Publications by authors named "Chiara Bovolenta"

20 Publications

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Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1.

Sci Transl Med 2017 Oct;9(411)

San Raffaele Telethon Institute for Gene Therapy, 20132 Milan, Italy.

Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the -edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases.
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http://dx.doi.org/10.1126/scitranslmed.aan0820DOI Listing
October 2017

Vectofusin-1 Promotes RD114-TR-Pseudotyped Lentiviral Vector Transduction of Human HSPCs and T Lymphocytes.

Mol Ther Methods Clin Dev 2017 Jun 8;5:22-30. Epub 2017 Mar 8.

MolMed S.p.A., 20132 Milan, Italy.

Ex vivo transduction of human CD34 hematopoietic stem/progenitor cells (hCD34 HSPCs) and T lymphocytes is a key process that requires high efficiency and low toxicity to achieve effective clinical results. So far, several enhancers have been used to improve this process. Among them, Retronectin highly meliorates VSV-G and RD114-TR pseudotyped lentiviral vector delivery in hCD34 HSPCs and T lymphocytes. However, Retronectin is expensive and requires pre-coating of culture dishes or bags before cell seeding, resulting in a cumbersome procedure. Recently, an alternative transduction adjuvant has been developed, named Vectofusin-1, whose effect has been demonstrated on gene delivery to cell lines and primary hCD34 HSPCs by lentiviral vectors pseudotyped with different envelope glycoproteins. In this study, we have focused our analysis on the effect of Vectofusin-1 on the transduction of hCD34 HSPCs and T lymphocytes by using mostly RD114-TR pseudotyped lentivectors and clinical transduction protocols. Here, we have proved that Vectofusin-1 reproducibly enhances gene delivery to hCD34 HSPCs and activated T cells without cell toxicity and with efficacy comparable to that of Retronectin. The use of Vectofusin-1 will therefore help to shorten and simplify clinical cell manipulation, especially if automated systems are planned for transducing large-scale clinical lots.
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http://dx.doi.org/10.1016/j.omtm.2017.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5415310PMC
June 2017

Codon Optimization Leads to Functional Impairment of RD114-TR Envelope Glycoprotein.

Mol Ther Methods Clin Dev 2017 Mar 11;4:102-114. Epub 2017 Jan 11.

MolMed S.p.A., 20132 Milano, Italy.

Lentiviral vectors (LVs) are a highly valuable tool for gene transfer currently exploited in basic, applied, and clinical studies. Their optimization is therefore very important for the field of vectorology and gene therapy. A key molecule for LV function is the envelope because it guides cell entry. The most commonly used in transiently produced LVs is the vesicular stomatitis virus glycoprotein (VSV-G) envelope, whose continuous expression is, however, toxic for stable LV producer cells. In contrast, the feline endogenous retroviral RD114-TR envelope is suitable for stable LV manufacturing, being well tolerated by producer cells under constitutive expression. We have previously reported successful, transient and stable production of LVs pseudotyped with RD114-TR for good transduction of T lymphocytes and CD34 cells. To further improve RD114-TR-pseudotyped LV cell entry by increasing envelope expression, we codon-optimized the RD114-TR open reading frame (ORF). Here we show that, despite the RD114-TRco precursor being produced at a higher level than the wild-type counterpart, it is unexpectedly not duly glycosylated, exported to the cytosol, and processed. Correct cleavage of the precursor in the functional surface and transmembrane subunits is prevented in vivo, and, consequently, the unprocessed precursor is incorporated into LVs, making them inactive.
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http://dx.doi.org/10.1016/j.omtm.2017.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363313PMC
March 2017

Lentiviral Vector Gene Therapy Protects XCGD Mice From Acute Staphylococcus aureus Pneumonia and Inflammatory Response.

Mol Ther 2016 10 26;24(10):1873-1880. Epub 2016 Jul 26.

San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), San Raffaele Scientific Institute, Milan, Italy.

