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Alessandra RECCHIA

Ricercatore Universitario presso: Dipartimento di Scienze della Vita sede ex-Scienze Biomediche

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2020 - Erratum: Genomic analysis of sleeping beauty transposon integration in human somatic cells (PLoS ONE (2014) 9:11 (e112712) DOI: 10.1371/journal.pone.0112712) [Articolo su rivista]
Turchiano, G.; Latella, M. C.; Doring, A. G.; Cattoglio, C.; Mavilio, F.; Izsvak, Z.; Ivics, Z.; Recchia, A.

The following information is missing from the Funding statement: Dr. Zsuzsanna Izsvák was funded by European Research Council-2011-ADG-TRANSPOSOstress- 294742.

2019 - AAV-CRISPR Persistence in the Eye of the Beholder [Recensione in Rivista]
Recchia, A.

Despite advances in genome editing technologies based on the adeno-associated virus (AAV)-CRISPR system, there are still concerns about the long-term persistence of recombinant AAV vectors in several organs (liver, muscle, eye) possibly leading to cytotoxicity or genotoxicity related to off-target effects. Indeed, there are still unanswered questions about long-lasting in vivo AAV persistence as a linear or circular DNA that is not targeted by epigenetic silencing in many tissues. In 2017, Kim et al.1 reported an editing approach based on AAV-CjCas9 to downregulate Vegfa or the hypoxia-inducible transcription factor Hif1a in mice displaying age-related macular degeneration (AMD)-related pathological choroidal neovascularization (CNV) induced by laser treatment. Although partial knockdown of either Vegfa or Hif1a provided benefits and reduced the area of CNV, local opsin dysfunction near the Vegfa-edited cells of murine retinal pigment epithelium (RPE) was observed. Conversely, no cone dysfunction was reported upon Hif1a partial knockdown. Lastly, no genome-wide off-target indels, evaluated 6 weeks after intravitreal injection of AAV-CjCas9 vector, were scored, indicating that prolonged expression of AAV-CjCas9 in vivo did not aggravate the genotoxic risk associated with the CjCas9 nuclease. In this issue of Molecular Therapy, the authors now report a long-term (14 months) safety study on C57BL/6J mice intravitreally injected with AAV-CjCas9 vectors targeting Vegfa or Hif1a genes.2 The findings continue to show that the AAV-CRISPR system in the eyes is long lasting, effective, and safe.

2019 - CRISPR/Cas9 gene editing in vitro and in retinal cells in vivo [Capitolo/Saggio]
Benati, D.; Marigo, V.; Recchia, A.

CRISPR/Cas9 is an efficient tool to knock down specific genes in various organisms. In this chapter, we describe how to assess knock-down of human Rhodopsin (RHO) gene carrying the P23H mutation in vitro, in engineered HeLa cells and in vivo, in P23H RHO transgenic mice. To this aim, we report two molecular assays: site-specific PCR on P23H RHO cells treated with CRISPR/Cas9 and Western blotting analysis on retinal cells prepared from P23H RHO transgenic mice electroporated with CRISPR/Cas9 and GFP plasmids.

2019 - Gene editing prospects for treating inherited retinal diseases [Recensione in Rivista]
Benati, D.; Patrizi, C.; Recchia, A.

Retinal diseases (RD) include inherited retinal dystrophy (IRD), for example, retinitis pigmentosa and Leber's congenital amaurosis, or multifactorial forms, for example, age-related macular degeneration (AMD). IRDs are clinically and genetically heterogeneous in nature. To date, more than 200 genes are known to cause IRDs, which perturb the development, function and survival of rod and cone photoreceptors or retinal pigment epithelial cells. Conversely, AMD, the most common cause of blindness in the developed world, is an acquired disease of the macula characterised by progressive visual impairment. To date, available therapeutic approaches for RD include nutritional supplements, neurotrophic factors, antiangiogenic drugs for wet AMD and gene augmentation/interference strategy for IRDs. However, these therapies do not aim at correcting the genetic defect and result in inefficient and expensive treatments. The genome editing technology based on clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas) and an RNA that guides the Cas protein to a predetermined region of the genome, represents an attractive strategy to tackle IRDs without available cure. Indeed, CRISPR/Cas system can permanently and precisely replace or remove genetic mutations causative of a disease, representing a molecular tool to cure a genetic disorder. In this review, we will introduce the mechanism of CRISPR/Cas system, presenting an updated panel of Cas variants and delivery systems, then we will focus on applications of CRISPR/Cas genome editing in the retina, and, as emerging treatment options, in patient-derived induced pluripotent stem cells followed by transplantation of retinal progenitor cells into the eye.

2019 - MSC-delivered soluble TRAIl and paclitaxel as novel combinatory treatment for pancreatic adenocarcinoma [Articolo su rivista]
Rossignoli, Filippo; Spano, Carlotta; Grisendi, Giulia; Foppiani, Elisabetta Manuela; Golinelli, Giulia; Mastrolia, Ilenia; Bestagno, Marco; Candini, Olivia; Petrachi, Tiziana; Recchia, Alessandra; Miselli, Francesca; Rovesti, Giulia; Orsi, Giulia; Veronesi, Elena; Medici, Gregorio; Petocchi, Benedetta; Pinelli, Massimo; Horwitz, Edwin M.; Conte, Pierfranco; Dominici, Massimo

Pancreatic cancer is the fourth leading cause of cancer death in western countries with more than 100,000 new cases per year in Europe and a mortality rate higher than 90%. In this scenario, advanced therapies based on gene therapies are emerging, thanks to a better understanding of tumour architecture and cancer cell alterations. We have demonstrated the efficacy of an innovative approach for pancreatic cancer based on mesenchymal stromal cells (MSC) genetically engineered to produce TNF-related Apoptosis Inducing Ligand (TRAIL). Here we investigated the combination of this MSC-based approach with the administration of a paclitaxel (PTX)-based chemotherapy to improve the potential of the treatment, also accounting for a possible resistance onset. Methods: Starting from the BXPC3 cell line, we generated and profiled a TRAIL-resistant model of pancreatic cancer, testing the impact of the combined treatment in vitro with specific cytotoxicity and metabolic assays. We then challenged the rationale in a subcutaneous mouse model of pancreatic cancer, assessing its effect on tumour size accounting stromal and parenchymal organization. Results: PTX was able to restore pancreatic cancer sensitivity to MSC-delivered TRAIL by reverting its pro-survival gene expression profile. The two compounds cooperate both in vitro and in vivo and the combined treatment resulted in an improved cytotoxicity on tumour cells. Conclusion: In summary, this study uncovers the potential of a combinatory approach between MSC-delivered TRAIL and PTX, supporting the combination of cell-based products and conventional chemotherapeutics as a tool to improve the efficacy of the treatments, also addressing possible mechanisms of resistance.

2019 - Soluble TRAIL Armed Human MSC As Gene Therapy For Pancreatic Cancer [Articolo su rivista]
Spano, Carlotta; Grisendi, Giulia; Golinelli, Giulia; Rossignoli, Filippo; Prapa, Malvina; Bestagno, Marco; Candini, Olivia; Petrachi, Tiziana; Recchia, Alessandra; Miselli, Francesca; Rovesti, Giulia; Orsi, Giulia; Maiorana, Antonino; Manni, Paola; Veronesi, Elena; Piccinno, Maria Serena; Murgia, Alba; Pinelli, Massimo; Horwitz, Edwin M.; Cascinu, Stefano; Conte, Pierfranco; Dominici, Massimo

Pancreatic ductal adenocarcinoma (PDAC) is still one of the most aggressive adult cancers with an unacceptable prognosis. For this reason novel therapies accounting for PDAC peculiarities, such as the relevant stromal reaction, are urgently needed. Here adipose mesenchymal stromal/stem cells (AD-MSC) have been armed to constantly release a soluble trimeric and multimeric variant of the known anti-cancer TNF-related apoptosis-inducing ligand (sTRAIL). This cancer gene therapy strategy was in vitro challenged demonstrating that sTRAIL was thermally stable and able to induce apoptosis in the PDAC lines BxPC-3, MIA PaCa-2 and against primary PDAC cells. sTRAIL released by AD-MSC relocated into the tumor stroma was able to significantly counteract tumor growth in vivo with a significant reduction in tumor size, in cytokeratin-7+ cells and by an anti-angiogenic effect. In parallel, histology on PDAC specimens form patients (n = 19) was performed to investigate the levels of TRAIL DR4, DR5 and OPG receptors generating promising insights on the possible clinical translation of our approach. These results indicate that adipose MSC can very efficiently vehicle a novel TRAIL variant opening unexplored opportunities for PDAC treatment.

2018 - An efficient in vitro transposition method by a transcriptionally regulated sleeping beauty system packaged into an integration defective lentiviral vector [Articolo su rivista]
Benati, Daniela; Cocchiarella, Fabienne; Recchia, Alessandra

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a "cut and paste" SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

2018 - Bimodal CD40/Fas-Dependent Crosstalk between iNKT Cells and Tumor-Associated Macrophages Impairs Prostate Cancer Progression [Articolo su rivista]
Cortesi, Filippo; Delfanti, Gloria; Grilli, Andrea; Calcinotto, Arianna; Gorini, Francesca; Pucci, Ferdinando; Lucianò, Roberta; ` Grioni, Matteo; Recchia, Alessandra; Benigni, Fabio; Briganti, Alberto; Salonia, Andrea; De Palma, Michele; Bicciato, Silvio; Doglioni, Claudio; Bellone, Matteo; Casorati, Giulia; Dellabona, Paolo.

