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Antonia Follenzi

Publications and source records attributed to Antonia Follenzi.

At least 19 recordsLinked to original sources
PubMed2006

Ex vivo gene therapy with lentiviral vectors rescues adenosine deaminase (ADA)-deficient mice and corrects their immune and metabolic defects.

Adenosine deaminase (ADA) deficiency is caused by a purine metabolic dysfunction, leading to severe combined immunodeficiency (SCID) and multiple organ damage. To investigate the efficacy of ex vivo gene therapy with self-inactivating lentiviral vectors (LVs) in correcting this complex phenotype, we used an ADA(-/-) mouse model characterized by early postnatal lethality. LV-mediated ADA gene transfer into bone marrow cells combined with low-dose irradiation rescued mice from lethality and restored their growth, as did transplantation of wild-type bone marrow. Mixed chimerism with multilineage engraftment of transduced cells was detected in the long term in animals that underwent transplantation. ADA activity was normalized in lymphocytes and partially corrected in red blood cells (RBCs), resulting in full metabolic detoxification and prevention of severe pulmonary insufficiency. Moreover, gene therapy restored normal lymphoid differentiation and immune functions, including antigen-specific antibody production. Similar degrees of detoxification and immune reconstitution were obtained in mice treated early after birth or after 1 month of enzyme-replacement therapy, mimicking 2 potential applications for ADA-SCID. Overall, this study demonstrates the efficacy of LV gene transfer in correcting both the immunological and metabolic phenotypes of ADA-SCID and supports the future clinical use of this approach.

Adenosine Deaminase↗
PubMed2005

Treatment of the mouse model of mucopolysaccharidosis type IIIB with lentiviral-NAGLU vector.

The Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is an autosomal recessive disorder due to mutations in the gene encoding NAGLU (alpha-N-acetylglucosaminidase), one of the enzymes required for the degradation of the GAG (glycosaminoglycan) heparan sulphate. No therapy exists for affected patients. We have shown previously the efficacy of lentiviral-NAGLU-mediated gene transfer in correcting in vitro the defect on fibroblasts of patients. In the present study, we tested the therapy in vivo on a knockout mouse model using intravenous injections. Mice (8-10 weeks old) were injected with one of the lentiviral doses through the tail vein and analysed 1 month after treatment. A single injection of lentiviral-NAGLU vector resulted in transgene expression in liver, spleen, lung and heart of treated mice, with the highest level reached in liver and spleen. Expression of 1% normal NAGLU activity in liver resulted in a 77% decrease in the GAG content; more remarkably, an expression of 0.16% normal activity in lung was capable of decreasing the GAG level by 29%. Long-term (6 months) follow up of the gene therapy revealed that the viral genome integration persisted in the target tissues, although the real-time PCR analysis showed a decrease in the vector DNA content with time. Interestingly, the decrease in GAG levels was maintained in liver, spleen, lung and heart of treated mice. These results show the promising potential and the limitations of lentiviral-NAGLU vector to deliver the human NAGLU gene in vivo.

Acetylglucosaminidase↗
PubMed2005

The MET oncogene drives a genetic programme linking cancer to haemostasis.

The close relationship between activation of blood coagulation and cancer is an old enigma. In 1865, migrans trombophlebitis ('a condition of the blood that predisposes it to spontaneous coagulation') was described as a forewarning of occult malignancy (Trousseau's sign). This pioneering observation emphasized the existence of haemostasis disorders associated with cancer onset; this phenomenon has since been extensively reported in clinical and epidemiological studies, but has so far resisted a mechanistic explanation. Here we report a mouse model of sporadic tumorigenesis based on genetic manipulation of somatic cells. Targeting the activated, human MET oncogene to adult liver caused slowly progressing hepatocarcinogenesis. This was preceded and accompanied by a syndrome manifesting first with blood hypercoagulation (venous thromboses), and then evolving towards fatal internal haemorrhages. The pathogenesis of this syndrome is driven by the transcriptional response to the oncogene, including prominent upregulation of plasminogen activator inhibitor type 1 (PAI-1) and cyclooxygenase-2 (COX-2) genes. In vivo analysis showed that both proteins support the thrombohaemorrhagic phenotype, thus providing direct genetic evidence for the long-sought-after link between oncogene activation and haemostasis.

