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1.
PLoS One ; 18(9): e0291315, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37713401

RESUMO

In a phase I dose escalation and safety study (NCT02555397), a replication-competent oncolytic adenovirus expressing yCD, TK and hIL-12 (Ad5-yCD/mutTKSR39rep-hIL-12) was administered in 15 subjects with localized recurrent prostate cancer (T1c-T2) at increasing doses (1 × 1010, to 1 × 1012 viral particles) followed by 7-day treatment of 5-fluorocytosine (5-FC) and valganciclovir (vGCV). The primary endpoint was toxicity through day 30 while the secondary and exploratory endpoints were quantitation of IL-12, IFNγ, CXCL10 and peripheral blood mononuclear cells (PBMC). The study maximum tolerated dose (MTD) was not reached indicating 1012 viral particles was safe. Total 115 adverse events were observed, most of which (92%) were grade 1/2 that did not require any treatment. Adenoviral DNA was detected only in two patients. Increase in IL-12, IFNγ, and CXCL10 was observed in 57%, 93%, and 79% patients, respectively. Serum cytokines demonstrated viral dose dependency, especially apparent in the highest-dose cohorts. PBMC analysis revealed immune system activation after gene therapy in cohort 5. The PSA doubling time (PSADT) pre and post treatment has a median of 1.55 years vs 1.18 years. This trial confirmed that replication-competent Ad5-IL-12 adenovirus (Ad5-yCD/mutTKSR39rep-hIL-12) was well tolerated when administered locally to prostate tumors.


Assuntos
Adenocarcinoma , Terapia Viral Oncolítica , Neoplasias da Próstata , Humanos , Masculino , Adenocarcinoma/terapia , Adenoviridae , Terapia Genética/efeitos adversos , Interleucina-12/genética , Leucócitos Mononucleares , Próstata , Neoplasias da Próstata/terapia , Genes Transgênicos Suicidas
2.
Cancer Biol Ther ; 24(1): 2232146, 2023 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-37439774

RESUMO

Gene edited and engineered cell-based therapies are a promising approach for treating a variety of disorders, including cancer. However, the ability of engineered cells to persist for prolonged periods along with possible toxicity raises concerns over the safety of these approaches. Although a number of different one-dimensional suicide systems have been incorporated into therapeutic cell types, the incorporation of a two-layered suicide system that allows controlled killing of therapeutic cells at different time points is needed. In this study, we engineered a variety of therapeutic cells to express two different kill switches, RapaCasp9 and HSV-TK and utilized Rapamycin and Ganciclovir respectively to activate these kill switches. We show that the function of both RapaCasp9 and HSV-TK molecules is preserved and can be activated to induce apoptosis detected early (24 h) and late (48 h) post-activation respectively, with no toxicity. In vivo, we show the eradication of a majority of cells after treatment in subcutaneous and orthotopic models. Furthermore, we demonstrate how both suicide switches work independently and can be activated sequentially for an improved killing, thus ensuring a failsafe mechanism in case the activation of a single one of them is not sufficient to eliminate the cells. Our findings highlight the reliability of the double suicide system, effective on a variety of cells with different biological characteristics, independent of their anatomic presence.


Assuntos
Genes Transgênicos Suicidas , Terapia Genética , Humanos , Terapia Genética/métodos , Reprodutibilidade dos Testes , Ganciclovir/farmacologia , Ganciclovir/uso terapêutico , Apoptose
3.
Cancer Gene Ther ; 30(1): 85-95, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36076062

RESUMO

Herpes simplex virus thymidine kinase (HSVTK)/ganciclovir (GCV) suicide gene therapy has a long history of treating malignant gliomas. Recently, stem cells from human exfoliated deciduous teeth (SHED), which are collected from deciduous teeth and have excellent harvestability, ethical aspects, and self-renewal, have been attracting attention mainly in the field of gene therapy. In the present study, we assessed SHED as a novel cellular vehicle for suicide gene therapy in malignant gliomas, as we have previously demonstrated with various cell types. SHED was transduced with the HSVTK gene (SHEDTK). In vitro experiments showed a significant bystander effect between SHEDTK and glioma cell lines in coculture. Furthermore, apoptotic changes caused by caspase 3/7 activation were simultaneously observed in SHEDTK and glioma cells. Mice implanted with a mixture of U87 and SHEDTK and treated with intraperitoneal GCV survived for longer than 100 days. Additionally, tumors in treatment model mice were significantly reduced in size during the treatment period. SHEDTK implanted at the contralateral hemisphere migrated toward the tumor crossing the corpus callosum. These results suggested that SHEDTK-based suicide gene therapy has potent tumor tropism and a bystander-killing effect, potentially offering a new promising therapeutic modality for malignant gliomas.