Heterotypic cellular and molecular interactions in the tumor microenvironment (TME) control cancer progression. Here, we show that CD1d-restricted invariant natural killer (iNKT) cells control prostate cancer (PCa) progression by sculpting the TME. In a mouse PCa model, iNKT cells restrained the proangiogenic and immunosuppressive capabilities of tumor-infiltrating immune cells by reducing proangiogenic TIE2+, M2-like macrophages (TEMs), and sustaining pro-inflammatory M1-like macrophages. iNKT cells directly contacted macrophages in the PCa stroma, and iNKT cell transfer into tumorbearing mice abated TEMs, delaying tumor progression. iNKT cells modulated macrophages through the cooperative engagement of CD1d, Fas, and CD40, which promoted selective killing of M2-like and survival of M1-like macrophages. Human PCa aggressiveness associate with reduced intra-tumoral iNKT cells, increased TEMs, and expression of pro-angiogenic genes, underscoring the clinical significance of this crosstalk. Therefore, iNKT cells may control PCa through mechanisms involving differential macrophage modulation, which may be harnessed for therapeutically reprogramming the TME.

2018 - CRISPR/Cas9-Mediated In Situ Correction of LAMB3 Gene in Keratinocytes Derived from a Junctional Epidermolysis Bullosa Patient [Articolo su rivista]
Benati, Daniela; Miselli, Francesca; Cocchiarella, Fabienne; Patrizi, Clarissa; Carretero, Marta; Baldassarri, Samantha; Ammendola, Virginia; Has, Cristina; Colloca, Stefano; Del Rio, Marcela; Larcher, Fernando; Recchia, Alessandra

Deficiency of basement membrane heterotrimeric laminin 332 component, coded by LAMA3, LAMB3, and LAMC2 genes, causes junctional epidermolysis bullosa (JEB), a severe skin adhesion defect. Herein, we report the first application of CRISPR/Cas9-mediated homology direct repair (HDR) to in situ restore LAMB3 expression in JEB keratinocytes in vitro and in immunodeficient mice transplanted with genetically corrected skin equivalents. We packaged an adenovector carrying Cas9/guide RNA (gRNA) tailored to the intron 2 of LAMB3 gene and an integration defective lentiviral vector bearing a promoterless quasi-complete LAMB3 cDNA downstream a splice acceptor site and flanked by homology arms. Upon genuine HDR, we exploited the in vitro adhesion advantage of laminin 332 production to positively select LAMB3-expressing keratinocytes. HDR and restored laminin 332 expression were evaluated at single-cell level. Notably, monoallelic-targeted integration of LAMB3 cDNA was sufficient to in vitro recapitulate the adhesive property, the colony formation typical of normal keratinocytes, as well as their cell growth. Grafting of genetically corrected skin equivalents onto immunodeficient mice showed a completely restored dermal-epidermal junction. This study provides evidence for efficient CRISPR/Cas9-mediated in situ restoration of LAMB3 expression, paving the way for ex vivo clinical application of this strategy to laminin 332 deficiency.

2018 - Inducible Caspase9-mediated suicide gene for MSC-based cancer gene therapy [Articolo su rivista]
Rossignoli, Filippo; Grisendi, Giulia; Spano, Carlotta; Golinelli, Giulia; Recchia, Alessandra; Rovesti, Giulia; Orsi, Giulia; Veronesi, Elena; Horwitz, Edwin M.; Dominici, Massimo

Cellular therapies based on mesenchymal stromal/stem cells (MSC) are promising strategies in regenerative medicine and oncology. Despite encouraging results, there is still some level of concerns on inoculating MSC in cancer patients. To face this issue, one possibility resides in engineering MSC by incorporating a suicide gene in order to control their fate once infused. Strategies based on Herpes Simplex Virus Thymidine Kinase (HSV-TK) and the Cytosine Deaminase genes have been developed and more recently a novel suicide gene, namely, iCasp9, has been proposed. This approach is based on a variant of human Caspase9 that binds with high affinity to a synthetic, bioinert small molecule (AP20187) leading to cell death. Based on this technology so far marginally applied to MSC, we tested the suitability of iCasp9 suicide strategy in MSC to further increase their safety. MSC have been transfected by a lentiviral vector carrying iCasp9 gene and then tested for viability after AP20187 treatment in comparison with mock-transfected cells. Moreover, accounting our anti-tumor approaches based on MSC expressing potent anti-cancer ligand TNF-Related Apoptosis-Inducing Ligand (TRAIL), we generated adipose MSC co-expressing iCasp9 and TRAIL successfully targeting an aggressive sarcoma type. These data show that anti-cancer and suicide mechanisms can coexist without affecting cells performance and hampering the tumoricidal activity mediated by TRAIL. In conclusion, this study originally indicates the suitability of combining a MSC-based anti-cancer gene approach with iCasp9 demonstrating efficiency and specificity.

2017 - CRISPR/Cas9-mediated specific knock-down of dominant mutations in Rhodopsin gene [Poster]
Benati, Daniela; Manel, Llado; Patrizi, Clarissa; Schiroli, Davide; Marigo, Valeria; Auricchio, Alberto; Recchia, Alessandra

Rhodopsin (RHO) mutations represent a common cause of blindness, accounting for 25% of autosomal dominant Retinitis Pigmentosa (RP) and 8-10% of all RP. Although gene therapy has been successfully applied to retinal degeneration caused by recessive mutations, therapeutic intervention for dominant mutations are still lagging behind. In this study, we explored the efficacy of newly described CRISPR/Cas9 variants with altered PAM specificity and nearly completely reduced off-target effects, to specifically inactivate two highly frequent dominant mutations, P23H and P347S, mapped in the N-terminal and the C-terminal region of the RHO gene, respectively. We designed gRNAs on the mutations to compare allele-specific targeting of the high fidelity SpCas9 (SpCas9-HF1), the respective VQR variant (SpCas9-VQR-HF1) or the SaCas9, and we tested gRNAs in vitro on HeLa clones stably expressing P23H, P347S or wild-type RHO. Analysis of insertions or deletions (Indels) in the genomic DNA specifically in the RHO gene, by Cel-I assay and sequencing, identified the most efficient and mutation-specific system able to induce Indels in the P23H or P347S RHO mutated allele, with almost undetectable editing of the wild-type allele. We are going to package the selected CRISPR/Cas9-gRNA in AAV2/8 particles to test this approach in P23H or P347S RHO transgenic mice, to evaluate retina functionality and vision recovery upon CRISPR/Cas9-mediated editing. Our results will provide clear evidences about the employment of CRISPR/Cas9 system to selectively target dominant mutations and the preclinical application of this strategy for patients affected by RP due to mutations in the RHO gene.

2017 - Correction of recessive dystrophic epidermolysis bullosa by transposon-mediated integration of COL7A1 in transplantable patient-derived primary keratinocytes. [Articolo su rivista]
Latella, Maria Carmela; Cocchiarella, Fabienne; DE ROSA, Laura; Turchiano, Giandomenico; Gonçalves, Manuel; Larcher, Fernando; DE LUCA, Michele; Recchia, Alessandra

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects in type-VII collagen (C7), a protein encoded by the COL7A1 gene and essential for anchoring fibril formation at the dermal-epidermal junction. Gene therapy of RDEB is based on transplantation of autologous epidermal grafts generated from genecorrected keratinocytes sustaining C7 deposition at the dermal-epidermal junction. Transfer of the COL7A1 gene is complicated by its very large size and repetitive sequence. This article reports a gene delivery approach based on the Sleeping beauty transposon, which allows integration of a full-length COL7A1 cDNA and secretion of C7 at physiological levels in RDEB keratinocytes without rearrangements or detrimental effects on their clonogenic potential. Skin equivalents derived from gene-corrected RDEB keratinocytes were tested in a validated preclinical model of xenotransplantation on immunodeficient mice, where they showed normal deposition of C7 at the dermal-epidermal junction and restoration of skin adhesion properties. These results indicate the feasibility and efficacy of a transposon-based gene therapy approach to RDEB.