Animals↗
PubMed2005

Hepatic targeting of transplanted liver sinusoidal endothelial cells in intact mice.

Targeting of cells to specific tissues is critical for cell therapy. To study endothelial cell targeting, we isolated mouse liver sinusoidal endothelial cells (LSEC) and examined cell biodistributions in animals. To identify transplanted LSEC in tissues, we labeled cells metabolically with DiI-conjugated acetylated low density lipoprotein particles (DiI-Ac-LDL) or (111)Indium-oxine, used LSEC from Rosa26 donors expressing beta-galactosidase or Tie-2-GFP donors with green fluorescent protein (GFP) expression, and tranduced LSEC with a GFP-lentiviral vector. LSEC efficiently incorporated (111)Indium and DiI-Ac-LDL and expressed GFP introduced by the lentiviral vector. Use of radiolabeled LSEC showed differences in cell biodistributions in relation to the cell transplantation route. After intraportal injection, LSEC were largely in the liver (60 +/- 13%) and, after systemic intravenous injection, in lungs (67 +/- 9%); however, after intrasplenic injection, only some LSEC remained in the spleen (29 +/- 10%; P < .01), whereas most LSEC migrated to the liver or lungs. Transplanted LSEC were found in the liver, lungs, and spleen shortly after transplantation, whereas longer-term cell survival was observed only in the liver. Transplanted LSEC were distinct from Kupffer cells with expression of Tie-2 promoter-driven GFP and of CD31, without F4/80 reactivity. In further studies using radiolabeled LSEC, we established that the manipulation of receptor-mediated cell adhesion in liver sinusoids or the manipulation of blood flow-dependent cell exit from sinusoids improved intrahepatic retention of LSEC to 89 +/- 7% and 89 +/- 5%, respectively (P < .01). In conclusion, the targeting of LSEC to the liver and other organs is directed by vascular bed-specific mechanisms, including blood flow-related processes, and cell-specific factors. These findings may facilitate analysis of LSEC for cell and gene therapy applications.

Animals↗
PubMed2005

Efficient Tet-dependent expression of human factor IX in vivo by a new self-regulating lentiviral vector.

Regulation of gene expression represents a long-sought goal of gene therapy. However, most viral vectors pose constraints on the incorporation of drug-dependent transcriptional regulatory systems. Here, by optimizing the design of self-regulating lentiviral vectors based on the tetracycline system, we have been able to overcome the limitations of previously reported constructs and to reach both robust expression and efficient regulation from a single vector. The improved performance allows us to report for the first time effective long-term in vivo regulation of a human clotting Factor IX (hF.IX) transgene upon systemic administration of a single vector to SCID mice. We showed that hF.IX expression in the plasma could be expressed to therapeutically significant concentrations, adjusted to different set levels by varying the tetracycline dose, rapidly turned off and on, and completely recovered after each treatment cycle. The new vector design was versatile, as it successfully incorporated a tissue-specific promoter that selectively targeted regulated expression to hepatocytes. Robust transgene expression in the systemic circulation coupled to the ability to switch off and even adjust the expression level may open the way to safer gene-based delivery of therapeutics.

Animals↗
PubMed2005

Axons mediate the distribution of arylsulfatase A within the mouse hippocampus upon gene delivery.

Axonal transport of the lysosomal enzyme arylsulfatase A (ARSA) may be an additional mechanism of enzyme distribution after in vivo brain gene transfer in an animal model of metachromatic leukodystrophy (MLD). Direct molecular demonstration of the movement of this lysosomal enzyme within axonal networks was missing. We generated lentiviral vectors carrying the ARSA cDNA tagged with hemagglutinin or the green fluorescent protein and examined the subcellular localization and anatomical distribution of the tagged enzymes within the MLD hippocampus after in vivo lentiviral gene transfer. The use of tagged ARSA allowed direct real-time observation and tracking of axon-dendritic transport of the enzyme after lentiviral gene therapy. Tagged ARSA was expressed in transduced pyramidal, granule, and hilar neurons within the lentiviral-injected side and was robustly contained in vesicles within ipsilateral axon-dendritic processes as well as in vesicles associated with contralateral axons and commissural axons of the ventral hippocampal commissure. Axonal transport of tagged ARSA led to the correction of hippocampal defects in long-term treated MLD mice, which was accompanied by enzyme uptake in nontransduced contralateral neurons, enzyme accumulation within the lysosomal compartment, and clearance of sulfatide storage deposits in this region of the MLD brain. These results contribute to the understanding of the mechanisms of distribution of lysosomal enzymes within the mammalian brain after direct gene therapy, demonstrating the use of neural processes for enzyme transport.