Chronic granulomatous disease (CGD) is a primary immunodeficiency due to a deficiency in one of the subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. CGD patients are characterized by an increased susceptibility to bacterial and fungal infections, and to granuloma formation due to the excessive inflammatory responses. Several gene therapy approaches with lentiviral vectors have been proposed but there is a lack of in vivo data on the ability to control infections and inflammation. We set up a mouse model of acute infection that closely mimic the airway infection in CGD patients. It involved an intratracheal injection of a methicillin-sensitive reference strain of S. aureus. Gene therapy, with hematopoietic stem cells transduced with regulated lentiviral vectors, restored the functional activity of NADPH oxidase complex (with 20-98% of dihydrorhodamine positive granulocytes and monocytes) and saved mice from death caused by S. aureus, significantly reducing the bacterial load and lung damage, similarly to WT mice even at low vector copy number. When challenged, gene therapy-treated XCGD mice showed correction of proinflammatory cytokines and chemokine imbalance at levels that were comparable to WT. Examined together, our results support the clinical development of gene therapy protocols using lentiviral vectors for the protection against infections and inflammation.
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http://dx.doi.org/10.1038/mt.2016.150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5112042PMC
October 2016

RD-MolPack technology for the constitutive production of self-inactivating lentiviral vectors pseudotyped with the nontoxic RD114-TR envelope.

Mol Ther Methods Clin Dev 2016 11;3:16033. Epub 2016 May 11.

Research Division, MolMed S.p.A , Milano, Italy.

To date, gene therapy with transiently derived lentivectors has been very successful to cure rare infant genetic diseases. However, transient manufacturing is unfeasible to treat adult malignancies because large vector lots are required. By contrast, stable manufacturing is the best option for high-incidence diseases since it reduces the production cost, which is the major current limitation to scale up the transient methods. We have previously developed the proprietary RD2-MolPack technology for the stable production of second-generation lentivectors, based on the RD114-TR envelope. Of note, opposite to vesicular stomatitis virus glycoprotein (VSV-G) envelope, RD114-TR does not need inducible expression thanks to lack of toxicity. Here, we present the construction of RD2- and RD3-MolPack cells for the production of self-inactivating lentivectors expressing green fluorescent protein (GFP) as a proof-of-concept of the feasibility and safety of this technology before its later therapeutic exploitation. We report that human T lymphocytes transduced with self-inactivating lentivectors derived from RD3-MolPack cells or with self-inactivating VSV-G pseudotyped lentivectors derived from transient transfection show identical T-cell memory differentiation phenotype and comparable transduction efficiency in all T-cell subsets. RD-MolPack technology represents, therefore, a straightforward tool to simplify and standardize lentivector manufacturing to engineer T-cells for frontline immunotherapy applications.
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http://dx.doi.org/10.1038/mtm.2016.33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863723PMC
May 2016

Production of lentiviral vectors.

Mol Ther Methods Clin Dev 2016 13;3:16017. Epub 2016 Apr 13.

New Technologies Unit, Research Division, MolMed S.p.A. , Milan, Italy.

Lentiviral vectors (LV) have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. This review presents the state of the art of the production of these vectors with particular emphasis on their large-scale production for clinical purposes. In contrast to oncoretroviral vectors, which are produced using stable producer cell lines, clinical-grade LV are in most of the cases produced by transient transfection of 293 or 293T cells grown in cell factories. However, more recent developments, also, tend to use hollow fiber reactor, suspension culture processes, and the implementation of stable producer cell lines. As is customary for the biotech industry, rather sophisticated downstream processing protocols have been established to remove any undesirable process-derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review compares published large-scale production and purification processes of LV and presents their process performances. Furthermore, developments in the domain of stable cell lines and their way to the use of production vehicles of clinical material will be presented.
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http://dx.doi.org/10.1038/mtm.2016.17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4830361PMC
April 2016

RD2-MolPack-Chim3, a packaging cell line for stable production of lentiviral vectors for anti-HIV gene therapy.

