RESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , DNA Viral/genética , Fase G2/fisiologia , Terapia Genética , HIV-1/fisiologia , Integração Viral , Produtos do Gene vif do Vírus da Imunodeficiência Humana/fisiologia , Southern Blotting , Linfócitos T CD4-Positivos/virologia , Sobrevivência Celular , Células Cultivadas , DNA Viral/metabolismo , Células-Tronco Hematopoéticas , Humanos , Imunoprecipitação , Replicação Viral , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/fisiologiaRESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/virologia , Terapia Genética/métodos , Infecções por HIV/terapia , HIV-1/crescimento & desenvolvimento , Macrófagos/virologia , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genética , Antígenos CD34/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/fisiologia , Diferenciação Celular/imunologia , Linhagem Celular , Sangue Fetal/citologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/imunologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Rim/citologia , Macrófagos/citologia , Macrófagos/fisiologia , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Transdução Genética , Integração Viral , Replicação Viral , Produtos do Gene vif do Vírus da Imunodeficiência Humana/químicaAssuntos
Antígenos CD13/análise , Integrina alfaVbeta3/análise , Ressonância Magnética Nuclear Biomolecular/métodos , Antígenos CD13/química , Antígenos CD13/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Integrina alfaVbeta3/química , Integrina alfaVbeta3/metabolismo , Ligação ProteicaRESUMO
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.
Assuntos
Síndrome da Imunodeficiência Adquirida/genética , Síndrome da Imunodeficiência Adquirida/terapia , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/genética , Infecções por HIV/terapia , HIV-1/genética , Animais , Terapia Genética/métodos , HumanosRESUMO
Poor penetration of antitumor drugs into the extravascular tumor tissue is often a major factor limiting the efficacy of cancer treatments. Our group has recently described a strategy to enhance tumor penetration of chemotherapeutic drugs through use of iRGD peptide (CRGDK/RGPDC). This peptide comprises two sequence motifs: RGD, which binds to αvß3/5 integrins on tumor endothelia and tumor cells, and a cryptic CendR motif (R/KXXR/K-OH). Once integrin binding has brought iRGD to the tumor, the peptide is proteolytically cleaved to expose the cryptic CendR motif. The truncated peptide loses affinity for its primary receptor and binds to neuropilin-1, activating a tissue penetration pathway that delivers the peptide along with attached or co-administered payload into the tumor mass. Here, we describe the design of a new tumor-penetrating peptide based on the current knowledge of homing sequences and internalizing receptors. The tumor-homing motif in the new peptide is the NGR sequence, which binds to endothelial CD13. The NGR sequence was placed in the context of a CendR motif (RNGR), and this sequence was embedded in the iRGD framework. The resulting peptide (CRNGRGPDC, iNGR) homed to tumor vessels and penetrated into tumor tissue more effectively than the standard NGR peptide. iNGR induced greater tumor penetration of coupled nanoparticles and co-administered compounds than NGR. Doxorubicin given together with iNGR was significantly more efficacious than the drug alone. These results show that a tumor-specific, tissue-penetrating peptide can be constructed from known sequence elements. This principle may be useful in designing tissue-penetrating peptides for other diseases.
Assuntos
Antineoplásicos/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , Neoplasias/tratamento farmacológico , Oligopeptídeos/uso terapêutico , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Desenho de Fármacos , Humanos , Camundongos , Ligação ProteicaRESUMO
Aminopeptidase-N (CD13) is an important target of tumor vasculature-targeting drugs. The authors investigated its expression by immunohistochemistry with three anti-CD13 monoclonal antibodies (WM15, 3D8, and BF10) in normal and pathological human tissues, including 58 normal, 32 inflammatory, and 149 tumor tissue specimens. The three antibodies stained vessels in most neoplastic tissues, interestingly with different patterns. As a matter of fact, WM15 stained almost all intratumor and peritumor capillaries and only partially large vessels, whereas BF10 and 3D8 reacted with arteries and venules and to a lesser extent with capillaries. These antibodies also stained the stroma in about half of neoplastic tissues. In inflammatory lesions, the three antibodies stained vessels and stroma, whereas in normal tissues, they stained a small percentage of blood vessels. Finally, the three antibodies failed to stain endothelial cells of normal colon, whereas they reacted with activated human umbilical vein endothelial cells and with endothelial cells of colon adenocarcinoma vessels. Overall, WM15 was the most specific antibody for angiogenic tumor vessels, suggesting that it may be a good tool for detecting the CD13 form associated with the tumor vasculature. This finding may be relevant for CD13-mediated vascular targeting therapies.