Animals↗
PubMed2005

RNAi technology and lentiviral delivery as a powerful tool to suppress Tpr-Met-mediated tumorigenesis.

Tpr-Met, the oncogenic counterpart of the Met receptor, has been detected in gastric cancers, as well as in precursor lesions and in the adjacent normal gastric mucosa. This has prompted the suggestion that Tpr-Met may predispose to the development of gastric tumors. Given the sequence specificity of RNA interference, oncogenes activated by point mutation or rearrangements can be targeted while spearing the product of the wild-type allele. In this work, we report specific suppression of Tpr-Met expression and inhibition of Tpr-Met-mediated transformation and tumorigenesis by means of a short interfering RNA (siRNA) directed toward the Tpr-Met junction (anti-TM2). When delivered by a lentiviral vector, anti-TM2 siRNA was effective also in mouse embryonal fibroblasts or epithelial cells expressing high levels of Tpr-Met. Our results suggest that lentiviral-mediated delivery of anti-TM2 siRNA may be developed into a powerful tool to treat Tpr-Met-positive cancers.

Animals↗
PubMed2005

Lentiviral transduction of primary myeloma cells with CD80 and CD154 generates antimyeloma effector T cells.

The development of immunotherapy approaches designed to obtain tumor-specific T cells might help eradicate residual malignant cells in multiple myeloma (MM) patients. To this end, we used autologous primary MM cells as antigen-presenting cells (APC). Gene transfer of both CD80 and CD154 by lentiviral vectors was necessary to significantly improve the APC function of human MM cells. Simultaneous CD80/CD154 expression on MM cells allowed the generation of CD8+ T cells that recognized unmodified MM cells in 11 of 16 cases, specifically in six of six patients with low-stage disease, but only in five of ten patients with advanced disease. The activity of CD8+ T cells was MHC restricted and MM specific. In seven of seven cases, CD8+ T cell activity was inhibited by monoclonal antibodies against HLA class I, and in four of four cases, CD8+ T cells recognized autologous MM cells but not autologous normal B and T lymphocytes nor bone marrow stromal cells. In addition, the activity of CD8+ T cells was directed against allogeneic MM cells that shared at least one MHC allele with the autologous counterpart, but not against MHC mismatched MM cells. These data lay the ground for the isolation of new MM antigens and for the design of vaccination protocols with primary MM cells genetically engineered to express immunostimulatory molecules.

Antigen-Presenting Cells↗
PubMed2005

Stability of lentiviral vector-mediated transgene expression in the brain in the presence of systemic antivector immune responses.

Lentiviral vectors are promising tools for gene therapy in the CNS. It is therefore important to characterize their interactions with the immune system in the CNS. This work characterizes transgene expression and brain inflammation in the presence or absence of immune responses generated after systemic immunization with lentiviral vectors. We characterized transduction with SIN-LV vectors in the CNS. A dose-response curve using SIN-LV-GFP demonstrated detectable transgene expression in the striatum at a dose of 10(2), and maximum expression at 10(6), transducing units of lentiviral vector, with minimal increase in inflammatory markers between the lowest and highest dose of vector injected. Our studies demonstrate that injection of a lentiviral vector into the CNS did not cause a measurable inflammatory response. Systemic immunization after CNS injection, with the lentiviral vector expressing the same transgene as a vector injected into the CNS, caused a decrease in transgene expression in the CNS, concomitantly with an infiltration of inflammatory cells into the CNS parenchyma at the injection site. However, peripheral immunization with a lentiviral vector carrying a different transgene did not diminish transgene expression, or cause CNS inflammation. Systemic immunization preceding injection of lentiviral vectors into the CNS determined that preexisting antilentiviral immunity, regardless of the transgene, did not affect transgene expression. Furthermore, we showed that the transgene, but not the virion or vector components, is responsible for providing antigenic epitopes to the activated immune system, on systemic immunization with lentivirus. Low immunogenicity and prolonged transgene expression in the presence of preexisting lentiviral immunity are encouraging data for the future use of lentiviral vectors in CNS gene therapy. In summary, the lentiviral vectors tested induced undetectable activation of innate immune responses, and stimulation of adaptive immune responses against lentiviral vectors was effective in causing a decrease in transgene expression only if the immune response was directed against the transgene. A systemic immune response against vector components alone did not cause brain inflammation, possibly because vector-derived epitopes were not being presented in the CNS.