Hum Gene Ther Methods 2013 Aug 3;24(4):228-40. Epub 2013 Aug 3.

MolMed S.p.A., Milano 20132, Italy.

Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.
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http://dx.doi.org/10.1089/hgtb.2012.190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753729PMC
August 2013

Therapeutic genes for anti-HIV/AIDS gene therapy.

Curr Pharm Biotechnol 2013 ;14(5):488-500

MolMed, SpA, Via Olgettina, 58, Milano, 20132, Italy.

The multiple therapeutic approaches developed so far to cope HIV-1 infection, such as anti-retroviral drugs, germicides and several attempts of therapeutic vaccination have provided significant amelioration in terms of life-quality and survival rate of AIDS patients. Nevertheless, no approach has demonstrated efficacy in eradicating this lethal, if untreated, infection. The curative power of gene therapy has been proven for the treatment of monogenic immunodeficiensies, where permanent gene modification of host cells is sufficient to correct the defect for life-time. No doubt, a similar concept is not applicable for gene therapy of infectious immunodeficiensies as AIDS, where there is not a single gene to be corrected; rather engineered cells must gain immunotherapeutic or antiviral features to grant either short- or long-term efficacy mostly by acquisition of antiviral genes or payloads. Anti-HIV/AIDS gene therapy is one of the most promising strategy, although challenging, to eradicate HIV-1 infection. In fact, genetic modification of hematopoietic stem cells with one or multiple therapeutic genes is expected to originate blood cell progenies resistant to viral infection and thereby able to prevail on infected unprotected cells. Ultimately, protected cells will re-establish a functional immune system able to control HIV-1 replication. More than hundred gene therapy clinical trials against AIDS employing different viral vectors and transgenes have been approved or are currently ongoing worldwide. This review will overview anti-HIV-1 infection gene therapy field evaluating strength and weakness of the transgenes and payloads used in the past and of those potentially exploitable in the future.
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http://dx.doi.org/10.2174/138920101405131111104009DOI Listing
October 2014

Chim3 confers survival advantage to CD4+ T cells upon HIV-1 infection by preventing HIV-1 DNA integration and HIV-1-induced G2 cell-cycle delay.

Blood 2010 May 10;115(20):4021-9. Epub 2010 Mar 10.

MolMed SpA, Milano, Italy.

The long-term expression and the ability of a therapeutic gene to confer survival advantage to transduced cells are mandatory requirements for successful anti-HIV gene therapy. In this context, we developed lentiviral vectors (LVs) expressing the F12-viral infectivity factor (Vif) derivative Chim3. We recently showed that Chim3 inhibits HIV-1 replication in primary cells by both blocking the accumulation of retrotranscripts, independently of either human APOBEC3G (hA3G) or Vif, and by preserving the antiviral function of hA3G. These results were predictive of long-lasting survival of Chim3(+) cells after HIV-1 infection. Furthermore, Vif, like Vpr, deregulates cell-cycle progression by inducing a delay in G(2) phase. Thus, the aim of this study was to investigate the role of Chim3 on both cell survival and cell-cycle regulation after HIV-1 infection. Here, we provide evidence that infected Chim3(+) T cells prevail over either mock- or empty-LV engineered cells, show reduced G(2) accumulation, and, as a consequence, ultimately extend their lifespan. Based on these findings, Chim3 rightly belongs to the most efficacious class of antiviral genes. In conclusion, Chim3 usage in anti-HIV gene therapy based on hematopoietic stem cell (HSC) modification has to be considered as a promising therapeutic intervention to eventually cope with HIV-1 infection.
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http://dx.doi.org/10.1182/blood-2009-09-243030DOI Listing
May 2010

Mutational analysis of the HIV-1 auxiliary protein Vif identifies independent domains important for the physical and functional interaction with HIV-1 reverse transcriptase.