2017 - Transcriptional and epigenetic analyses of the DMD locus reveal novel cis‑acting DNA elements that govern muscle dystrophin expression [Articolo su rivista]
Gherardi, Samuele; Bovolenta, Matteo; Passarelli, Chiara; Falzarano, Maria Sofia; Pigini, Paolo; Scotton, Chiara; Neri, Marcella; Armaroli, Annarita; Osman, Hana; Selvatici, Rita; Gualandi, Francesca; Recchia, Alessandra; Mora, Marina; Bernasconi, Pia; Maggi, Lorenzo; Morandi, Lucia; Ferlini, Alessandra; Perini, Giovanni

The dystrophin gene (DMD) is the largest gene in the human genome, mapping on the Xp21 chromosome locus. It spans 2.2 Mb and accounts for approximately 0,1% of the entire human genome. Mutations in this gene cause Duchenne and Becker Muscular Dystrophy, X-linked Dilated Cardiomyopathy, and other milder muscle phenotypes. Beside the remarkable number of reports describing dystrophin gene expression and the pathogenic consequences of the gene mutations in dystrophinopathies, the full scenario of the DMD transcription dynamics remains however, poorly understood. Considering that the full transcription of the DMD gene requires about 16 h, we have investigated the activity of RNA Polymerase II along the entire DMD locus within the context of specific chromatin modifications using a variety of chromatin-based techniques. Our results unveil a surprisingly powerful processivity of the RNA polymerase II along the entire 2.2 Mb of the DMD locus with just one site of pausing around intron 52. We also discovered epigenetic marks highlighting the existence of four novel cis‑DNA elements, two of which, located within intron 34 and exon 45, appear to govern the architecture of the DMD chromatin with implications on the expression levels of the muscle dystrophin mRNA. Overall, our findings provide a global view on how the entire DMD locus is dynamically transcribed by the RNA pol II and shed light on the mechanisms involved in dystrophin gene expression control, which can positively impact on the optimization of the novel ongoing therapeutic strategies for dystrophinopathies.

2016 - Closure of a Large Chronic Wound through Transplantation of Gene-Corrected Epidermal Stem Cells [Articolo su rivista]
Bauer, Johann; Koller, Josef; Murauer, Eva; DE ROSA, Laura; Enzo, Elena; Carulli, Sonia; Bondanza, Sergio; Recchia, Alessandra; Muss, Wolfgang; Diem, Anja; Mayr, Elisabeth; Schlager, Pamina; Gratz, Iris; Pellegrini, Graziella; DE LUCA, Michele

Generalized junctional epidermolysis bullosa (JEB) is caused by mutations in LAMA3,LAMB3,or LAMC2,which together encode laminin-332, a hetero-trimeric protein consisting ofa3,b3, andg2chain. In nonlethal generalized intermediate JEB, laminin-332 is highly reduced, and hemidesmosomes are rudimentary or completely absent, leading to blister formation within the lamina lucida of the basement membrane upon minor trauma. The resulting chronic skin wounds invariably develop recurrent infections and scarring, which greatly impair patients’ quality of life. We report on a patient in whom gene-corrected epidermal sheets were transplanted onto a large nonhealing epidermal ulceration following a good manufacturing practice protocol

2016 - In vitro and in vivo CRISPR/Cas9-mediated genome editing to downregulate dominant mutations in Rhodopsin gene [Poster]
Benati, Daniela; Latella, Maria Carmela; DI SALVO, MARIA TERESA; Cocchiarella, Fabienne; Marigo, Valeria; Recchia, Alessandra

Many progresses have been made in understanding the genetic basis for Retinitis Pigmentosa (RP), however therapeutic interventions are still lagging behind. Rhodopsin (RHO) mutations represent a common cause of RP, accounting for 25% of autosomal dominant RP and 8-10% of all RP (Hartong et al., 2006) with more than 100 different mutations identified so far. Here we show the application of CRISPR-Cas9 technology to knock out the RHO defective alleles by introducing double strand breaks into the target gene. We designed single or double gRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. The two gRNAs were tested singularly or together in vitro in HeLa clones stably expressing P23H RHO. Cel I assay, TIDE and sequencing analyses demonstrated insertions or deletions (indels) in the genomic DNA specifically in the RHO gene, which caused strong reduction of RHO expression up to 90%. The higher effect was obtained with two gRNAs together. The CRISPR/Cas9 plasmid expressing two gRNAs were then in vivo tested in P23H RHO transgenic mice by sub-retinal electroporation, together with EGFP expressing plasmids. Analysis of indels in FACS-sorted EGFP+ cells demonstrated up to 30% of in vivo genome editing of the human P23H RHO gene, without targeting of the murine Rho allele. We also detected reduction of RHO at mRNA and protein levels. Thus, successful in vivo application of the CRISPR/Cas9 system confirms its efficacy as a genetic engineering tool and its potential use in gene therapy.

2016 - In vitro and in vivo genome editing of the RHO gene to downregulate dominant mutations [Abstract in Rivista]
Marigo, Valeria; Latella, Maria Carmela; DI SALVO, MARIA TERESA; Recchia, Alessandra

Purpose: Although progresses have been made in the understanding of the genetic basis for Retinitis Pigmentosa (RP), the development of therapeutic intervention is still lagging behind. Gene therapy was successfully applied to retina degeneration but only to recessive mutations. Rhodopsin (RHO) mutations represent a common cause of RP, accounting for 25% of adRP and 8 to 10% of all RP. We aimed at developing genome editing tools to knock out the RHO defective alleles by introducing a double strand break (DSB) into the target gene. Methods: The CRISPR/Cas9 system was developed to target the first exon of the human RHO gene by designing 2 gRNAs. The system was tested in vitro in HeLa clones expressing RHO. The effects on the RHO gene were evaluated by sequencing and by analyses at mRNA and protein levels. We expressed by electroporation these 2 gRNAs and Cas9 in vivo in transgenic mice expressing human RHO with P23H mutation. Genome editing was evaluated by sequencing genomic DNA from targeted cells and by analyzing RHO mRNA and protein. Results: Two gRNAs were designed in the first exon of the RHO gene and one of them targeted the P23H mutation. The two gRNAs were tested singularly or together in vitro on HeLa clones stably expressing RHO. We demonstrated insertions or deletions (indels) in the genomic DNA specifically in the RHO gene. Indels caused strong reduction of the RHO mRNA and of RHO protein up to 90%. The higher effect was obtained with the two gRNAs together. The two gRNAs were then in vivo expressed with Cas9 in photoreceptors of transgenic mice by electroporation. Targeted cells were tracked by co-expression with EGFP. EGFP+ cells were FACS sorted and indels in the human P23H RHO gene were analyzed by sequencing. We were able to detect up to 30% of genome editing in vivo. We also detected reduction of human RHO mRNA expression as well as RHO protein. Conclusions: We developed new tools to downregulate mutant RHO in dominant forms of RP. The CRISPR/Cas9 system reveled a high efficiency and should be tested for knock-down followed by gene replacement approaches.

2016 - In vivo editing of the human mutant Rhodopsin gene by electroporation of plasmid-based CRISPR/Cas9 in the mouse retina [Articolo su rivista]
Latella, Maria Carmela; Di Salvo, Maria Teresa; Cocchiarella, Fabienne; Benati, Daniela; Grisendi, Giulia; Comitato, Antonella; Marigo, Valeria; Recchia, Alessandra

The bacterial CRISPR/Cas system has proven to be an efficient tool for genetic manipulation in various organisms. Here we show the application of CRISPR-Cas9 technology to edit the human Rhodopsin (RHO) gene in a mouse model for autosomal dominant Retinitis Pigmentosa. We designed single or double sgRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. By delivering Cas9 and sgRNAs in a single plasmid we induced an efficient gene editing in vitro, in HeLa cells engineered to constitutively express the P23H mutant RHO allele. Similarly, after subretinal electroporation of the CRISPR/Cas9 plasmid expressing two sgRNAs into P23H RHO transgenic mice, we scored specific gene editing as well as significant reduction of the mutant RHO protein. Successful in vivo application of the CRISPR/ Cas9 system confirms its efficacy as a genetic engineering tool in photoreceptor cells.

2016 - Transcriptionally regulated and nontoxic delivery of the hyperactive Sleeping Beauty Transposase [Articolo su rivista]
Cocchiarella, Fabienne; Latella, Maria Carmela; Basile, Valentina; Miselli, Francesca; Galla, Melanie; Imbriano, Carol; Recchia, Alessandra

The Sleeping Beauty (SB) transposase and, in particular, its hyperactive variant SB100X raises increasing interest for gene therapy application, including genome modification and, more recently, induced pluripotent stem cells (iPS) reprogramming. The documented cytotoxicity of the transposase, when constitutively expressed by an integrating retroviral vector (iRV), has been circumvented by the transient delivery of SB100X using retroviral mRNA transfer. In this study, we developed an alternative, safe, and efficient transposase delivery system based on a tetracycline-ON regulated expression cassette and the rtTA2(S)-M2 transactivator gene transiently delivered by integration-defective lentiviral vectors (IDLVs). Compared with iRV-mediated delivery, expression of tetracycline-induced SB100X delivered by an IDLV results in more efficient integration of a GFP transposon and reduced toxicity. Tightly regulated expression and reactivation of the transposase was achieved in HeLa cells as wells as in human primary keratinocytes. Based on these properties, the regulated transposase-IDLV vectors may represent a valuable tool for genetic engineering and therapeutic gene transfer.

