitution was transient, gene-corrected peripheral T cells harboring approximately 1 AAV genome per cell persisted for more than 40 weeks, and AAV vector integration was detected.CONCLUSIONS: Intrathymic AAV-transduced progenitors promote a rapid restoration of the thymic architecture, with a single wave of thymopoiesis generating long-term peripheral T-cell function.

International audience; Liver-directed gene therapy for the coagulation disorder hemophilia showed safe and effective results in clinical trials using adeno-associated viral vectors to replace a functional coagulation factor, although some unmet needs remain. Lentiviral vectors (LVs) may address some of these hurdles because of their potential for stable expression and the low prevalence of preexisting viral immunity in humans. However, systemic LV administration to hemophilic dogs was associated to mild acute toxicity and low efficacy at the administered doses. Here, exploiting intravital microscopy and LV surface engineering, we report a major role of the human phagocytosis inhibitor CD47, incorporated into LV cell membrane, in protecting LVs from uptake by professional phagocytes and innate immune sensing, thus favoring biodistribution to hepatocytes after systemic administration. By enforcing high CD47 surface content, we generated phagocytosis-shielded LVs which, upon intravenous administration to nonhuman primates, showed selective liver and spleen targeting and enhanced hepatocyte gene transfer compared to parental LV, reaching supraphysiological activity of human coagulation factor IX, the protein encoded by the transgene, without signs of toxicity or clonal expansion of transduced cells.

STUDY QUESTION Given the relevant role of the extracellular microenvironment in regulating tissue homeostasis, is testicular bacterial microbiome (BM) associated with germ cell aplasia in idiopathic non-obstructive azoospermia (iNOA)? SUMMARY ANSWER A steady increase of dysbiosis was observed among testis with normal spermatogenesis vs. iNOA with positive sperm retrieval and iNOA with complete germ cell aplasia. WHAT IS KNOWN ALREADY Tissue-associated BM has been reported to be a biologically important extracellular microenvironment component for numerous body habitats, but not yet for the human testis. STUDY DESIGN, SIZE, DURATION Cross-sectional study, investigating tissue-associated BM in the testis of (i) five men with iNOA and negative sperm retrieval at microdissection testicular sperm extraction (microTESE); (ii) five men with iNOA and positive sperm retrieval at microTESE; and (iii) five normozoospermic men upon orchiectomy. Every testicular specimen was histologically classified and analyzed in terms of bacterial community. PARTICIPANTS/MATERIALS, SETTING, METHODS Massive ultra-deep pyrosequencing was applied to investigate testis microbiome. Metagenome was analyzed using Quantitative Insights Into Microbial Ecology (QIIME). Tissue-associated bacterial load was quantified by digital droplet PCR. MAIN RESULTS AND THE ROLE OF CHANCE Normozoospermic men showed small amounts of bacteria in the testis, with Actinobacteria, Bacteroidetes, Firmicutes Proteobacteria as the dominating phyla; iNOA individuals had increased amounts of bacterial DNA (P = 0.02), associated with decreased taxa richness due to the lack of Bacteroidetes and Proteobacteria (P = 2 × 10−5). Specimens with negative sperm retrieval at microTESE depicted complete germ cell aplasia and a further decrease in terms of Firmicutes and Clostridia (P < 0.05), a complete lack of Peptoniphilus asaccharolyticus, but increased amount of Actinobacteria. LIMITATIONS, REASONS FOR CAUTION The limited number of specimens analyzed in this preliminary study deserves external validation. The paraneoplastic microenvironment could have an impact on the residential bacterial flora. WIDER IMPLICATION OF THE FINDINGS Human testicular microenvironment is not microbiologically sterile, containing low amounts of Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. A dysbiotic bacterial community was associated with iNOA and complete germ cell aplasia. Novel findings on testicular BM could support future translational therapies of male-factor infertility. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by URI-Urological Research Institute free funds. Authors declared no conflict of interest. TRIAL REGISTRATION NUMBER N/A.

Improving hematopoietic stem and progenitor cell (HSPC) permissiveness to HIV-derived lentiviral vectors (LVs) remains a challenge for the field of gene therapy as high vector doses and prolonged ex vivo culture are still required to achieve clinically relevant transduction levels. We report here that Cyclosporin A (CsA) and Rapamycin (Rapa) significantly improve LV gene transfer in human and murine HSPC. Both compounds increased LV but not gammaretroviral transduction and acted independently of calcineurin and autophagy. Improved gene transfer was achieved across all CD34(+) subpopulations, including in long-term SCID repopulating cells. Effects of CsA were specific of HSPC and opposite to its known impact on HIV replication. Mutating the Cyclophilin A binding pocket of the viral capsid (CA) further improved transduction in combination with CsA. Tracking of the LV genome fate revealed that CsA relieves a CA-dependent early block and increases integration, while Rapa acts early in LV infection independently of the viral CA. In agreement, only Rapa was able to improve transduction by an integrase-defective LV harboring wild-type CA. Overall, our findings pave the way for more efficient and sustainable LV gene therapy in human HSPCs and shed light on the multiple innate barriers specifically hampering LV transduction in these cells.