Nucleic Acids Res 2009 Jun 15;37(11):3660-9. Epub 2009 Apr 15.

Institute of Molecular Genetics IGM-CNR, via Abbiategrasso 207, I-27100 Pavia, Italy.

The HIV-1 accessory protein Vif plays a dual role: it counteracts the natural restriction factors APOBEC3G and 3F and ensures efficient retrotranscription of the HIV-1 RNA genome. We have previously shown that Vif can act as an auxiliary factor for HIV-1 reverse transcriptase (RT), increasing its rate of association to RNA or DNA templates. Here, by using seven different Vif mutants, we provide in vitro evidences that Vif stimulates HIV-1 RT through direct protein-protein interaction, which is mediated by its C-terminal domain. Physical interaction appears to require the proline-rich region comprised between amino acid (aa) 161 and 164 of Vif, whereas the RT stimulatory activity requires, in addition, the extreme C-terminal region (aa 169-192) of the Vif protein. Neither the RNA interaction domain, nor the Zn(++)-binding domain of Vif are required for its interaction with the viral RT. Pseudotyped HIV-1 lentiviral vectors bearing Vif mutants deleted in the RNA- or RT-binding domains show defects in retrotranscription/integration processes in both permissive and nonpermissive cells. Our results broaden our knowledge on how three important functions of Vif (RNA binding, RT binding and stimulation and Zn(++) binding), are coordinated by different domains.
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http://dx.doi.org/10.1093/nar/gkp226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2699511PMC
June 2009

Transcriptional enhancers induce insertional gene deregulation independently from the vector type and design.

Mol Ther 2009 May 17;17(5):851-6. Epub 2009 Mar 17.

Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy.

The integration characteristics of retroviral (RV) vectors increase the probability of interfering with the regulation of cellular genes, and account for a tangible risk of insertional mutagenesis in treated patients. To assess the potential genotoxic risk of conventional or self-inactivating (SIN) gamma-RV and lentiviral (LV) vectors independently from the biological consequences of the insertion event, we developed a quantitative assay based on real-time reverse transcriptase--PCR on low-density arrays to evaluate alterations of gene expression in individual primary T-cell clones. We show that the Moloney leukemia virus long terminal repeat (LTR) enhancer has the strongest activity in both a gamma-RV and a LV vector context, while an internal cellular promoter induces deregulation of gene expression less frequently, at a shorter range and to a lower extent in both vector types. Downregulation of gene expression was observed only in the context of LV vectors. This study indicates that insertional gene activation is determined by the characteristics of the transcriptional regulatory elements carried by the vector, and is largely independent from the vector type or design.
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http://dx.doi.org/10.1038/mt.2009.51DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2835143PMC
May 2009

The F12-Vif derivative Chim3 inhibits HIV-1 replication in CD4+ T lymphocytes and CD34+-derived macrophages by blocking HIV-1 DNA integration.

Blood 2009 Apr 11;113(15):3443-52. Epub 2009 Feb 11.

MolMed S.p.A., Milano, Italy.

The viral infectivity factor (Vif) is essential for HIV-1 infectivity and hence is an ideal target for promising anti-HIV-1/AIDS gene therapy. We previously demonstrated that F12-Vif mutant inhibits HIV-1 replication in CD4(+) T lymphocytes. Despite macrophage relevance to HIV-1 pathogenesis, most gene therapy studies do not investigate macrophages because of their natural resistance to genetic manipulation. Here, we confirm the F12-Vif antiviral activity also in macrophages differentiated in vitro from transduced CD34(+) human stem cells (HSCs). Moreover, we identified the 126- to 170-amino-acid region in the C-terminal half of F12-Vif as responsible for its antiviral function. Indeed, Chim3 protein, containing this 45-amino-acid region embedded in a WT-Vif backbone, is as lethal as F12-Vif against HIV-1. Of major relevance, we demonstrated a dual mechanism of action for Chim3. First, Chim3 functions as a transdominant factor that preserves the antiviral function of the natural restriction factor APOBEC3G (hA3G). Second, Chim3 blocks the early HIV-1 retrotranscript accumulation and thereby HIV-1 DNA integration regardless of the presence of WT-Vif and hA3G. In conclusion, by impairing the early steps of HIV-1 life cycle, Chim3 conceivably endows engineered cells with survival advantage, which is required for the efficient immune reconstitution of patients living with HIV/AIDS.
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http://dx.doi.org/10.1182/blood-2008-06-158790DOI Listing
April 2009