Animals↗
PubMed2004

Robust in vivo gene transfer into adult mammalian neural stem cells by lentiviral vectors.

Stable genetic modification of adult stem cells is fundamental for both developmental studies and therapeutic purposes. Using in vivo marking studies, we showed that injection of lentiviral vectors (LVs) into the subventricular zone of the adult mouse brain enables efficient gene transfer into long-term self-renewing neural precursors and steady, robust vector expression in their neuronal progeny throughout the subventricular zone and its rostral extension, up to the olfactory bulb. By clonal and population analysis in culture, we proved that in vivo-marked neural precursors display self-renewal and multipotency, two essential characteristics of neural stem cells (NSCs). Thus, LVs efficiently target long-term repopulating adult NSCs, and the effect of the initial transduction is amplified by the continuous generation of NSC-derived, transduced progeny. LVs may thus allow novel studies on NSCs' physiology in vivo, and introduction of therapeutic genes into NSCs may allow the development of novel approaches for untreatable CNS diseases.

Animals↗
PubMed2004

Lentiviral vector-mediated gene transfer in T cells from Wiskott-Aldrich syndrome patients leads to functional correction.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with a median survival below the age of 20 due to infections, severe hemorrhage, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but is available for a minority of patients. Transplantation of genetically corrected autologous hematopoietic stem cells or T cells could represent an alternative treatment applicable to all patients. We investigated whether WAS gene transfer with MMLV-based oncoretroviral and HIV-based lentiviral vectors could restore normal functions of patients' T cells. T cells transduced either with lentiviral vectors expressing the WAS protein (WASP) from the ubiquitous PGK promoter or the tissue-specific WASP promoter or with an oncoretroviral vector expressing WASP from the LTR, reached normal levels of WASP with correction of functional defects, including proliferation, IL-2 production, and lipid raft upregulation. Lentiviral vectors transduced T cells from WAS patients at higher rates, compared to oncoretroviral vectors, and efficiently transduced both activated and naive WAS T cells. Furthermore, a selective growth advantage of T cells corrected with the lentiviral vectors was demonstrated. The observation that lentiviral vector-mediated gene transfer results in correction of T cell defects in vitro supports their application for gene therapy in WAS patients.

Antibodies↗
PubMed2003

Targeting lentiviral vector expression to hepatocytes limits transgene-specific immune response and establishes long-term expression of human antihemophilic factor IX in mice.

Stable gene replacement by in vivo administration of lentiviral vectors (LVs) has therapeutic potential for metabolic disorders and other systemic diseases. We studied the expression of intracellular and secreted proteins by LVs in immunocompetent mice. Liver, spleen, and bone marrow cells were efficiently transduced. However, transgene expression, driven by a ubiquitous promoter, was limited by transgene-specific cellular and humoral immune responses, leading to the clearance of transduced cells. After green fluorescent protein (GFP) gene transfer, the liver showed infiltration of CD8(+) cytotoxic T cells, and GFP-specific CD8(+) T cells were isolated from the spleen. After human factor IX (hF.IX) gene transfer, anti-hF.IX antibodies were induced. These immune responses were not detected in mice injected with heat-inactivated or genome-lacking LVs or in GFP-transgenic mice, indicating that they were specifically triggered by transgene expression in vivo. Intriguingly, selective targeting of LV expression to hepatocytes limited the immune responses to the transgenes. By this approach, high levels of hF.IX, potentially in the therapeutic range, were reached and maintained long term in immunocompetent mice, without inducing antibody formation. These results prompt further studies in relevant animal models to explore the potential of in vivo LV administration for the gene therapy of hemophilias and other liver-based diseases.