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

In hematopoietic stem progenitor cell (HSPC) gene therapy (GT) applications for the treatment of genetic diseases, retroviral vectors (RVs) are used to efficiently transduce and integrate therapeutic genes in the genome of patient-derived HSPCs, which, upon reinfusion, reconstruct the entire hematopoietic system and restore the correct hematopoietic functions, or deliver the therapeutic factor to different tissues. However, in initial HSPC-GT clinical trials using early-generation γ-RVs, vector insertions near proto-oncogenes triggered their overexpression and induced leukemia in some of the transplanted patients. These unexpected adverse events have prompted the development of highly sensitive preclinical assays to test the genotoxic potential of different GT vector types and designs, and the development of powerful PCR-based techniques, combined with next generation sequencing (NGS) and bioinformatics analyses, have allowed to study integration sites (ISs) present in leukemic and dominant expanding cells, identify the genes targeted by insertions and to investigate the clonal composition of complex vector-marked cell populations. The positive safety data obtained from the testing in highly sensitive preclinical models and, successively, in clinical trials, of the more advanced lentiviral vectors (LVs) with self-inactivating (SIN) long terminal repeats (LTRs), have reduced the concerns related to insertional mutagenesis, encouraging the adoption of this vector platform in GT protocols for the treatment of many other diseases. Nevertheless, the evidences collected from genotoxicity assays and a β-thalassemia clinical trial, during which a vector-driven clonal dominance event has occurred, point to the fact that even SIN LVs insertions are not entirely neutral, and thus to the importance of a continuous effort to improve both the design of GT vectors, and the sensitivity of preclinical assays aimed at assessing their residual genotoxicity.

HIV-1 insertions targeting BACH2 or MLK2 are enriched and persist for decades in hematopoietic cells from patients under combination antiretroviral therapy. However, it is unclear how these insertions provide such selective advantage to infected cell clones. Here, we show that in 30/87 (34%) patients under combination antiretroviral therapy, BACH2, and STAT5B are activated by insertions triggering the formation of mRNAs that contain viral sequences fused by splicing to their first protein-coding exon. These chimeric mRNAs, predicted to express full-length proteins, are enriched in T regulatory and T central memory cells, but not in other T lymphocyte subsets or monocytes. Overexpression of BACH2 or STAT5B in primary T regulatory cells increases their proliferation and survival without compromising their function. Hence, we provide evidence that HIV-1-mediated insertional activation of BACH2 and STAT5B favor the persistence of a viral reservoir in T regulatory cells in patients under combination antiretroviral therapy., HIV insertions in hematopoietic cells are enriched in BACH2 or MLK2 genes, but the selective advantages conferred are unknown. Here, the authors show that BACH2 and additionally STAT5B are activated by viral insertions, generating chimeric mRNAs specifically enriched in T regulatory cells favoring their persistence.

Transposons and gamma-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells and that hamper the identification of early cancer-driving events among bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVVs) by which we could efficiently induce hepatocellular carcinoma (HCC) in three different mouse models. By virtue of the LVV's replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of four previously unknown liver cancer-associated genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. The newly identified genes are likely to play a role in human cancer because they are upregulated, amplified and/or deleted in human HCCs and can predict clinical outcomes of patients.

Gamma-retroviral/lentiviral vectors (γRV/LV) with self-inactivating (SIN) long terminal repeats (LTRs) and internal moderate cellular promoters pose a reduced risk of insertional mutagenesis when compared with vectors with active LTRs. Yet, in a recent LV-based clinical trial for β-thalassemia, vector integration within the HMGA2 gene induced the formation of an aberrantly spliced mRNA form that appeared to cause clonal dominance. Using a method that we developed, cDNA linear amplification-mediated PCR, in combination with high-throughput sequencing, we conducted a whole transcriptome analysis of chimeric LV-cellular fusion transcripts in transduced human lymphoblastoid cells and primary hematopoietic stem/progenitor cells. We observed a surprising abundance of read-through transcription originating outside and inside the provirus and identified the vector sequences contributing to the aberrant splicing process. We found that SIN LV has a sharply reduced propensity to engage in aberrant splicing compared with that of vectors carrying active LTRs. Moreover, by recoding the identified vector splice sites, we reduced residual read-through transcription and demonstrated an effective strategy for improving vectors. Characterization of the mechanisms and genetic features underlying vector-induced aberrant splicing will enable the generation of safer vectors, with low impact on the cellular transcriptome.

A recent clinical trial for adrenoleukodystrophy (ALD) showed the efficacy and safety of lentiviral vector (LV) gene transfer in hematopoietic stem progenitor cells. However, several common insertion sites (CIS) were found in patients' cells, suggesting that LV integrations conferred a selective advantage. We performed high-throughput LV integration site analysis on human hematopoietic stem progenitor cells engrafted in immunodeficient mice and found the same CISs reported in patients with ALD. Strikingly, most CISs in our experimental model and in patients with ALD cluster in megabase-wide chromosomal regions of high LV integration density. Conversely, cancer-triggering integrations at CISs found in tumor cells from γretroviral vector–based clinical trials and oncogene-tagging screenings in mice always target a single gene and are contained in narrow genomic intervals. These findings imply that LV CISs are produced by an integration bias toward specific genomic regions rather than by oncogenic selection.

Lentiviral vectors with self-inactivating (SIN) long terminal repeats (LTRs) are promising for safe and sustained transgene expression in dividing as well as quiescent cells. As genome organization and transcription substantially differs between actively dividing and postmitotic cells in vivo, we hypothesized that genomic vector integration preferences might be distinct between these biological states. We performed integration site (IS) analyses on mouse dividing cells (fibroblasts and hematopoietic progenitor cells (HPCs)) transduced ex vivo and postmitotic cells (eye and brain) transduced in vivo. As expected, integration in dividing cells occurred preferably into gene coding regions. In contrast, postmitotic cells showed a close to random frequency of integration into genes and gene spare long interspersed nuclear elements (LINE). Our studies on the potential mechanisms responsible for the detected differences of lentiviral integration suggest that the lowered expression level of Psip1 reduce the integration frequency in vivo into gene coding regions in postmitotic cells. The motif TGGAA might represent one of the factors for preferred lentiviral integration into mouse and rat Satellite DNA. These observations are highly relevant for the correct assessment of preclinical biosafety studies, indicating that lentiviral vectors are well suited for safe and effective clinical gene transfer into postmitotic tissues.