2015 - A single epidermal stem cell strategy for safe ex vivo gene therapy [Articolo su rivista]
Droz‐Georget Lathion, Stéphanie; Rochat, Ariane; Knott, Graham; Recchia, Alessandra; Martinet, Danielle; Benmohammed, Sara; Grasset, Nicolas; Zaffalon, Andrea; Besuchet Schmutz, Nathalie; Savioz‐dayer, Emmanuelle; Beckmann, Jacques Samuel; Rougemont, Jacques; Mavilio, Fulvio; Barrandon, Yann

There is a widespread agreement from patient and professional organisations alike that the safety of stem cell therapeutics is of paramount importance, particularly for ex vivo autologous gene therapy. Yet current technology makes it difficult to thoroughly evaluate the behaviour of genetically corrected stem cells before they are transplanted. To address this, we have developed a strategy that permits transplantation of a clonal population of genetically corrected autologous stem cells that meet stringent selection criteria and the principle of precaution. As a proof of concept, we have stably transduced epidermal stem cells (holoclones) obtained from a patient suffering from recessive dystrophic epidermolysis bullosa. Holoclones were infected with self‐inactivating retroviruses bearing a COL7A1 cDNA and cloned before the progeny of individual stem cells were characterised using a number of criteria. Clonal analysis revealed a great deal of heterogeneity among transduced stem cells in their capacity to produce functional type VII collagen (COLVII). Selected transduced stem cells transplanted onto immunodeficient mice regenerated a non‐blistering epidermis for months and produced a functional COLVII. Safety was assessed by determining the sites of proviral integration, rearrangements and hit genes and by whole‐genome sequencing. The progeny of the selected stem cells also had a diploid karyotype, was not tumorigenic and did not disseminate after long‐term transplantation onto immunodeficient mice. In conclusion, a clonal strategy is a powerful and efficient means of by‐passing the heterogeneity of a transduced stem cell population. It guarantees a safe and homogenous medicinal product, fulfilling the principle of precaution and the requirements of regulatory affairs. Furthermore, a clonal strategy makes it possible to envision exciting gene‐editing technologies like zinc finger nucleases, TALENs and homologous recombination for next‐generation gene therapy.

2015 - Design of a regulated lentiviral vector for hematopoietic stem cell gene therapy of globoid cell leukodystrophy [Articolo su rivista]
Ungari, S.; Montepeloso, A.; Morena, F.; Cocchiarella, Fabienne; Recchia, Alessandra; Martino, S.; Gentner, B.; Naldini, L.; Biffi, A.

Globoid cell leukodystrophy (GLD) is a demyelinating lysosomal storage disease due to the deficiency of the galactocerebrosidase (GALC) enzyme. The favorable outcome of hematopoietic stem and progenitor cell (HSPC)-based approaches in GLD and other similar diseases suggests HSPC gene therapy as a promising therapeutic option for patients. The path to clinical development of this strategy was hampered by a selective toxicity of the overexpressed GALC in the HSPC compartment. Here, we presented the optimization of a lentiviral vector (LV) in which miR-126 regulation was coupled to codon optimization of the human GALC cDNA to obtain a selective and enhanced enzymatic activity only upon transduced HSPCs differentiation. The safety of human GALC overexpression driven by this LV was extensively demonstrated in vitro and in vivo on human HSPCs from healthy donors. No perturbation in the content of proapoptotic sphingolipids, gene expression profile, and capability of engraftment and mutlilineage differentiation in chimeric mice was observed. The therapeutic potential of this LV was then assessed in a severe GLD murine model that benefited from transplantation of corrected HSPCs with longer survival and ameliorated phenotype as compared to untreated siblings. This construct has thus been selected as a candidate for clinical translation

2015 - Nuclear architecture dictates HIV-1 integration site selection [Articolo su rivista]
Marini, Bruna; Kertesz Farkas, Attila; Ali, Hashim; Lucic, Bojana; Lisek, Kamil; Manganaro, Lara; Pongor, Sandor; Luzzati, Roberto; Recchia, Alessandra; Mavilio, Fulvio; Giacca, Mauro; Lusic, Marina

Long-standing evidence indicates that human immunodeficiency virus type 1 (HIV-1) preferentially integrates into a subset of transcriptionally active genes of the host cell genome1–4. However, the reason why the virus selects only certain genes among all transcriptionally active regions in a target cell remains largely unknown. Here we show that HIV-1 integration occurs in the outer shell of the nucleus in close correspondence with the nuclear pore. This regioncontains a seriesof cellular genes,which arepreferentially targeted by the virus, and characterized by the presence of active transcription chromatin marks before viral infection. In contrast, the virus strongly disfavours the heterochromatic regions in the nuclear lamin-associateddomains5 and other transcriptionally active regions located centrally in the nucleus. Functional viral integrase and the presence of the cellularNup153 and LEDGF/p75 integration cofactors are indispensable for the peripheral integration of the virus. Once integrated at the nuclear pore, the HIV-1 DNA makes contact with various nucleoporins; this association takes part in the transcriptional regulation of the viral genome.These results indicate that nuclear topography is an essential determinant of the HIV-1 life cycle.

2014 - Genomic Analysis of Sleeping Beauty Transposon Integration in Human Somatic Cells [Articolo su rivista]
Turchiano, Giandomenico; Latella, Maria Carmela; Gogol Doring, Andreas; Cattoglio, Claudia; Mavilio, Fulvio; Izsvak, Zsuzsanna; Ivics, Zoltan; Recchia, Alessandra

The Sleeping Beauty (SB) transposon is a non-viral integrating vector system with proven efficacy for gene transfer and functional genomics. However, integration efficiency is negatively affected by the length of the transposon. To optimize the SB transposon machinery, the inverted repeats and the transposase gene underwent several modifications, resulting in the generation of the hyperactive SB100X transposase and of the high-capacity ‘‘sandwich’’ (SA) transposon. In this study, we report a side-by-side comparison of the SA and the widely used T2 arrangement of transposon vectors carrying increasing DNA cargoes, up to 18 kb. Clonal analysis of SA integrants in human epithelial cells and in immortalized keratinocytes demonstrates stability and integrity of the transposon independently from the cargo size and copy number-dependent expression of the cargo cassette. A genome-wide analysis of unambiguously mapped SA integrations in keratinocytes showed an almost random distribution, with an overrepresentation in repetitive elements (satellite, LINE and small RNAs) compared to a library representing insertions of the first-generation transposon vector and to gammaretroviral and lentiviral libraries. The SA transposon/SB100X integrating system therefore shows important features as a system for delivering large gene constructs for gene therapy applications

2014 - Long-Term Skin Regeneration From a Gene-Targeted Human Epidermal Stem Cell Clone [Articolo su rivista]
Duarte, Blanca; Miselli, Francesca; Murillas, Rodolfo; Espinosa Hevia, Luis; Cruz Cigudosa, Juan; Recchia, Alessandra; Del Río, Marcela; Larcher, Fernando

Ex vivo gene therapy is one of the current strategies being tested to treat genodermatoses such as epidermolysis bullosa (EB).1 In fact, Mavilio et al. proved the feasibility of this therapeutic modality in a patient with the junctional form of EB (JEB).2 Efforts are now being directed toward the development of efficient approaches minimizing potential genotoxic effects due to vector-induced insertional mutagenesis. Gene correction by gene editing through nucleasefacilitated homologous recombination (HR) has recently been proven to be achievable on recessive dystrophic EB cells that were subsequently reprogrammed to induced pluripotent stem cells (iPSCs) and differentiated to collagen VII–expressing keratinocytes.3 We have also demonstrated the feasibility of zinc-finger nuclease–facilitated, HR-mediated insertion of a marker gene into the intron 1 of the PPP1R12C gene (AAVS1 locus) in a limited number of human epidermal repopulating cells that, upon grafting, persisted as small foci in skin regenerated in immunodeficient mice.4 In this study we report that engraftment and persistent skin regeneration can be achieved with an expanded stem cell clone isolated from AAVS1 gene–targeted human keratinocytes.

2014 - Long-term stability and safety of transgenic cultured epidermal stem cells in gene therapy of junctional epidermolysis bullosa. [Articolo su rivista]
DE ROSA, Laura; Carulli, S; Cocchiarella, Fabienne; Quaglino, Daniela; Enzo, Elena; Franchini, Eleonora; Giannetti, A; DE SANTIS, Giorgio; Recchia, Alessandra; Pellegrini, Graziella; DE LUCA, Michele

We report a long-term follow-up (6.5 years) of a phase I/II clinical trial envisaging the use of autologous genetically modified cultured epidermal stem cells for gene therapy of junctional epidermolysis bullosa, a devastating genetic skin disease. The critical goals of the trial were to evaluate the safety and long-term persistence of genetically modified epidermis. A normal epidermal-dermal junction was restored and the regenerated transgenic epidermis was found to be fully functional and virtually indistinguishable from a normal control. The epidermis was sustained by a discrete number of long-lasting, self-renewing transgenic epidermal stem cells that maintained the memory of the donor site, whereas the vast majority of transduced transit-amplifying progenitors were lost within the first few months after grafting. These data pave the way for the safe use of epidermal stem cells in combined cell and gene therapy for genetic skin diseases.

2013 - Collagen VII gene delivery via Sleeping Beauty transposon in COL7A1-deficient keratinocytes from epidermolysis bullosa patients [Abstract in Atti di Convegno]
Latella, Maria Carmela; Cocchiarella, Fabienne; Turchiano, Giandomenico; Gonçalves, Manuel; Larcher, Fernando; Mavilio, Fulvio; Izsvak, Zsuzsanna; Ivics, Zoltan; Recchia, Alessandra

Autosomal recessive epidermolysis bullosa (RDEB) is a genetic skin adhesion defect caused by mutations in the type VII collagen gene (COL7A1). Although full-length type-VII collagen can be successfully produced in human keratinocytes following retroviral vector transduction, genetic instability due to the large size (9kb) and highly repetitive nature of the gene sequence remains problematic. The Sleeping Beauty (SB) transposon-based integration system can potentially overcome these issues by taking advantage of the hyperactive SB100X transposase in combination with the pT2 transposon..

