Stefania Merella

Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations

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.

Uncovering and Dissecting the Genotoxicity of Self-inactivating Lentiviral Vectors In Vivo

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.

Brain conditioning is instrumental for successful microglia reconstitution following hematopoietic stem cell transplantation

The recent hypothesis that postnatal microglia are maintained independently of circulating monocytes by local precursors that colonize the brain before birth has relevant implications for the treatment of various neurological diseases, including lysosomal storage disorders (LSDs), for which hematopoietic cell transplantation (HCT) is applied to repopulate the recipient myeloid compartment, including microglia, with cells expressing the defective functional hydrolase. By studying wild-type and LSD mice at diverse time-points after HCT, we showed the occurrence of a short-term wave of brain infiltration by a fraction of the transplanted hematopoietic progenitors, independently from the administration of a preparatory regimen and from the presence of a disease state in the brain. However, only the use of a conditioning regimen capable of ablating functionally defined brain-resident myeloid precursors allowed turnover of microglia with the donor, mediated by local proliferation of early immigrants rather than entrance of mature cells from the circulation.

Lentiviral vector–based insertional mutagenesis identifies genes associated with liver cancer

Transposons and γ-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 LVVs 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.

The combination of transcriptomics and informatics identifies pathways targeted by miR-204 during neurogenesis and axon guidance

Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by in vivo functional studies. Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development.

VISPA: a computational pipeline for the identification and analysis of genomic vector integration sites

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.

Mutant uromodulin expression leads to altered homeostasis of the endoplasmic reticulum and activates the unfolded protein response

Uromodulin is the most abundant urinary protein in physiological conditions. It is exclusively produced by renal epithelial cells lining the thick ascending limb of Henle’s loop (TAL) and it plays key roles in kidney function and disease. Mutations in UMOD, the gene encoding uromodulin, cause autosomal dominant tubulointerstitial kidney disease uromodulin-related (ADTKD-UMOD), characterised by hyperuricemia, gout and progressive loss of renal function. While the primary effect of UMOD mutations, retention in the endoplasmic reticulum (ER), is well established, its downstream effects are still largely unknown. To gain insight into ADTKD-UMOD pathogenesis, we performed transcriptional profiling and biochemical characterisation of cellular models (immortalised mouse TAL cells) of robust expression of wild type or mutant GFP-tagged uromodulin. In this model mutant uromodulin accumulation in the ER does not impact on cell viability and proliferation. Transcriptional profiling identified 109 genes that are differentially expressed in mutant cells relative to wild type ones. Up-regulated genes include several ER resident chaperones and protein disulphide isomerases. Consistently, pathway enrichment analysis indicates that mutant uromodulin expression affects ER function and protein homeostasis. Interestingly, mutant uromodulin expression induces the Unfolded Protein Response (UPR), and specifically the IRE1 branch, as shown by an increased splicing of XBP1. Consistent with UPR induction, we show increased interaction of mutant uromodulin with ER chaperones Bip, calnexin and PDI. Using metabolic labelling, we also demonstrate that while autophagy plays no role, mutant protein is partially degraded by the proteasome through ER-associated degradation. Our work demonstrates that ER stress could play a central role in ADTKD-UMOD pathogenesis. This sets the bases for future work to develop novel therapeutic strategies through modulation of ER homeostasis and associated protein degradation pathways.

Abstract 3169: Lentiviral vector-based insertional mutagenesis identifies new clinically relevant liver cancer genes.

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 CCl 4 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. Bartholoma, 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

Abstract 104: New liver cancer genes identified by lentiviral vector-based insertional mutagenesis in mice are associated to differential survival in hepatocellular carcinoma patients

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 CCl 4 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

Assessing the impact of lentiviral vector integration on splicing of cellular genes at the genome-wide level

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.