It has been recently suggested that HIV-1 by integrating near cancer-associated genes could promote the expansion and persistence of infected cells in patients under Anti Retroviral Therapy (ART). However, the molecular mechanism/s of insertional mutagenesis used and the physiological impact on the cells harboring these integrations are completely unknown.Here, we found that in the peripheral blood mononuclear cells from 54 HIV-1 infected patients under ART, BACH2 and STAT5B were targeted by a significant number of integrations compared to other lentiviral integration datasets (p

Genotoxicity assays based on systemic vector injection into newborn tumor-prone Cdkn2a−/− and Cdkn2a+/− mice has shown that self-inactivating (SIN) lentiviral vector (LV) harboring strong or moderate enhancer/promoters in internal position caused acceleration in hematopoietic tumor onset compared to control mice. Integration site (IS) analysis in vector-induced tumors showed that oncogene activation or tumor suppressor inactivation occurs by mechanisms of aberrant splicing and/or enhancer-mediated overexpression of cellular genes. Although oncogene activation may be reduced by the use of SIN design, moderate cellular promoters and insulator sequences, how to reduce genotoxic splicing-capture events and aberrant transcript formation triggered by vector integration is still unclear. Here, we specifically designed SINLVs harboring sequences complementary to microRNAs (mirT sequence) which are active in hematopoietic cells (mir223 and mir142-3p) within the SIN LTR (mirsT-LTR.LV) or in the vector backbone and outside the gene expression cassette (mirT-LV). In our rationale, the mirT sequences when incorporated in an aberrantly generated mRNA would be selectively degraded through the miRNA pathway. Thus, by taking advantage of our in vivo models, we assessed the genotoxicity of these LVs with mirT sequences. Systemic injection of mirsT-LTR.LV (N=34) and mirT-LV (N=39) in Cdkn2a−/− mice did not cause any significant acceleration in hematopoietic tumor onset compared to un-injected mice (N=37) or mice injected with a SINLV that does not harbor mirT sequences (N=24). Similar results have been obtained after injection of the same vectors in Cdkn2a+/− mice (N=29 mirsT-LTR.LV, N=25 mirT-LV, N=40 un-injected and N=15 injected control mice). To gain additional information on the safety profile of these vectors, we performed IS analysis (N>10,000) in tumor-derived DNA. By this analysis, we previously found that Map3k8 activation by LV insertions was the major mechanism of genotoxicity when prototypical SINLVs were injected into Cdkn2a−/− mice. Now, we found that mice treated with mirsT-LTR.LV and mirT-LV did not show any Map3k8 activating insertions, suggesting that the new vectors are efficient in preventing its activation and confirming their superior safety profile. Furthermore, as expected, Pten was the most frequently targeted gene in tumors derived from Cdkn2a−/− mice injected with the LVs harboring mirT sequences. Pten insertions mainly targeted exons, suggesting the potential inactivation of its transcription unit. Finally, we found that Sfi1 was the major Common Insertion Site (CIS) in Cdkn2a+/− mice injected with LVs harboring mirT sequences. This CIS gene however appears to be the product of an intrinsic bias of LV integration, rather than the result of a selection process. Overall, our studies showed that these new advanced design LVs have a significantly improved safety profile and could represent the vector design of choice in future gene therapy applications.

Since its identification, HIV-1 remains a global health threat responsible for a world-wide pandemic. The introduction of Anti-Retroviral Therapy (ART) greatly extended patients survival; however ART can control but not cure HIV infection. It has been recently suggested that HIV-1 by integrating near cancer-associated genes could promote the expansion and persistence of infected cells in patients under ART. However, the molecular mechanism/s of insertional mutagenesis used and the physiological impact on the cells harboring these integrations are completely unknown.Here, we found that in peripheral blood mononuclear cells (PBMC) from 54 HIV-1 infected patients under ART, BACH2 and STAT5B were targeted by a significantly higher number of integrations and with the same orientation of gene transcription compared to other lentiviral integration (LV) datasets (p

The analysis of the genomic distribution of viral vector genomic integration sites is a key step in hematopoietic stem cell-based gene therapy applications, allowing to assess both the safety and the efficacy of the treatment and to study the basic aspects of hematopoiesis and stem cell biology. Identifying vector integration sites requires ad-hoc bioinformatics tools with stringent requirements in terms of computational efficiency, flexibility, and usability. We developed VISPA (Vector Integration Site Parallel Analysis), a pipeline for automated integration site identification and annotation based on a distributed environment with a simple Galaxy web interface. VISPA was successfully used for the bioinformatics analysis of the follow-up of two lentiviral vector-based hematopoietic stem-cell gene therapy clinical trials. Our pipeline provides a reliable and efficient tool to assess the safety and efficacy of integrating vectors in clinical settings. Electronic supplementary material The online version of this article (doi:10.1186/s13073-014-0067-5) contains supplementary material, which is available to authorized users.

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency caused by reduced or absent expression of the WAS protein (WASP). WAS patients are affected by microthrombocytopenia, recurrent infections, eczema, autoimmune diseases, and malignancies. Although immune deficiency has been proposed to play a role in tumor pathogenesis, there is little evidence on the correlation between immune cell defects and tumor susceptibility. Taking advantage of a tumor-prone model, we show that the lack of WASP induces early tumor onset because of defective immune surveillance. Consistently, the B16 melanoma model shows that tumor growth and the number of lung metastases are increased in the absence of WASP. We then investigated the in vivo contribution of Was(-/-) NK cells and DCs in controlling B16 melanoma development. We found fewer B16 metastases developed in the lungs of Was(-/-) mice that had received WT NK cells as compared with mice bearing Was(-/-) NK cells. Furthermore, we demonstrated that Was(-/-) DCs were less efficient in inducing NK-cell activation in vitro and in vivo. In summary, for the first time, we demonstrate in in vivo models that WASP deficiency affects resistance to tumor and causes impairment in the antitumor capacity of NK cells and DCs.