Animals↗
PubMed2003

Deletion in a (T)8 microsatellite abrogates expression regulation by 3'-UTR.

A high level of genetic instability might cause mutations to accumulate in tumours. Microsatellite instability (MSI), due to defects of the DNA mismatch repair system, affects in particular repeat sequences (microsatellites) scattered throughout the genome. By scanning transcriptome databases, we found that microsatellites in the human genome are less numerous in coding DNA than in the 3'-untranslated region (UTR), known to mediate control of gene expression. By mutation analysis, we identified a 1 bp deletion in a (T)(8) microsatellite embedded in the 1801 nucleotide long 3'-UTR of CEACAM1 gene, thought to be involved in tumour onset and progression. By Lentiviral Vector- mediated gene transfer, we showed that the wild-type but not the mutated CEACAM1 3'-UTR greatly decreased transgene expression at both mRNA and protein level. Messenger RNA abundance was fully regulated by the most 3' region of CEACAM1 3'-UTR. This region includes the (T)(8) microsatellite but not any known classified regulatory element. These data show that CEACAM1 3'-UTR contains non-canonical elements contributing to mRNA regulation, among which a short repeat sequence could play a critical regulatory function. This suggests that, in cancer cells, a single mutation in a 3'-UTR short microsatellite might strongly affect gene expression.

3' Untranslated Regions↗
PubMed2003

Efficiency of onco-retroviral and lentiviral gene transfer into primary mouse and human B-lymphocytes is pseudotype dependent.

B lymphocytes are attractive targets for gene therapy of genetic diseases associated with B-cell dysfunction and for immunotherapy. Transduction of B lymphocytes was evaluated using green fluorescent protein (GFP)-encoding onco-retroviral and HIV-derived lentiviral vectors which were pseudotyped with ecotropic, amphotropic or vesicular stomatitis virus (VSV-G) envelopes. Transduction of mouse B lymphocytes activated with lipopolysaccharides (LPS) or by cross-linking CD40 in conjunction with interleukin-4 (IL-4) was significantly more efficient (p < 0.003) with ecotropic (11%) than with VSV-G pseudotyped onco-retroviral vectors (1%). Using high-titer cell-free ecotropic viral supernatant or by coculture with ecotropic onco-retroviral vector-producing cells, transduction efficiency increased significantly (p < 0.001) to approximately 50%, whereas transduction efficiency by coculture with VSV-G pseudotyped vector-producing cells remained low (< 2%). Similarly, transduction of mouse B lymphocytes was significantly more efficient (twofold, p < 0.01) with the ecotropic (7%) than with the VSV-G pseudotyped lentiviral vectors although gene transfer efficiency remained low because of dose-limiting toxicity of the concentrated vector preparations on the LPS-activated murine B cells. Consistent with murine B-cell transduction, human B cells activated with CD40L and IL-4 were also found to be relatively refractory to VSV-G pseudotyped onco-retroviral vectors (< 1%). However, higher transduction efficiencies could be achieved in activated primary human B lymphocytes using VSV-G pseudotyped lentiviral vectors instead (5%-6%). Contrary to the significant increase in mouse B-cell transduction efficiency with ecotropic vectors, the use of amphotropic onco-retroviral or lentiviral vectors did not increase transduction efficiency in primary human B cells. The present study shows that the transduction efficiency of onco-retroviral and lentiviral vectors in human and mouse B lymphocytes is pseudotype-dependent and challenges the widely held assumption that VSV-G pseudotyping facilitates gene transfer into all cell types.

Animals↗
PubMed2002

A human immunodeficiency virus type 1 pol gene-derived sequence (cPPT/CTS) increases the efficiency of transduction of human nondividing monocytes and T lymphocytes by lentiviral vectors.

