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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.

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.

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.

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.

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.

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

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.

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.

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.

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.