2013 - Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells [Articolo su rivista]
Holkers, M; Maggio, I; Liu, J; Janssen, Jm; Miselli, F; Mussolino, C; Recchia, Alessandra; Cathomen, T; Gonçalves, Ma

The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 'safe harbor' locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform

2013 - Genotoxic signature in cord blood cells of newborns exposed in utero to a zidovudine-based antiretroviral combination [Articolo su rivista]
Isabelle Andre, Schmutz; Liliane Dal, Cortivo; Emmanuelle, Six; Sophie, Kaltenbach; Fabienne, Cocchiarella; Jerome Le, Chenadec; Nicolas, Cagnard; Anne Gael, Cordier; Alexandra, Benachi; Laurent, Mandelbrot; Elie, Azria; Naima, Bouallag; Sonia, Luce; Brigitte, Ternaux; Christian, Reimann; Patrick, Revy; Isabelle Radford, Weiss; Cristina, Leschi; Recchia, Alessandra; Mavilio, Fulvio; Marina Cavazzana, Calvo*; Stéphane, Blanche

Background: Zidovudine experimental genotoxicity has been established. Objective of the study was to identify genotoxicity markers in cord blood cells from newborns exposed in utero to ARV combinations containing zidovudine. Methods: Cells were investigated by karyotyping and gene expression analysis of the CD34+ hematopoietic stem/progenitor cell compartment (HPC). Results: Karyotyping of the cord blood cells from 15 ARV-exposed newborns and 12 controls revealed a higher proportion of aneuploid cells in the exposed group (median [IQR]: 18.8% [10.0-26.7] vs. 6.6 % [3.1-11.7], p<0.001). All chromosomes were involved with a random distribution of these alterations. Gene expression profiling of CD34+ HPCs from seven ARV-exposed and six control newborns revealed >300 genes significantly up- or down-regulated (p<0.05) by at least 1.5-fold in the exposed group. Significant alterations of genes involved in the control of cell cycle, mitotic check-points and DNA repair were identified. Although this study does not allow discrimination between the roles of each of the three drugs, both cytogenetic and transcriptional findings are similar to those in cellular experiments using zidovudine alone. Conclusion: The cord blood cells —including hematopoietic stem cells— from newborns exposed in utero to a zidovudine-based ARV combination present cytogenetic and transcriptional abnormalities compatible with DNA damage.

2013 - IL-7 and IL-15 instruct the generation of human memory stem T cells from naïve precursors [Articolo su rivista]
N., Cieri; B., Camisa; Cocchiarella, Fabienne; Forcato, Mattia; G., Oliveira; E., Provasi; A., Bondanza; C., Bordignon; J., Peccatori; F., Ciceri; M. T., Lupo Stanghellini; Mavilio, Fulvio; A., Mondino; Bicciato, Silvio; Recchia, Alessandra; C., Bonini

Long-living memory stem T cells (TSCM) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here we show that it is possible to differentiate in vitro, expand and gene modify in clinically compliant conditions CD8+ TSCM lymphocytes starting from naïve precursors. Requirements for the generation of this T-cell subset, described as CD62L+CCR7+CD45RA+CD45R0+IL- 7Rα+CD95+, are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly TSCM accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T lymphocyte subset, intermediate between naïve and central memory cells. When transplanted in immunodeficient mice, gene-modified naïve-derived TSCM prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GvHD. Furthermore, gene-modified TSCM are the only T-cell subset able to expand and mediate GvHD upon serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for TSCM generation and pave the way for their clinical rapid exploitation in adoptive cell therapy

2013 - Targeted Gene Addition in Human Epithelial Stem Cells by Zinc-finger Nuclease-mediated Homologous Recombination [Articolo su rivista]
Coluccio, Andrea; Miselli, Francesca; Angelo, Lombardo; Marconi, Alessandra; Guidantonio Malagoli, Tagliazucchi; Manuel A., Gonçalves; Pincelli, Carlo; Giulietta, Maruggi; Marcela Del, Rio; Luigi, Naldini; Fernando, Larcher; Mavilio, Fulvio; Recchia, Alessandra

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a “safe harbor” locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of >20% in a human keratinocyte cell line, >10% in immortalized keratinocytes, and <1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.

2013 - Targeted integration of a large transgene cassette by TALEN and ZFN-mediated homologous recombination. [Poster]
Marchetti, Marco; Miselli, Francesca; Holkers, Maarten; Mussolino, Claudio; Gonçalves, Manuel; Cathomen, Toni; Recchia, Alessandra

Targeted transgene integration by homologous recombination (HR) represents a promising strategy for gene therapy as it may overcome the issue of insertional mutagenesis associated with retroviral vectors. We recently published the feasibility of using adenoviral vectors (Ad) to package and deliver functional TALEN genes into human cells, demonstrating that Ad-TALEN-mediated transduction results in efficient site-specific DSB formation at the chromosomal safe harbor site AAVS1. Moreover, we demonstrated efficient targeting at AAVS1 in human repopulating epidermal stem cells upon Ad-ZFN cleavage.

2013 - Transcriptionally regulated and non-toxic delivery of the hyperactive Sleeping Beauty Transposase [Abstract in Atti di Convegno]
Cocchiarella, Fabienne; Latella, Maria Carmela; Basile, Valentina; Galla, Melanie; Imbriano, Carol; Ivics, Zoltan; Izsvak, Zsuzsanna; Recchia, Alessandra

The Sleeping Beauty (SB) transposase and, in particular, its hyperactive variant SB100X raised increasing interest for gene therapy application, genomic modification and, more recently, iPS reprogramming. The documented cytotoxicity of the transposase, when constitutively expressed by a gamma retroviral vector (iRV) has been circumvented by transduction of SB100X mRNA packaged into retrovirus particles (Galla et al. NAR 2011, Vol. 39, No. 16). In this study, we developed an alternative, safe and efficient transposase delivery system based on the tetracycline-ON regulatory system and on integrase defective lentiviral vectors (IDLV). The un-integrated and transcriptionally regulated expression of SB100X may become crucial in gene therapy and in iPS reprogramming, where permanent transposase expression could result in uncontrollable transposition. We generated two IDLV vectors, one carrying the rtTA2-M2 transactivator and the second bearing the tightly regulated, drug-inducible SB100X expression cassette.

2012 - Non viral gene transfer via Sleeping beauty transposon for Collagen VII delivery in human primary keratinocytes. [Poster]
Latella, Maria Carmela; Turchiano, Giandomenico; Cocchiarella, Fabienne; Izsvak, Zsuzsanna; Ivics, Zoltan; Mavilio, Fulvio; Recchia, Alessandra

Autosomal recessive epidermolysis bullosa (RDEB) is a genetic skin adhesion defect caused by mutations in the type VII collagen gene (COL7A1). Although full-length type-VII collagen is successfully produced in human keratinocytes by retroviral vectors, genetic instability due to the large size (9kb) and the highly repeated nature of the gene sequence is a persistent problem. The Sleeping-Beauty (SB) transposon-based integration system can potentially overcome these issues by taking advantage of the hyperactive SB100X transposase in combination with the wild-type (pT2) transposon or the “sandwich” version (pSA) that showed robust transposition efficiency in human cells. We molecularly characterized the “sandwich” SB-mediated integrants in epithelial cell lines and in primary keratinocytes. Co-transfecting the transposase together with 10kb-transposon (pT2 or pSA) we observed up to 37% of transposition in HaCaT and in GABEB (generalized atrophic benign epidermolysis bullosa keratinocytes) cells with both transposons. Clonal analysis demonstrated that the transposition events occur with a minimal risk of rearrangements (<3%). LM-PCR based bi-directional sequencing of the transposon-genome junctions shows genuine “cut and paste” activity of the SB hyperactive transposase.