Self-inactivating (SIN) lentiviral vectors (LV) have an excellent therapeutic potential as demonstrated in preclinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could still pose a significant risk in clinical applications. Taking advantage of novel in vivo genotoxicity assays, we tested a battery of LV constructs, including some with clinically relevant designs, and found that oncogene activation by promoter insertion is the most powerful mechanism of early vector-induced oncogenesis. SIN LVs disabled in their capacity to activate oncogenes by promoter insertion were less genotoxic and induced tumors by enhancer-mediated activation of oncogenes with efficiency that was proportional to the strength of the promoter used. On the other hand, when enhancer activity was reduced by using moderate promoters, oncogenesis by inactivation of tumor suppressor gene was revealed. This mechanism becomes predominant when the enhancer activity of the internal promoter is shielded by the presence of a synthetic chromatin insulator cassette. Our data provide both mechanistic insights and quantitative readouts of vector-mediated genotoxicity, allowing a relative ranking of different vectors according to these features, and inform current and future choices of vector design with increasing biosafety.

The excellent therapeutic potential of self-inactivating (SIN) lentiviral vectors (LV) has been demonstrated in pre-clinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could endanger their clinical applications. Systemic vector injection into newborn tumor-prone Cdkn2a-/- and Cdkn2a+/-mice, conducted in our previous work, demonstrated that SINLVs harboring strong or moderate enhancer/promoters in internal position caused acceleration in hematopoietic tumor onset with respect to control mice. Integration sites analyses of vector-induced tumor showed that oncogene activations or tumor suppressor inactivation by LV integrations occur by combining mechanisms of transcript truncation, induction of aberrant splicing and/or enhancer-mediated overexpression of cellular transcription units. Although oncogene activation may be reduced by the use of self-inactivating design, moderate cellular promoters and insulator sequences how to reduce genotoxic splicing-capture events and aberrant transcript formation triggered by vector integration is still unclear.From this and a previous study, we identified the LV sequences most frequently involved in chimeric transcript formation. In our rationale, these LV sequences could be tagged by sequences complementary to microRNAs (mirT sequence) active in hematopoietic cells in order to allow selective degradation, through the miRNA pathway, of vector-mediated aberrantly spliced transcripts. Hence, we specifically designed SIN LVs harboring mirT sequences recognized by mir223 and mir142-3p (that are expressed in hematopoietic lineages) within the SIN LTR (mirsT-LTR LV) or in the vector backbone and outside the gene expression cassette (mirT LV). We then assessed the genotoxicity of the SIN LVs harboring mirT sequences by taking advantage of our in vivo models. Interestingly, injection of mirsT-LTR LV (N=73) and mirT LV (N=73) in Cdkn2a-/- mice did not caused any significant acceleration in hematopoietic tumor onset compared to control un-injected mice (N=40). Similar results have been obtained after injection in Cdkn2a+/- mice (N=28 for mirsT-LTR LV, N=26 for mirT LV and N=34 un-injected mice). We are currently performing integration site analyses in Cdkn2a-/- and Cdkn2a+/- treated mice to dissect if and how the integrated mirsT-LTR LV and mirT LV proviral genome interacts with the surrounding cellular genome.Overall, these studies show that this new advanced design lentiviral vectors completely abrogated residual vector genotoxicity in highly sensitive mouse models and could represent the vector design of choice in future gene therapy applications.

Integrative viral vectors are able to efficiently transduce hematopoietic stem progenitor cells allowing stable transgene expression in the entire hematopoietic system upon transplant in conditioned recipients. For these reasons, integrative vectors based on γ-retroviruses and lentiviruses have been successfully used in gene therapy clinical trials for the treatment of genetic diseases, especially blood disorders. However, in different γ-retroviral-based clinical trials, vector integration into the host cell genome triggered oncogenesis by a mechanism called insertional mutagenesis. Thus, a thorough reassessment of the safety of available gene transfer systems is a crucial outstanding issue for the whole gene therapy field. Sensitive preclinical models of vector genotoxicity are instrumental to achieve a more detailed understanding of the factors that modulate the risks of insertional mutagenesis. Here, we will describe the methodologies used to address the mutagenesis risk of vector integration using a murine in vivo genotoxicity assay based on transduction and transplantation of tumor-prone hematopoietic stem and progenitor cells.

Integrative viral vectors are able to efficiently transduce hematopoietic stem progenitor cells allowing stable transgene expression in the entire hematopoietic system upon transplant in conditioned recipients. For these reasons, integrative vectors based on γ-retroviruses and lentiviruses have been successfully used in gene therapy clinical trials for the treatment of genetic diseases, especially blood disorders. However, in different γ-retroviral-based clinical trials, vector integration into the host cell genome triggered oncogenesis by a mechanism called insertional mutagenesis. Thus, a thorough reassessment of the safety of available gene transfer systems is a crucial outstanding issue for the whole gene therapy field. Sensitive preclinical models of vector genotoxicity are instrumental to achieve a more detailed understanding of the factors that modulate the risks of insertional mutagenesis. Here, we will describe the methodologies used to address the mutagenesis risk of vector integration using a murine in vivo genotoxicity assay based on transduction and transplantation of tumor-prone hematopoietic stem and progenitor cells.