Naturally occurring C-terminally truncated STAT5 is a negative regulator of HIV-1 expression.

Blood 2007 Jun 1;109(12):5380-9. Epub 2007 Mar 1.

AIDS Immunopathogenesis Unit and the Division of Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy.

CD4(+) cells of most individuals infected with HIV-1 harbor a C-terminally truncated and constitutively activated form of signal transducer and activator of transcription-5 (STAT5 Delta). We report that the chronically HIV-infected U1 cell line expresses STAT5 Delta but not full-length STAT5. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of U1 cells promoted early activation of STAT5 Delta and of extracellular signal regulated kinases (ERKs), followed by later activation of activator protein 1 (AP-1) and HIV expression. Inhibition of ERK/AP-1 by PD98,059 abolished, whereas either tyrphostin AG490 or a STAT5 small interfering RNA (siRNA) enhanced, virion production in GM-CSF-stimulated U1 cells. Chromatin immunoprecipitation demonstrated the induction of STAT5 Delta binding to STAT consensus sequences in the HIV-1 promoter together with a decreased recruitment of RNA polymerase II after 1 hour of GM-CSF stimulation of U1 cells. Down-regulation of STAT5 Delta by siRNA resulted in the up-regulation of both HIV-1 gag-pol RNA and p24 Gag antigen expression in CD8-depleted leukocytes of several HIV-positive individuals cultivated ex vivo in the presence of interleukin-2 but not of interleukin-7. Thus, the constitutively activated STAT5 Delta present in the leukocytes of most HIV-positive individuals acts as a negative regulator of HIV expression.
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http://dx.doi.org/10.1182/blood-2006-08-042556DOI Listing
June 2007

Heterogeneity of signal transducer and activator of transcription binding sites in the long-terminal repeats of distinct HIV-1 subtypes.

Open Virol J 2007 20;1:26-32. Epub 2007 Oct 20.

AIDS Immunopathogenesis, San Raffaele Scientific Institute, Milano, Italy.

HIV-1 can be subdivided into distinct subtypes; the consequences of such a genomic variability remain largely speculative. The long terminal repeats (LTR) control HIV transcription and reflect the major differences of distinct viral subtypes. Three regions in the HIV-1 subtype B LTR are close matches to the Signal Transducer and Activator of Transcription (STAT) consensus sequence. Here, we show heterogeneity in these putative STAT binding sites among HIV-1 LTR subtypes A through G. Transfection of constitutively activated STAT5 lead to transcriptional activation of HIV-1 expression in 293T cells transfected with a reporter assay driven by HIV-1 LTR subtype B. Constitutively activated STAT5 transactivated the LTR of various subtypes in U937 cells with different potency. These findings support and expand the potential relevance of STAT5 activation in HIV infection and may bear relevance for a differential regulation of latency and expression of different subtypes of HIV-1.
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http://dx.doi.org/10.2174/1874357900701010026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2675545PMC
June 2009

T Lymphocytes transduced with a lentiviral vector expressing F12-Vif are protected from HIV-1 infection in an APOBEC3G-independent manner.

Mol Ther 2005 Oct;12(4):697-706

Molmed SpA, Milan, Italy.