2012 - Targeted gene integration in human epidermal stem cells by Zinc-finger nuclease-mediated homologous recombination. [Abstract in Atti di Convegno]
Coluccio, Andrea; Lombardo, Angelo; Miselli, Francesca; Holmes, Michael; Gregory, Philip D.; Naldini, Luigi; Recchia, Alessandra; Mavilio, Fulvio

Transplantation of autologous, genetically corrected epidermal stem cells (EpSC) is a potential treatment for junctional epidermolysis bullosa, a genetic skin adhesion disorder. Targeted transgene integration overcomes the issue of random insertional mutagenesis associated with retroviral vectors, and may thus provides a safer gene transfer alternative. We developed a gene-targeting platform based on the use of zinc-finger nucleases (ZFNs) and integrase-defective lentiviral vectors (IDLVs) to insert a transgene by homologous recombination (HR) into the AAVS1 locus on chromosome 19. We evaluated the targeting efficiency in a keratinocyte cell line (HaCaT) by IDLV-mediated delivery of an AAVS1-specific ZFN pair together with an HR construct driving the insertion of a GFP expression cassette into the site of cleavage. We achieved up to 25% of targeted insertion of single copies or concatamers of the GFP cassette into the AAVS1 locus, as analyzed by PCR, Southern blotting and sequencing on individual HaCaT cell clones. Evidence of HR-mediated targeted integration was also obtained at a lower but significant frequency in human primary keratinocyte cultures, by using ZFNs-expressing Adeno vector together with IDLV carrying the GFP expression cassette flanked by AAVS1 homology arms. We observed up to 9% disruption of the ZFN target site, repaired by non-homologous end joining, as evaluated by Cel-1 assay and pyrosequencing. These data suggest that the major limitation of ZFN-mediated targeted integration is represented by poor induction of the HR-dependent DNA repair pathway. To study gene targeting in repopulating keratinocyte stem cells, human skin equivalents derived from keratinocytes co-infected with IDLV donor and AdZFNs vectors were grafted onto immunodeficient (nu/nu) mice. GFP-positive spots were observed for at least 10 weeks in the grafted tissue, confirming that stable integration occurred in transplantable keratinocyte stem cells.

2011 - Defining the lentiviral integrome in human hematopoietic cells [Abstract in Atti di Convegno]
Recchia, Alessandra; Cattoglio, Claudia; Pellin, Danilo; Rizzi, Ermanno; Corti, Giorgio; Di Serio, Clelia; Malani, Nirav; Bushman, Frederick; De Bellis, Gianluca; Mavilio, Fulvio

Retroviral integration is a non-random process, whereby pre-integration complexes of different viruses recognize components or features of the host cell chromatin in a specific fashion. By using deep sequencing technology, we mapped >60,000 MLV and HIV integration sites in the genome of human CD34+ hematopoietic stem/progenitor cells and >16,000 sites in peripheral blood T-lymphocytes, and defined genome-wide integration maps in both cell types. MLV integrations cluster around regulatory elements (promoters, enhancers, and evolutionarily conserved non-coding regions) of genes involved in hematopoietic functions, and to chromatin regions bearing epigenetic marks of active or poised transcription. On the contrary, HIV integrations are clustered in regions marked by histone modifications associated to the body of transcribed genes (H3K36me3 and H2BK5me1), and are under-represented in regulatory regions. Although >90% of the genes targeted by HIV integration are expressed by Affymetrix analysis, expressed genes are not equally targeted. By a rigorous statistical analysis, we define a set of <400 genes that are targeted by HIV at significantly higher frequency than matched random controls after normalization for gene length, and a smaller set of genes that are targeted at significantly lower frequency. Functional clustering analysis shows that highly targeted genes are involved in chromatin remodeling and transcription, and are enriched in housekeeping functions. This analysis identifies a set of “high-risk” genes in hematopoietic cells, the function of which is more likely to be influenced by lentiviral vector integration in clinical gene therapy. Many of these genes are over-represented in collections of lentiviral vector integrations from patients treated by gene therapy, indicating that lentiviral “common integration sites” are determined by the HIV target site selection rather than clonal dominance in vivo.

2011 - Development of a Sleeping Beauty transposon-based integration system for gene transfer in human epithelial cells [Abstract in Atti di Convegno]
Turchiano, Giandomenico; Latella, Maria Carmela; Izsvak, Zsuzsanna; Ivics, Zoltan; Mavilio, Fulvio; Recchia, Alessandra

Transplantation of autologous, genetically corrected epidermal stem cells (EpSC) was successfully used to treat junctional epidermolysis bullosa (EB), a genetic skin adhesion disorder. The dystrophic forms of EB is caused by mutations in the type-VII collagen gene (COL7A1) Delivering the >9 kb COL7A1 cDNA by a retroviral vector is problematic, due to the large size and highly repeated nature of its sequence, which induce genetic rearrangements during reverse transcription and integration. We tested the feasibility of using a non-viral vector system based on Sleeping Beauty (SB)-derived transposons, taking advantages of the recently developed, high-capacity “sandwich” version of the SB transposon and the “hyperactive” SB 100X transposase, which showed high transposition efficiency in human stem cells. We tested the system in HeLa cells and in a keratinocyte cell line (HaCaT), which were co-transfected with the SB 100X transposase and either the normal or the sandwich version of the SB transposon containing a small-size reporter gene (Venus or GFP) expression cassette. In both cell lines, transposition was obtained in up to 80% of the transfected cells with the sandwich transposon, compared to ~50% obtained with the older version. Transposed HaCaT cells were cloned and analysed for integration events. Individual clones carried several copies of the integrated transposon of the predicted size. High-throughput sequencing is under way to analyze the sandwich SB transposon integration characteristics. We then tested a transposon carrying an 8.8-kb cassette, which again showed up to 80% transposition efficiency in transfected cells. Finally, we generated sandwich transposons containing an expression cassette for the COL7A1 cDNA under the control of a constitutive (PGK) or a keratinocyte-specific (K14) promoter, which are currently being tested for integration in HaCaT cells. The SB-based gene delivery system will finally be tested in human primary keratinocyte cultures.

2011 - Site-Specific Integration by the Adeno-associated Virus Rep Protein. [Articolo su rivista]
RECCHIA, Alessandra; MAVILIO, Fulvio

Inserting genetic information at precise locations into the human genome has been the goal of gene transfertechnology for almost two decades. The spectacular progress of mammalian genetics in the last two decades has led to thedevelopment of technology for genome editing and homologous recombination in human somatic cells that is finally approachingefficiency compatible with clinical application. Site-specific integration, or the insertion of genes at known locationsby enzymes with target recognition capacity, has progressed slowly but steadily in recent years, and could verywell be the basis of the next generation of gene transfer technology. This review focuses on the use of Rep, the replicase/integrase of the adeno-associated virus (AAV), to insert genes at the natural AAV integration site on human chromosome19. This region (AAVS1) has characteristics that make it an ideal target for somatic transgenesis

2010 - High-definition mapping of retroviral integration sites defines the fate of allogeneic T cells after donor lymphocyte infusion. [Articolo su rivista]
C., Cattoglio C; G., Maruggi; C., Bartholomae; N., Malani; D., Pellin; Cocchiarella, Fabienne; Z., Magnani; F., Ciceri; A., Ambrosi; C., von Kalle; Bushman, F. D.; C., Bonini; M., Schmidt; Mavilio, Fulvio; Recchia, Alessandra

The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34+ hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction.

2009 - Comprehensive genomic access to vector integration in clinical gene therapy. [Articolo su rivista]
Gabriel, R; Eckenberg, R; Paruzynski, A; Bartholomae, Cc; Nowrouzi, A; Arens, A; Howe, Sj; Recchia, Alessandra; Cattoglio, C; Wang, W; Faber, K; Schwarzwaelder, K; Kirsten, R; Deichmann, A; Ball, Cr; Balaggan, Ks; Yáñez Muñoz, Rj; Ali, Rr; Gaspar, Hb; Biasco, L; Aiuti, A; Cesana, D; Montini, E; Naldini, L; Cohen Haguenauer, O; Mavilio, Fulvio; Thrasher, Aj; Glimm, H; von Kalle, C; Saurin, W; Schmidt, M.

Retroviral vectors have induced subtle clonal skewing inmany gene therapy patients and severe clonal proliferationand leukemia in some of them, emphasizing the needfor comprehensive integration site analyses to assess thebiosafety and genomic pharmacokinetics of vectors andclonal fate of gene-modified cells in vivo. Integration siteanalyses such as linear amplification–mediated PCR(LAM-PCR) require a restriction digest generating unevenlysmall fragments of the genome. Here we show that eachrestriction motif allows for identification of only a fractionof all genomic integrants, hampering the understanding andprediction of biological consequences after vector insertion.We developed a model to define genomic access to theviral integration site that provides optimal restriction motifcombinations and minimizes the percentage of nonaccessibleinsertion loci. We introduce a new nonrestrictive LAM-PCRapproach that has superior capabilities for comprehensiveunbiased integration site retrieval in preclinical andclinical samples independent of restriction motifs andamplification inefficiency

2009 - Tracking gene-modified T cells in vivo. [Capitolo/Saggio]
Recchia, Alessandra; Mavilio, Fulvio

Identification, monitoring, and analysis of genetically modified cells in the peripheral blood are an important component of the clinical follow-up of patients treated by hematopoietic cell gene therapy. Analysis of gene-marked peripheral blood cells provides crucial information on gene transfer efficiency as well as on the nature and characteristics of the genetically modified cells, and may provide early evidence of the occurrence of potentially detrimental side effects. T lymphocytes are a convenient target for this type of analysis, due to their abundance and their relatively long life span in vivo. Tracking of gene-marked T cells is based on relatively simple, FACS- and PCR-based techniques, which may be applied to monitoring genetically modified T cells as well as T cells derived from transplanted, genetically modified hematopoietic stem cells. This chapter provides a description of these techniques and clues to their rational use in a clinical setting.