Oncogenesis induced by insertional mutagenesis with gene therapy vectors occurs mainly by activation of proto-oncogenes found at or nearby the insertion site. This activation often occurs by an enhancer-mediated mechanism or by a process of splicing capture which generates chimeric transcripts comprising portions of vector and cellular mRNAs. Although the activation of oncogenes may be reduced by the use of self-inactivating design and moderate cellular promoters, how to reduce genotoxic splicing capture events and aberrant transcript formation triggered by vector integration is still unclear. We developed a modified Linear Amplification-Mediated (LAM) PCR technique, named cDNA LAM PCR (cLAM-PCR), aimed at retrieving, from the whole transcriptome of LV-transduced cells aberrantly spliced mRNAs that contain lentiviral vector (LV) sequences fused with cellular transcripts in a high-throughput fashion. The sequences of cLAM-PCR products were obtained by 454 pyrosequencing and analyzed by dedicated high-throughput computational pipeline running in a computer cluster that use a dynamic analysis process composed by different steps based on a map-reduce parallelization model. Thus, chimeric LV-genome sequences are recognized, the nucleotide position of the fused sequence is identified (the splice site), and the remaining portion mapped on the appropriate genome assembly by BLAST. We identified several established and previously unknown splice sites within the LV backbone that participate in the aberrant splicing process with variable efficiency. Results obtained with different LV designs show that integrated LVs can perturb the processing of cellular transcripts by interacting with the cellular splicing machinery and fusing with its own splice sites to cellular splice sites both upstream and downstream the integration site. So far, 70 different fusion transcripts could be identified in total, 84% of which were fused to known splice sites of gene exons, 6% were fused to uncharacterized cryptic splice sites located in introns and the remaining 10% were fused to genomic sequences not corresponding to any annotated gene. This analysis allows identifying also several different slice sites within the LV backbone that participated to the aberrabt splicing process. Quantitative PCR on different LV portions within the LV backbone allow measuring the relative contribution to the aberrant splicing process of each splice site identified. Interestingly, the amount of transcription occurring in regions outside the expression cassette reaches the 3% of the entire transgene expression. The cLAM-PCR technique, coupled to high-throughput sequencing and the computational power of our specialized data analysis pipeline allows gaining insights into the biology of vector-mediated splicing alteration. Since this process could induce neoplastic transformation by the generation of aberrant oncogenic protein, its in-depth characterization is instrumental in the development of next-generation LV with a higher safety profile.

Retroviral vectors have induced subtle clonal skewing in many gene therapy patients and severe clonal proliferation and leukemia in some of them, emphasizing the need for comprehensive integration site analyses to assess the biosafety and genomic pharmacokinetics of vectors and clonal fate of gene-modified cells in vivo. Integration site analyses such as linear amplification-mediated PCR (LAM-PCR) require a restriction digest generating unevenly small fragments of the genome. Here we show that each restriction motif allows for identification of only a fraction of all genomic integrants, hampering the understanding and prediction of biological consequences after vector insertion. We developed a model to define genomic access to the viral integration site that provides optimal restriction motif combinations and minimizes the percentage of nonaccessible insertion loci. We introduce a new nonrestrictive LAM-PCR approach that has superior capabilities for comprehensive unbiased integration site retrieval in preclinical and clinical samples independent of restriction motifs and amplification inefficiency.

gamma-Retroviral vectors (gamma RVs), which are commonly used in gene therapy, can trigger oncogenesis by insertional mutagenesis. Here, we have dissected the contribution of vector design and viral integration site selection (ISS) to oncogenesis using an in vivo genotoxicity assay based on transplantation of vector-transduced tumor-prone mouse hematopoietic stem/progenitor cells. By swapping genetic elements between gamma RV and lentiviral. vectors (LVs), we have demonstrated that transcriptionally active long terminal repeats (LTRs) are major determinants of genotoxicity even when reconstituted in LVs and that self-inactivating (SIN) LTRs enhance the safety of gamma RVs. By comparing the genotoxicity of vectors with matched active LTRs, we were able to determine that substantially greater LV integration loads are required to approach the same oncogenic risk as gamma RVs. This difference in facilitating oncogenesis is likely to be explained by the observed preferential targeting of cancer genes by gamma RVs. This integration-site bias was intrinsic to gamma RVs, as it was also observed for SIN gamma RVs that lacked genotoxicity in our model. Our findings strongly support the use of SIN viral vector platforms and show that ISS can substantially modulate genotoxicity.

Notch signalling plays an important role in hematopoiesis and in the pathogenesis of T-ALL. Notch is known to interact with Ras and PTEN/PI3K (phosphoinositide-3 kinase)/Akt pathways. We investigated the interaction of Notch with these pathways and the possible reciprocal regulation of these signalling systems in T-ALL cells in vitro. Our analyses indicate that the PI3K/Akt pathway is constitutively active in the four T-ALL cell lines tested. Akt phosphorylation was not altered by the sequestration of growth factors, that is, Akt activation seems to be less dependent on but not completely independent of growth factors, possibly being not subject to negative feedback regulation. PTEN expression was not detected in 3/4 cell lines tested, suggesting the loss of PTEN-mediated Akt activation. Inhibition of the PI3K/Akt pathway arrests growth and enhances apoptosis, but with no modulation of expression of Bax-α and Bcl-2 proteins. We analysed the relationship between Notch-1 and the PI3K/Akt signalling and show that inhibition of the Akt pathway changes Notch expression; Notch-1 protein decreased in all the cell lines upon treatment with the inhibitor. Our studies strongly suggest that Notch signalling interacts with PI3K/Akt signalling and further that this occurs in the absence of PTEN expression. The consequences of this to the signalling outcome are yet unclear, but we have uncovered a significant inverse relationship between Notch and PI3K/Akt pathway, which leads us to postulate the operation of a reciprocal regulatory loop between Notch and Ras-PI3K/Akt in the pathogenesis of T-ALL. J. Cell. Biochem. 103: 1405–1412, 2008. © 2007 Wiley-Liss, Inc.