We have investigated the capacity of two human immunodeficiency virus type 1-derived lentivectors, differing in the presence of a 118-bp pol fragment containing the cPPT/CTS element, to transduce human normal primary cells of different hematopoietic lineages. Infection of resting monocytes with a high multiplicity of infection (MOI > 10) revealed that the lentivirus carrying the pol fragment (cPPT) is effective, transducing 75% of cells compared with 36% for the no-cPPT vector. Even at low MOIs (< or =1) the cPPT vector still shows a better transduction efficiency than the no-cPPT vector. Moreover, transduction does not require dendritic cell differentiation. In contrast, infection of nonactivated T lymphocytes showed that both vectors, tested at high MOIs, can transduce a small, although measurable, percentage of cells (up to 10%), which may correspond to G(1a) "activated" cells as detected by simultaneous staining of DNA and RNA, in our cultures in the presence of medium alone. Furthermore, we show that the sole addition of interleukin 2 or interleukin 15 represents a full proliferative signal under our conditions and permits high transduction efficiency (up to 30% with the cPPT vector and 15% with the no-cPPT vector). Still higher transduction of T lymphocytes can be achieved after stimulation with phytohemagglutinin and interleukin 2 (up to 78% with the cPPT vector vs. 42% with the no-cPPT vector). Finally, both viruses do not transduce either resting or proliferating tonsillar B lymphocytes.

B-Lymphocytes↗
PubMed2002

Lentiviral vectors containing the human immunodeficiency virus type-1 central polypurine tract can efficiently transduce nondividing hepatocytes and antigen-presenting cells in vivo.

High-titer self-inactivating human immunodeficiency virus type-1 (HIV-1)-based vectors expressing the green fluorescent protein reporter gene that contained the central polypurine and termination tract and the woodchuck hepatitis virus posttranscriptional regulatory element were constructed. Transduction efficiency and biodistribution were determined, following systemic administration of these improved lentiviral vectors. In adult severe combined immunodeficiency (SCID) mice, efficient stable gene transfer was achieved in the liver (8.0% +/- 6.0%) and spleen (24% +/- 3%). Most transduced hepatocytes and nonhepatocytes were nondividing, thereby obviating the need to induce liver cell proliferation. In vivo gene transfer with this improved lentiviral vector was relatively safe since liver enzyme concentration in the plasma was only moderately and transiently elevated. In addition, nondividing major histocompatibility complex class II-positive splenic antigen-presenting cells (APCs) were efficiently transduced in SCID and normal mice. Furthermore, B cells were efficiently transduced, whereas T cells were refractory to lentiviral transduction in vivo. However, in neonatal recipients, lentiviral transduction was more widespread and included not only hepatocytes and splenic APCs but also cardiomyocytes. The present study suggests potential uses of improved lentiviral vectors for gene therapy of genetic blood disorders resulting from serum protein deficiencies, such as hemophilia, and hepatic disease. However, the use of liver-specific promoters may be warranted to circumvent inadvertent transgene expression in APCs. In addition, these improved lentiviral vectors could potentially be useful for genetic vaccination and treatment of perinatal cardiac disorders.

Animals↗
PubMed2002

Correction of mucopolysaccharidosis type IIIb fibroblasts by lentiviral vector-mediated gene transfer.

Mucopolysaccharidosis type IIIB (MPS IIIB; or Sanfilippo syndrome type B) is a lysosomal disease, due to glycosaminoglycan storage caused by mutations on the alpha-N-acetylglucosaminidase (NAGLU) gene. The disease is characterized by neurological dysfunction but relatively mild somatic manifestations. No effective treatment is available for affected patients. In the present study, we evaluated the role of a lentiviral vector as the transducing agent of NAGLU cDNA in MPS IIIB fibroblasts. The vector expressed high transduction efficiency and high levels of enzymic activity, 20-fold above normal levels, persisting for at least 2 months. PCR experiments confirmed the integration of the viral vector into the target genome. The NAGLU activity restored by virus infection was sufficient to normalize glycosaminoglycan accumulation, which is directly responsible for the disease phenotype. Metabolic labelling experiments on transduced fibroblasts exhibited, in the medium and in cellular lysates, polypeptide forms of 84 and 80 kDa respectively related to the precursor and mature forms of the enzyme. The enzyme secreted by transduced MPS IIIB fibroblasts was endocytosed in deficient cells by the mannose 6-phosphate system. Thus we show that lentiviral vectors may provide a therapeutic approach for the treatment of MPS IIIB disease.

Acetylglucosaminidase↗