2009 - Transcription factor binding sites are genetic determinants of retroviral integration in the human genome. [Articolo su rivista]
Felice, B; Cattoglio, C; Cittaro, D; Testa, Anna; Miccio, Annarita; Ferrari, G; Luzi, L; Recchia, Alessandra; Mavilio, Fulvio

Gamma-retroviruses and lentiviruses integrate non-randomly in mammalian genomes, with specific preferences for active chromatin, promoters and regulatory regions. Gene transfer vectors derived from gamma-retroviruses target at high frequency genes involved in the control of growth, development and differentiation of the target cell, and may induce insertional tumors or pre-neoplastic clonal expansions in patients treated by gene therapy. The gene expression program of the target cell is apparently instrumental in directing gamma-retroviral integration, although the molecular basis of this phenomenon is poorly understood. We report a bioinformatic analysis of the distribution of transcription factor binding sites (TFBSs) flanking >4,000 integrated proviruses in human hematopoietic and non-hematopoietic cells. We show that gamma-retroviral, but not lentiviral vectors, integrate in genomic regions enriched in cell-type specific subsets of TFBSs, independently from their relative position with respect to genes and transcription start sites. Analysis of sequences flanking the integration sites of Moloney leukemia virus (MLV)- and human immunodeficiency virus (HIV)-derived vectors carrying mutations in their long terminal repeats (LTRs), and of HIV vectors packaged with an MLV integrase, indicates that the MLV integrase and LTR enhancer are the viral determinants of the selection of TFBS-rich regions in the genome. This study identifies TFBSs as differential genomic determinants of retroviral target site selection in the human genome, and suggests that transcription factors binding the LTR enhancer may synergize with the integrase in tethering retroviral pre-integration complexes to transcriptionally active regulatory regions. Our data indicate that gamma-retroviruses and lentiviruses have evolved dramatically different strategies to interact with the host cell chromatin, and predict a higher risk in using gamma-retroviral vs. lentiviral vectors for human gene therapy applications.

2009 - Transcriptional enhancers induce insertional gene deregulation independently from the vector type and design [Articolo su rivista]
Maruggi, G; Porcellini, S; Facchini, G; Perna, Sk; Cattoglio, C; Sartori, D; Ambrosi, A; Schambach, A; Baum, C; Bonini, C; Bovolenta, C; Mavilio, Fulvio; Recchia, Alessandra

The integration characteristics of retroviral (RV) vectorsincrease the probability of interfering with the regulationof cellular genes, and account for a tangible riskof insertional mutagenesis in treated patients. To assessthe potential genotoxic risk of conventional or self-inactivating(SIN) γ-RV and lentiviral (LV) vectors independentlyfrom the biological consequences of the insertionevent, we developed a quantitative assay based on realtimereverse transcriptase—PCR on low-density arraysto evaluate alterations of gene expression in individualprimary T-cell clones. We show that the Moloney leukemiavirus long terminal repeat (LTR) enhancer has thestrongest activity in both a γ-RV and a LV vector context,while an internal cellular promoter induces deregulationof gene expression less frequently, at a shorter range andto a lower extent in both vector types. Downregulationof gene expression was observed only in the context ofLV vectors.This study indicates that insertional gene activationis determined by the characteristics of the transcriptionalregulatory elements carried by the vector, andis largely independent from the vector type or design.

2008 - Absence of an intrathecal immune reaction to a helper-dependent adenoviral vector delivered into the cerebrospinal fluid of non-human primates. [Articolo su rivista]
Butti, E; Bergami, A; Recchia, Alessandra; Brambilla, E; Franciotta, D; Cattalini, A; Stornaiuolo, A; Lachapelle, F; Comi, G; Mavilio, Fulvio; Martino, G; Furlan, R.

Inflammation and immune reaction, or pre-existing immunity towards commonly used viral vectors for gene therapy severely impair long-term gene expression in the central nervous system (CNS), impeding the possibility to repeat the therapeutic intervention. Here, we show that injection of a helper-dependent adenoviral (HD-Ad) vector by lumbar puncture into the cerebrospinal fluid (CSF) of non-human primates allows long-term (three months) infection of neuroepithelial cells, also in monkeys bearing a pre-existing anti-adenoviral immunity. Intrathecal injection of the HD-Ad vector was not associated with any sign of systemic or local toxicity, nor by signs of a CNS-specific immune reaction towards the HD-Ad vector. Injection of HD-Ad vectors into the CSF circulation may thus represent a valuable approach for CNS gene therapy allowing for long-term expression and re-administration.

2008 - IL4 gene delivery to the CNS recruits regulatory T cells and induces clinical recovery in mouse models of multiple sclerosis [Articolo su rivista]
Butti, E; Bergami, A; Recchia, Alessandra; Brambilla, E; Del Carro, U; Amadio, S; Cattalini, A; Esposito, M; Stornaiuolo, A; Comi, G; Pluchino, S; Mavilio, Fulvio; Martino, G; Furlan, R.

Central nervous system (CNS) delivery of anti-inflammatory cytokines, such as interleukin 4 (IL4), holds promise as treatment for multiple sclerosis (MS). We have previously shown that short-term herpes simplex virus type 1-mediated IL4 gene therapy is able to inhibit experimental autoimmune encephalomyelitis (EAE), an animal model of MS, in mice and non-human primates. Here, we show that a single administration of an IL4-expressing helper-dependent adenoviral vector (HD-Ad) into the cerebrospinal fluid (CSF) circulation of immunocompetent mice allows persistent transduction of neuroepithelial cells and long-term (up to 5 months) CNS transgene expression without toxicity. Mice affected by chronic and relapsing EAE display clinical and neurophysiological recovery from the disease once injected with the IL4-expressing HD-Ad vector. The therapeutic effect is due to the ability of IL4 to increase, in inflamed CNS areas, chemokines (CCL1, CCL17 and CCL22) capable of recruiting regulatory T cells (CD4+CD69-CD25+Foxp3+) with suppressant functions. CSF delivery of HD-Ad vectors expressing anti-inflammatory molecules might represent a valuable therapeutic option for CNS inflammatory disorders.

2008 - Relative influence of the adeno-associated virus (AAV) type 2 p5 element for recombinant AAV vector site-specific integration. [Articolo su rivista]
Guilbaud, M; Chadeuf, G; Avolio, F; François, A; Moullier, P; Recchia, Alessandra; Salvetti, A.

The p5 promoter region of the adeno-associated virus type 2 (AAV-2) rep gene has been described as essential for Rep-mediated site-specific integration (RMSSI) of plasmid sequences in human chromosome 19. We report here that insertion of a full-length or minimal p5 element between the viral inverted terminal repeats does not significantly increase RMSSI of a recombinant AAV (rAAV) vector after infection of growth-arrested or proliferating human cells. This result suggests that the p5 element may not improve RMSSI of rAAV vectors in vivo.

2007 - Beta1 integrin activates Rac1 in Schwann cells to generate radial lamellae during axonal sorting and myelination [Articolo su rivista]
A., Nodari; D., Zambroni; A., Quattrini; Court, F. A.; A., D'Urso; Recchia, Alessandra; Vl, Tybulewicz; L., Wrabetz; Feltri, M. L.

Myelin is a multi-spiraled extension of glial membrane that surrounds axons.How glia extend a surface many-fold larger than their body is poorly understood.Schwann cells are peripheral glia and insert radial cytoplasmic extensions into bundles of axons in order to sort, ensheath and myelinate them. Laminins and β1integrins are required for axonal sorting, but the downstream signals are unknown.Here we show that Schwann cells devoid of β1 integrin migrate to and elongate on axons, but cannot extend radial lamellae of cytoplasm, similar to cells with low Rac1 activation. Accordingly, active Rac1 is decreased in β1 integrin-null nerves,inhibiting Rac1 activity decreases radial lamellae in Schwann cells, and ablating Rac1 in Schwann cells of transgenic mice delays axonal sorting and impairs myelination.Finally, expressing active Rac1 in β1 integrin-null nerves improves sorting. Thus, increase activation of Rac1 by β1 integrins allows Schwann cells to switch from migration/elongation to the extension of radial membranes required for axonal sorting and myelination.

2007 - Hot spots of retroviral integrations in human CD34+ hematopoietic cells [Articolo su rivista]
Cattoglio, C; Facchini, Giulia; Sartori, D; Antonelli, A; Miccio, Annarita; Cassani, B; Schmidt, M; VON KALLE, C; Howe, S; THRASHER A., J; Aiuti, A; Ferrari, G; Recchia, Alessandra; Mavilio, Fulvio

Insertional oncogenesis is a possible consequence of the integration of gamma-retroviral (RV) or lentiviral (LV) vectors into the human genome. RV common insertion sites (CISs) have been identified in hematopoietic malignancies and in the nonmalignant progeny of transduced hematopoietic stem/progenitor cells (HSCs), possibly as a consequence of clonal selection in vivo. We have mapped a large number of RV and LV integrations in human CD34+ HSCs, transduced in vitro and analyzed without selection. Recurrent insertion sites (hot spots) account for more than 21% of the RV integration events, while they are significantly less frequent in the case of LV vectors. RV but not LV hot spots are highly enriched in proto-oncogenes, cancer-associated CISs, and growth-controlling genes, indicating that at least part of the biases observed in the HSC progeny in vivo are characteristics of RV integration, already present in nontransplanted cells. Genes involved in hematopoietic and immune system development are targeted at high frequency and enriched in hot spots, suggesting that the CD34+ gene expression program is instrumental in directing RV integration. The lower propensity of LV vectors for integrating in potentially dangerous regions of the human genome may be a factor determining a better safety profile for gene therapy applications.