The viral infectivity factor (Vif) is an essential component of the HIV-1 infectious cycle. Vif counteracts the action of the cytidine deaminase APOBEC3G (AP3G), which confers nonimmune antiviral defense against HIV-1 to T lymphocytes. Disabling or interfering with the function of Vif could represent an alternative therapeutic approach to AIDS. We have expressed a natural mutant of Vif, F12-Vif, in a VSV-G-pseudotyped lentiviral vector under the Tat-inducible control of the HIV-1 LTR. Conditional expression of F12-Vif prevents replication and spreading of both CXCR4 and CCR5 strains of HIV-1 in human primary T lymphocyte and T cell lines. T cells transduced with F12-Vif release few HIV-1 virions and with reduced infectivity. Several lines of evidence indicate that HIV-1 interference requires the presence of both wild-type and F12-Vif proteins, suggesting a dominant-negative feature of the F12-Vif mutant. Surprisingly, however, the F12-Vif-mediated inhibition does not depend on the reestablishment of the AP3G function.
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http://dx.doi.org/10.1016/j.ymthe.2005.05.014DOI Listing
October 2005

Blocking HIV-1 Vif restores a natural mechanism of intracellular antiviral defense.

Authors:
Chiara Bovolenta

Curr Drug Targets Immune Endocr Metabol Disord 2004 Dec;4(4):257-63

Molmed S.p.A., via Olgettina, 58, 20132 Milano, Italy.

AIDS has become the greatest pandemic in the human history counting approximately 40 millions people worldwide. To purge HIV-1 infection, new therapeutic approaches need to be searched in alternative and/or in addition to the current pharmacological ones. Recently, several independent laboratories have unveiled a non-immune intracellular anti-HIV-1 defense strategy based on the cytidine deaminase APOBEC3G, which restricts HIV-1 production by directly mutating the proviral DNA in infected cells. To counteract this defense pathway, HIV-1 has developed an evasion strategy by acquiring the accessory protein Vif, which blocks the action of APOBEC3G by inducing its proteasome-mediated degradation.
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http://dx.doi.org/10.2174/1568008043339758DOI Listing
December 2004

IFN-gamma and IL-12 differentially regulate CC-chemokine secretion and CCR5 expression in human T lymphocytes.

J Leukoc Biol 2002 Oct;72(4):735-42

Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy.

Interleukin (IL)-12, especially in the presence of neutralizing anti-IL-4 monoclonal antibodies, primed CD45RO(-) T clones for high CCL3/macrophage-inflammatory protein-1alpha (MIP-1alpha) and CCL4/MIP-1beta levels. In CD4(+) and CD8(+) clones from two patients deficient for IL-12Rbeta1 (IL-12Rbeta1(-/-)), production of CCL3/MIP-1alpha and CCL4/MIP-1beta was defective. CD4(+) clones from two patients deficient for interferon-gamma (IFN-gamma) R1 (IFN-gammaR1(-/-)) produced somewhat decreased CCL4/MIP-1beta levels. IL-12 failed to prime CD4(+) or CD8(+) healthy clones for high CCL5/regulated on activation, normal T expressed and secreted (RANTES) production, although its secretion was impaired in CD4(+) clones from IL-12Rbeta1(-/-) and IFN-gammaR1(-/-) patients. CCR5 surface expression was up-regulated in resting peripheral blood mononuclear cells and CD4(+) clones from both kinds of patients, rendering them more susceptible to CCR5-dependent (R5) HIV-1 infection. Neutralization of IFN-gamma increased CCR5 expression and decreased CC-chemokine secretion by CD4(+) clones from healthy and IL-12Rbeta1(-/-) individuals, suggesting an IFN-gamma-dependent control of CCR5 expression. These data provide the first documented analysis of chemokine secretion and chemokine receptor expression on T cells from IL-12 and IFN-gamma receptor-deficient patients and dissect the role of IL-12 and IFN-gamma on inducing inflammatory chemokine secretion and down-regulating CCR5 expression in human T cells.
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October 2002

Retroviral interference on STAT activation in individuals coinfected with human T cell leukemia virus type 2 and HIV-1.