Chromatin insulators (CI) have been proposed as safety features to increase the safety of self-inactivating (SIN) lentiviral vectors (LV) for gene therapy applications.By taking advantage of an in vivo genotoxicity assay based on the systemic injection of LVs in newborn tumor-prone Cdkn2a-/- mice we were able to measure vector-induced genotoxicity as an accelerated tumor onset that was proportional to the genotoxic potential of the tested LV. Importantly, we took advantage of integration sites (IS) analysis to qualitatively characterize CI that were shown by other in vitro and ex vivo studies to function as insulators. Recently we showed for the first time that a CAAT-box binding Nuclear factor 1 (CTF/NF1)-based CI, when cloned in the LTRs of a SIN.LV with a strong SFFV enhancer-promoter in internal position, significantly reduced the frequency of tumors harboring integrations activating Map3k8 oncogene accompanied by a marked skewing towards tumors harboring inactivating insertions targeting Pten.Here by using this stringent in vivo genotoxicity assay and IS analysis in tumors we expanded our studies towards other CI sequences whose function is regulated by the binding of the CCCTC-binding factor (CTCF), the best characterized insulator protein in vertebrates.Each CTCF-based insulating cassette was cloned in the LTRs of a LV construct containing the SFFV promoter in internal position (CTCF.SIN.LVs) and injected in Cdkn2a-/- mice. Interestingly, mice treated with some of the CTCF.SIN.LVs tested displayed an increased median survival time (ranging from 193.5 to 214 days) compared to mice treated with the uninsulated parental SIN.LV (186 days). Importantly, our preliminary IS analysis in tumors (881 IS) showed that two CTCF.SIN.LVs did not target Map3k8 oncogene while Pten was often disrupted by exonic insertions, an escape genotoxicity mechanism on which CI cannot act.These data confirm that the inclusion of two novel CTCF-based CIs of human origin completely abrogated the formation of tumors caused by enhancer-mediated activation of an oncogene in vivo.The ability of these two new insulator elements to block the crosstalk between powerful vector enhancers and cellular regulatory elements increase the safety of SIN LVs and justify their prompt adoption in future gene therapy applications.

Insertional mutagenesis represents a major hurdle to gene therapy and necessitates sensitive preclinical genotoxicity assays. Cdkn2a(-/-) mice are susceptible to a broad range of cancer-triggering genetic lesions. We exploited hematopoietic stem cells from these tumor-prone mice to assess the oncogenicity of prototypical retroviral and lentiviral vectors. We transduced hematopoietic stem cells in matched clinically relevant conditions, and compared integration site selection and tumor development in transplanted mice. Retroviral vectors triggered dose-dependent acceleration of tumor onset contingent on long terminal repeat activity. Insertions at oncogenes and cell-cycle genes were enriched in early-onset tumors, indicating cooperation in tumorigenesis. In contrast, tumorigenesis was unaffected by lentiviral vectors and did not enrich for specific integrants, despite the higher integration load and robust expression of lentiviral vectors in all hematopoietic lineages. Our results validate a much-needed platform to assess vector safety and provide direct evidence that prototypical lentiviral vectors have low oncogenic potential, highlighting a major rationale for application to gene therapy.

Next generation sequencing approaches are identifying a plethora of mutations in a variety of human cancers. However, the clinical implications of these new findings are still limited, since the identification of cancer driving mutations is hampered by the co-occurrence of several bystander and progression related events. Here we developed a forward genetics approach based on a new lentiviral vector-based insertional mutagen aimed at identifying cancer initiating genes that are relevant in human hepatocarcinogenesis. We generated a replication-defective lentiviral vector (LV) engineered with long terminal repeats carrying hepatospecific enhancers able to deregulate genes upon integration. Differently from retroviruses and transposons, our replication-defective LV provides a single round of integration shortly after the administration, thus tagging mainly early events in carcinogenesis. A single administration of LV in newborn mice was able to induce hepatocellular carcinoma (HCC) in 3 clinically relevant models of hepatocarcinogenesis: in 30% of Cdkn2a deficient mice (P=0.005 Vs untreated), in 27% of liver-specific Pten deficient mice (P=0.04) and in 75% of wild type mice coupled to CCl4 administration (P=0.002). From 30 LV-induced HCCs we retrieved 172 LV integrations that allowed the identification of Braf, Fign, Sos1 and Rtl1 as candidate cancer loci. The causative role of these 4 genes in HCC was experimentally validated in vivo by forced expression in the mouse liver. Whole transcriptome gene expression analysis allowed unveiling the molecular pathways deregulated by the newly identified cancer genes. HCC induced by integration at the paternally expressed gene Rtl1 (mapping within the imprinted Dlk1-Dio3 region) displayed the peculiar upregulation of oxidative phosphorylation genes. Conversely, LV-mediated upregulation of Braf and Fign caused the overexpression of the maternally expressed microRNAs encoded within the Dlk1-Dio3 region in HCCs which display the downregulation of oxidative phosphorylation genes. These findings highlighted a relevant role of Dlk1-Dio3 region in HCCs and in the regulation of metabolism. Additionally, we showed that WNT pathway may represent a target of the new enigmatic oncogene Fign. We then analyzed different human HCCs collections induced by HBV or HCV (N tot of HCC=221). We found that all the newly identified cancer genes were significantly upregulated and amplified or deleted in human HCCs. Moreover, we showed that the expression level of the new liver cancer genes or the specific gene expression signature caused by their upregulation can efficiently distinguish HCC patients characterized by poor survival. Overall, we developed a new insertional mutagen by which we identified new clinically relevant liver cancer genes that may provide novel prognostic markers and therapeutic targets for the diagnosis and treatment of human HCCs. Citation Format: Marco Ranzani, Daniela Cesana, Cynthia C. Bartholomä, Francesca Sanvito, Michela Riba, Mauro Pala, Fabrizio Benedicenti, Pierangela Gallina, Stefano Annunziato, Lucia Sergi Sergi, Stefania Merella, Alessandro Bulfone, Claudio Doglioni, Christof von Kalle, Yoon Jun Kim, Manfred Schmidt, Elia Stupka, Giovanni Tonon, Luigi Naldini, Eugenio Montini. Lentiviral vector-based insertional mutagenesis identifies new clinically relevant liver cancer genes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3169. doi:10.1158/1538-7445.AM2013-3169