2007 - Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy [Articolo su rivista]
Aiuti, A; Cassani, B; Andolfi, G; Mirolo, M; Biasco, L; Recchia, Alessandra; Urbinati, Fabrizia; Valacca, C; Scaramuzza, S; Cazzola, M; Sartori, D; Ambrosi, A; DI SERIO, C; RONCAROLO M., G; Mavilio, Fulvio; AND BORDIGNON, C.

Gene transfer into HSCs is an effective treatment for SCID, although potentially limited by the risk of insertional mutagenesis. We performed a genome-wide analysis of retroviral vector integrations in genetically corrected HSCs and their multilineage progeny before and up to 47 months after transplantation into 5 patients with adenosine deaminase–deficient SCID. Gene-dense regions, promoters, and transcriptionally active genes were preferred retroviral integrations sites (RISs) both in preinfusion transduced CD34+ cells and in vivo after gene therapy. The occurrence of insertion sites proximal to protooncogenes or genes controlling cell growth and self renewal, including LMO2, was not associated with clonal selection or expansion in vivo. Clonal analysis of long-term repopulating cell progeny in vivo revealed highly polyclonal T cell populations and shared RISs among multiple lineages, demonstrating the engraftment of multipotent HSCs. These data have important implications for the biology of retroviral vectors, the dynamics of genetically modified HSCs, and the safety of gene therapy.

2006 - Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. [5YIF: 27.14; Citations: 298] [Articolo su rivista]
Mavilio, Fulvio; Pellegrini, Graziella; Ferrari, S.; DI NUNZIO, Francesca; Di Iorio, E.; Recchia, Alessandra; Maruggi, Giulietta; Ferrari, G.; Provasi, E.; Bonini, C.; Capurro, S.; Conti, A.; Magnoni, Cristina; Giannetti, Alberto; DE LUCA, Michele

The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-beta3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-beta3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.

2006 - Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells [Articolo su rivista]
Recchia, Alessandra; C., Bonini; Z., Magnani; Urbinati, Fabrizia; D., Sartori; S., Muraro; Tagliafico, Enrico; A., Bondanza; Mtl, Stanghellini; M., Bernardi; A., Pescarollo; F., Ciceri; C., Bordignon; Mavilio, Fulvio

The use of retroviral vectors in gene therapy has raised safety concerns for the genotoxic risk associated with their uncontrolled insertion into the human genome. We have analyzed the consequences of retroviral transduction in T cells from leukemic patients treated with allogeneic stem cell transplantation and donor lymphocytes genetically modified with a suicide gene (HSV-TK). Retroviral vectors integrate preferentially within or near transcribed regions of the genome, with a preference for sequences around promoters and for genes active in T cells at the time of transduction. Quantitative transcript analysis shows that one fifth of these integrations affect the expression of nearby genes. However, transduced T cell populations maintain remarkably stable gene expression profiles, phenotype, biological functions, and immune repertoire in vivo, with no evidence of clonal selection up to 9 yr after administration. Analysis of integrated proviruses in transduced cells before and after transplantation indicates that integrations interfering with normal T cell function are more likely to lead to clonal ablation than expansion in vivo. Despite the potentially dangerous interactions with the T cell genome, retroviral integration has therefore little consequence on the safety and efficacy of T cell transplantation.

2006 - Site-specific integration into the human genome: Ready for clinical application? [Articolo su rivista]
Recchia, Alessandra; Mavilio, Fulvio

Inserting genetic information at precise locations into the human genome has been the goal of the gene therapy community for almost two decades. Despite their spectacular progress in many fields of mammalian genetics, genome editing and homologous recombination are still too inefficient to be applied to human primary cells and tissues, the targets of any medical application. Site-specific integration, or the insertion of genes at known locations by enzymes that target recognition capacity, has progressed slowly but steadily in recent years, and could very well be the basis of the next generation of gene transfer technology.

2004 - Site-specific integration of functional transgenes into the human genome by adeno/AAV hybrid vectors [Articolo su rivista]
Recchia, Alessandra; L., Perani; D., Sartori; L., Olgiati; Mavilio, Fulvio

Uncontrolled insertion of gene transfer vectors into the human genome is raising significant safety concerns for their clinical use. The wild-type adeno-associated virus (AAV) can insert its genome at a specific site in human chromosome 19 (AAVS1) through the activity of a specific replicase/integrase protein (Rep) binding both the AAVS1 and the viral inverted terminal repeats (ITRs). AAV-derived vectors, however, do not carry the rep gene and cannot maintain site-specific integration properties. We describe a novel hybrid vector carrying an integration cassette flanked by AAV ITRs and a tightly regulated, drug-inducible Rep expression cassette in the framework of a high-capacity, helper-dependent adenoviral (Ad) vector. Rep-dependent integration of ITR-flanked cassettes of intact size and function was obtained in human primary cells and cell lines in the absence of selection. The majority of integrations were site specific and occurred within a 1000-bp region of the AAVS1. Genome-wide sequencing of integration junctions indicates that nonspecific integrations occurred predominantly in intergenic regions. Site-specific integration was obtained also in vivo, in an AAVS1 transgenic mouse model: upon a single tail vein administration of a nontoxic dose of Ad/AAV vectors, AAVS1-specific integrations were detected and sequenced in DNA obtained from the liver of all animals in which Rep expression was induced by drug treatment. Nonrandom integration of double-stranded DNA can therefore be obtained ex vivo and in vivo by the use of hybrid Ad/AAV vectors, in the absence of toxicity and with efficiency compatible with gene therapy applications.

2004 - The Journal of Gene Medicine European Society of Gene Therapy Young Investigator Award 2004 [Articolo su rivista]
Recchia, A.

1999 - Site-specific integration mediated by a hybrid adenovirus/adeno-associated virus vector [Articolo su rivista]
Recchia, Alessandra; R. J., Parks; S., Lamartina; C., Toniatti; L., Pieroni; F., Palombo; G., Ciliberto; R., Cortese; N. LA MONICA AND S., Colloca

Adenovirus (Ad) and adeno-associated virus(AAV) have attractive and complementary properties that canbe exploited for gene transfer purposes. Ad vectors are probablythe most efficient vehicles to deliver foreign genes both invitro and in vivo. AAV exhibits the unique ability to establishlatency by efficiently integrating at a specific locus of humanchromosome 19 (AAVS1). Two viral elements are necessaryfor the integration at AAVS1: Rep68y78 and the invertedterminal repeats (AAV-ITRs). In this study, we report thedevelopment of two helper-dependent adenoviral (HD) vectors,one carrying the Rep78 gene, the other an AAV-ITRflankedtransgene. Although Rep proteins have been demonstratedto interfere with Ad replication, HD Rep78 vector wassuccessfully amplified on serial passages in 293CRE4 cellswith a yield of 50–100 transducing units per cell. DNAintegration at the AAVS1 site also was demonstrated inhepatoma cells coinfected with the HD-expressing Rep78 andwith the second HD vector carrying a transgene flanked byAAV-ITRs. The high transduction efficiency, large cloningcapacity, and high titer of the HD, combined with the sitespecificintegration machinery provided by AAV-derived components,make the AdyAAV hybrid viruses a promising vehiclefor gene therapy

1998 - Site-specific integration in mammalian cells mediated by a new hybrid baculovirus-adeno-associated virus vector [Articolo su rivista]
F., Palombo; A., Monciotti; Recchia, Alessandra; R., Cortese; G. CILIBERTO AND N., LA MONICA

Baculovirus can transiently transduce primary human and rat hepatocytes, as well as a subset of stable celllines. To prolong transgene expression, we have developed new hybrid vectors which associate key elementsfrom adeno-associated virus (AAV) with the elevated transducing capacity of baculovirus. The hybrid vectorscontain a transgene cassette composed of the !-galactosidase (!-Gal) reporter gene and the hygromycin resistance(Hygr) gene flanked by the AAV inverted terminal repeats (ITRs), which are necessary for AAV replicationand integration in the host genome. Constructs were derived both with and without the AAV rep geneunder the p5 and p19 promoters cloned in different positions with respect to the baculovirus polyheidrinpromoter. A high-titer preparation of baculovirus-AAV (Bac-AAV) chimeric virus containing the ITR–Hygr–!-Gal sequence was obtained with insect cells only when the rep gene was placed in an antisense orientationto the polyheidrin promoter. Infection of 293 cells with Bac-AAV virus expressing the rep gene results in a 10-to 50-fold increase in the number of Hygr stable cell clones. Additionally, rep expression determined the localizationof the transgene cassette in the aavs1 site in approximately 41% of cases as detected by bothSouthern blotting and fluorescent in situ hybridization analysis. Moreover, site-specific integration of the ITRflankedDNA was also detected by PCR amplification of the ITR-aavs1 junction in transduced human fibroblasts.These data indicate that Bac-AAV hybrid vectors can allow permanent, nontoxic gene delivery of DNAconstructs for ex vivo treatment of primary human cells.