J Immunol 2002 Oct;169(8):4443-9

Department of Immunology and Infectious Disease, San Raffaele Scientific Institute, via Olgettina 58, 20132 Milan, Italy.

Human T cell leukemia virus (HTLV) type-2 is a human retrovirus whose infection has not been tightly linked to human diseases. However, the fairly high prevalence of this infection among HIV-1-positive individuals indicates the importance of better understanding the potential interference of HTLV-2 infection on HIV-1 infection and AIDS. We previously demonstrated that one signature of PBMC freshly derived from HIV-1-infected individuals is the constitutive activation of a C-terminal truncated STAT5 (STAT5Delta). Therefore, we analyzed the potential activation of STATs in HTLV-2 monoinfected and HTLV-2/HIV-1 dually infected individuals. We observed that PBMC of HTLV-2-infected individuals do not show STAT activation unless they are cultivated ex vivo, in the absence of any mitogenic stimuli, for at least 8 h. The emergence of STAT activation, namely of STAT1, in culture was mostly related to the secretion of IFN-gamma. Of note, this phenomenon is not only a characteristic feature of HTLV-2-infected individuals but also occurred with PBMC of HIV-1(+) individuals. Surprisingly, HTLV-2/HIV-1 coinfection resulted in low/absent STAT activation in vivo that paralleled a diminished secretion of IFN-gamma after ex vivo cultivation. Our findings indicate that both HTLV-2 and HIV-1 infection prime T lymphocytes for STAT1 activation, but they also highlight an interference exerted by HTLV-2 on HIV-1-induced STAT1 activation. Although the nature of such a phenomenon is unclear at the present, these findings support the hypothesis that HTLV-2 may interfere with HIV-1 infection at multiple levels.
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http://dx.doi.org/10.4049/jimmunol.169.8.4443DOI Listing
October 2002

Human T-cell leukemia virus type 2 induces survival and proliferation of CD34(+) TF-1 cells through activation of STAT1 and STAT5 by secretion of interferon-gamma and granulocyte macrophage-colony-stimulating factor.

Blood 2002 Jan;99(1):224-31

AIDS Immunopathogenesis Unit, San Raffaele Scientific Institute, Milan, Italy.

Human T-cell leukemia-lymphoma virus (HTLV) type-2 can induce the survival and proliferation of CD34(+) TF-1 cells deprived of interleukin (IL)-3. This effect did not require productive infection and occurred when HTLV-2 was produced from T cells (CMo), but not from B cells (BMo), unless the latter virus was complexed with anti-HLA-DR monoclonal antibodies (mAbs). Cellular and molecular mechanisms triggered by HTLV-2 interaction with TF-1 cells were here investigated. Activation of signal transducer and activator of transcription (STAT) 5 protein occurred in TF-1 cells incubated either with IL-3 or with HTLV-2/CMo; in addition the virus, but not IL-3, activated STAT1. The effect of HTLV-2 required several hours, suggesting dependence on the induction of cellular factors. By screening a panel of secreted factors, granulocyte macrophage-colony-stimulating factor (GM-CSF), interferon (IFN)-gamma, and stem cell factor (SCF) were found induced by HTLV-2 in TF-1 cells. Of note is the fact that these molecules induce a variety of biologic effects through the activation of STAT proteins, including STAT1 and STAT5. Neutralization experiments indicated that GM-CSF and IFN-gamma, but not SCF, were responsible for HTLV-2-induced STAT activation, whereas anti-GM-CSF antibodies greatly inhibited TF-1 cell proliferation. Finally, incubation of BMo virus with anti-HLA-DR mAb rescued TF-1 cell survival in the absence of IL-3. Thus, HTLV-2 interaction with CD34(+) precursor cells may lead to the expression of cytokines that, by inducing autocrine activation of STATs, may influence the host's regenerative capacity and immune response to HTLV-2 and to other infectious agents.
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http://dx.doi.org/10.1182/blood.v99.1.224DOI Listing
January 2002