In mouse, retroviral infection triggers leukemias by activation of cellular proto-oncogenes. This is usually mediated by promoter/enhancer sequences in the proviral LTR that recruit ubiquitous and/or cell type-specific transcription factors, and up-regulate expression of flanking genes, up to 300 Kb away from the integration site, and independently of orientation. Unfortunately, Retroviral Vectors (RV) used in gene therapy also triggered unwanted oncogenesis, indicating that the risk of RV insertional mutagenesis had been underestimated, and raising concerns for their further use in gene therapy. In addition RV, Lentiviral (LV) and Adeno-Associated vectors have the tendency to integrate in the proximity of expressed genes. Thus, these safety concerns apply to all integrative vectors, in particular to the closely related LV that have been proposed as improved versions of RV for several applications. The actual frequency and extent of transcriptional de-regulation occurring at randomly picked retroviral integration sites is currently unknown. In fact, integration sites retrieved from tumors are likely the result of in vivo selection for proto-oncogene activation, and it has proven very difficult to estimate the frequency of such events in unselected target cells. This information is critical to properly assess the insertional mutagenesis risk of each vector. Thus, we embarked in testing and comparing the effect of non-selected RV and LV integration on the expression of flanking genes. We generated three panels of hematopoietic cell clones, each carrying a single insertion of either a conventional RV expressing GFP from the LTR, a self inactivating (SIN)-RV expressing GFP from an internal PGK promoter, and a third-generation SIN-LV expressing GFP from the same PGK promoter. For each vector, we isolated 150 clones, and retrieved genomic DNA flanking the proviral insertion in 50 of them by linear amplification-mediated PCR. So far, sequence analysis has allowed in silico mapping of 40 single integrations. Even if our study requires a relatively small sample size, it is apparent that integration site selection differed between RV and LV; RV integrated within regions of lower gene density with respect to LV; 66% of RV insertions were intergenic, while 83 % of LV insertions were intragenic; 50% of RV insertions were found between 0.5–5kb from the closest transcription start site, as compared to only 5% of LV insertions. We then selected all the genes (SeqRef database) contained in a 600 kb interval centered on each integration site (344 up to now). We are currently comparing their relative expression levels in all cell clones by microarray technology, using custom-made microchips spotted with the selected sequences, and probing with total cDNA obtained from each clone and a pool of control untransduced ones. This analysis will indicate the frequency and extent of transcriptional perturbation around RV and LV integration. We expect to find significant differences between each vector, according to its viral origin and design. If so, our data will be critical to estimate the relative risk of oncogene activation in gene therapy applications.

Next generation sequencing approaches are identifying a plethora of mutations in a variety of human cancers. However, the potential clinical implications of these new findings are still limited, since the identification of cancer driving mutations is hampered by the co-occurrence of several bystander and progression related events. Here we developed a forward genetics approach based on a new lentiviral vector-based insertional mutagen aimed at identifying liver cancer initiating genes that are relevant in human hepatocarcinogenesis. We generated a replication-defective lentiviral vector (LV) engineered with long terminal repeats carrying hepatospecific enhancers capable to induce hepatocellular carcinoma (HCC) upon a single administration in 3 clinically relevant mouse models of hepatocarcinogenesis. LV injection in newborn mice was able to induce HCC in 30% of Cdkn2a deficient mice (P=0.005 Vs untreated), in 27% of liver-specific Pten deficient mice (P=0.04) and in 75% of wild type mice coupled to CCl4 administration (P=0.002). From 30 LV-induced HCCs we could retrieve LV integrations that allowed the identification of Braf, Fign, Sos1 and Dlk1-Dio3 region as candidate cancer loci. The causative role of these genes in HCC was experimentally validated in vivo by forced expression in the mouse liver. Whole transcriptome gene expression analysis allowed unveiling the molecular pathways on which the new cancer genes have an impact. We showed that tumors with integration within Dlk1-Dio3 imprinted region displayed the overexpression of the paternally expressed gene Rtl1 encoded within the region and a peculiar upregulation of oxidative phosphorylation genes. Conversely, LV-mediated upregulation of Braf and Fign caused the overexpression of all maternal genes from Dlk1-Dio3 region in HCCs which display the downregulation of oxidative phosphorylation genes. Additionally, upregulation of Fign was associated to the deregulation of Wnt pathway in HCCs. We found that all the newly identified cancer genes were significantly upregulated and amplified or deleted in the human HCCs, highlighting a relevant role of these genes in human hepatocarcinogenesis. We also identified the specific molecular signature resulting from the activation of each cancer gene found in murine HCCs. Remarkably, gene expression analyses performed on 3 different human HCC collections showed that the signatures of all the 4 new cancer genes: 1) are present also in human HCCs; 2) can distinguish different HCC stages; 3) can identify different prognostic groups of patients. Moreover, SOS1 expression levels alone can discriminate HCC patients with good or poor overall survival. This study identified new clinically relevant liver cancer genes that may provide novel prognostic markers and therapeutic targets for the diagnosis and treatment of human HCCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 104. doi:1538-7445.AM2012-104

Retrovirus or transposon-based in vivo insertional mutagenesis triggers cancer by activating nearby oncogenes upon integration and led to the discovery of many candidate oncogenes important in human cancer. However, these insertional mutagens constantly undergo integration cycles throughout the life of the animal, thus resulting in several bystander and progression-related integrations that hamper the identification of cancer-initiating events. To overcome these limitations, we generated a replication-defective lentiviral vector (LV) engineered with long terminal repeats carrying hepatospecific enhancers capable to induce hepatocellular carcinoma (HCC) upon a single administration in 3 different mouse models of hepatocarcinogenesis. LV injection in newborn mice was able to induce HCC in 30% of Cdkn2a deficient mice (0 in controls, P=0.005), in 27% of liver-specific Pten deficient mice (6% in controls, P=0.04) and in 75% of wild type mice coupled to CCl4 administration (0 in controls, P=0.002). From 30 LV-induced HCCs we could retrieve 172 unique integrations that allowed the identification of Braf, Fign, Sos1 and Dlk1-Dio3 region as candidate cancer loci. The causative role of these genes in HCC was experimentally validated in vivo by forced expression in the mouse liver. The LV integration in Braf and Sos1 generated constitutively active proteins previously found in human tumors from tissues other than liver. Genome-wide gene expression analyses identified the specific molecular signature resulting from the activation of each cancer gene found in murine HCCs. Remarkably, gene expression analyses performed on 2 different human HCC collections showed that these signatures: 1) are present also in human HCCs; 2) discriminate among normal liver, non-tumoral diseased liver and HCC; 3) can distinguish different HCC stages. All the newly identified cancer genes were significantly upregulated and/or mutated in the human HCCs, underscoring a relevant role of these genes in human hepatocarcinogenesis. Integration of LV at Dlk1-Dio3 locus resulted in the upregulation of the paternally expressed gene Rtl1 and induced HCCs characterized by a peculiar upregulation of oxidative phosphorylation genes. Oppositely, LV-mediated upregulation of Braf and Fign caused the overexpression of all maternal genes from Dlk1-Dio3 locus in HCCs which display a canonical dowregulation of oxidative phosphorylation genes. The latter expression pattern was confirmed in a cohort of human HCCs. Our data indicate that the Dlk1-Dio3 locus plays a relevant role in hepatocarcinogenesis, energy metabolism and stem cell maintenance in mice and humans.This study uncovered a role in human HCCs of some previously known and other completely new oncogenes that altogether may provide novel candidate therapeutic targets and diagnostic markers for human HCCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4982. doi:10.1158/1538-7445.AM2011-4982

Insertional mutagenesis represents a major hurdle to successful gene therapy and mandates for sensitive pre-clinical assays of genotoxicity. Cdkn2a−/ − mice are defective for p53 and Rb pathways, and are susceptible to a broad range of cancer-triggering genetic lesions. We developed an in-vivo genotoxicity assay, based on transplantation of Cdkn2a−/ − hematopoietic stem cells (HSCs), treated or not with prototypical retroviral (RV) and lentiviral (LV) vectors. In our rationale if RV or LV treatment is genotoxic, transplanted mice will show a significantly earlier tumor onset. Using this approach, we detected a dose-dependent acceleration in tumor onset in the mice transplanted with RV treated cells. We compared the RV and LV integration site distribution in pre-transplant cells and tumors from the transplanted mice. As expected, RV integrates close to gene promoters in pre-transplant cells and tumors. Moreover, we found that RV preferentially targeted genes encoding for transcription factors, kinases and genomic positions previously described as Common Integration Sites (CIS). CIS are genomic regions targeted at high frequency in tumors by retroviral integrations and probably map within or close to proto-oncogenes activated upon integration. Interestingly, in tumors, RV insertions at CIS and cell cycle genes were further enriched and associated to early lymphomagenesis. Remarkably, LV tested in the same conditions, did not show any tumor acceleration, and targeted CIS much less frequently than RV in pre-transplant cells or tumors, and did not show selection for integrations at any specific gene class. This is the first evidence that prototypic LV have low oncogenic potential, and provides a major rationale for their application to HSC gene therapy. To dissect the role in lymphomagenesis of the strong enhancers in the RV LTRs and the different integration site selection of each vector, we tested an RV with enhancer deleted LTRs (SIN RV) and a moderate promoter in internal position, an LV with a strong RV-like enhancer-promoter into the LTRs or in internal position. Our results show that: SIN RV shows reduced effect on lymphomagenesis acceleration with respect the conventional RV; LV with RV like LTRs treatment significantly accelerates lymphomagenesis, underlining important role of RV-LTRs in oncogenesis; LV with RV-like enhancer in internal position show a reduced genotoxicity. These data show that the type of genetic elements used (strong enhancers or moderate promoter) and their position in the vector genome (LTR or internal positions) significantly influence the vector safety profile. We are currently mapping the integration sites in pre-transplant cells and tumors marked by each vector to identify the genes targeted by the integrations and elucidate the potential mechanism of deregulation. The described model provide an important tool to compare the risk of insertional mutagenesis of different integrating vectors and provides a platform to test different vector types, designs and safety improvements.

Insertional mutagenesis represents a major hurdle to successful gene therapy and mandates for sensitive pre-clinical assays of genotoxicity. Cdkn2a|[minus]|/|[minus]| mice are defective for p53 and Rb pathways, and are susceptible to a broad range of cancer-triggering genetic lesions. We exploited the sensitivity of these tumor-prone mice to develop an in-vivo genotoxicity assay, based on transplantation of Cdkn2a|[minus]|/|[minus]| hematopoietic stem cells (HSC), treated or not with prototypical retroviral (RV) and lentiviral (LV) vectors. In our rationale if RV or LV treatment is genotoxic, then transplanted mice will show a significantly earlier tumor onset. The sensitivity of the model was shown by the ability to detect a vector dose-dependent acceleration in tumor onset in mice transplanted with RV-treated cells. Such acceleration, as in previous studies, is consequent to genetic lesions, produced by vector integration, that cooperate with the germ-line mutation, and is contingent on LTR activity.

Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.