Assuntos
Ganciclovir , Terapia Genética , Glioma , Animais , Humanos , Camundongos , Efeito Espectador/genética , Ganciclovir/farmacologia , Terapia Genética/métodos , Glioma/terapia , Glioma/tratamento farmacológico , Simplexvirus/genética , Células-Tronco , Timidina Quinase/genética , Dente Decíduo , Genes Transgênicos Suicidas
4.
Front Immunol ; 13: 975233, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36189285

RESUMO

Gene-modified cellular therapies carry inherent risks of severe and potentially fatal adverse events, including the expansion of alloreactive cells or malignant transformation due to insertional mutagenesis. Strategies to mitigate uncontrolled proliferation of gene-modified cells include co-transfection of a suicide gene, such as the inducible caspase 9 safety switch (ΔiC9). However, the activation of the ΔiC9 fails to completely eliminate all gene-modified cells. Therefore, we tested a two suicide gene system used independently or together, with the goal of complete cell elimination. The first approach combined the ΔiC9 with an inducible caspase 8, ΔiC8, which lacks the endogenous prodomain. The rationale was to use a second caspase with an alternative and complementary mechanism of action. Jurkat cells co-transduced to co-express the ΔiC8, activatable by a BB homodimerizer, and the ΔiC9 activatable by the rapamycin analog sirolimus were used in a model to estimate the degree of inducible cell elimination. We found that both agents could activate each caspase independently, with enhanced elimination with superior reduction in cell regrowth of gene-modified cells when both systems were activated simultaneously. A second approach was employed in parallel, combining the ΔiC9 with the RQR8 compact suicide gene. RQR8 incorporates a CD20 mimotope, targeted by the anti-CD20 monoclonal antibody rituxan, and the QBend10, a ΔCD34 selectable marker. Likewise, enhanced cell elimination with superior reduction in cell regrowth was observed when both systems were activated together. A dose-titration effect was also noted utilizing the BB homodimerizer, whereas sirolimus remained very potent at minimal concentrations. Further in vivo studies are needed to validate these novel combination systems, which may play a role in future cancer therapies or regenerative medicine.


Assuntos
Genes Transgênicos Suicidas , Sirolimo , Caspase 8/genética , Caspase 9/genética , Caspase 9/metabolismo , Genes Transgênicos Suicidas/genética , Humanos , Rituximab , Sirolimo/farmacologia
5.
PLoS One ; 17(2): e0264001, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35167620

RESUMO

The role played by certain domestic species such as dogs as a translational model in comparative oncology shows great interest to develop new therapeutic strategies in brain tumors. Gliomas are a therapeutic challenge that represents the most common form of malignant primary brain tumors in humans and the second most common form in dogs. Gene-directed enzyme/prodrug therapy using adipose mesenchymal stem cells (Ad-MSCs) expressing the herpes simplex virus thymidine kinase (TK) has proven to be a promising alternative in glioblastoma therapy, through its capacity to migrate and home to the tumor and delivering local cytotoxicity avoiding other systemic administration. In this study, we demonstrate the possibility for canine Ad-MSCs (cAd-MSCs) to be genetically engineered efficiently with a lentiviral vector to express TK (TK-cAd-MSCs) and in combination with ganciclovir (GCV) prodrug demonstrated its potential antitumor efficacy in vitro and in vivo in a mice model with the human glioblastoma cell line U87. TK-cAd-MSCs maintained cell proliferation, karyotype stability, and MSCs phenotype. Genetic modification significantly affects its secretory profile, both the analyzed soluble factors and exosomes. TK-cAd-MSCs showed a high secretory profile of some active antitumor immune response cytokines and a threefold increase in the amount of secreted exosomes, with changes in their protein cargo. We also found that the prodrug protein is not released directly into the culture medium by TK-cAd-MSCs. We believe that our work provides new perspectives for glioblastoma gene therapy in dogs and a better understanding of this therapy in view of its possible implantation in humans.


Assuntos
Neoplasias Encefálicas/terapia , Ganciclovir/administração & dosagem , Glioblastoma/terapia , Herpes Simples/enzimologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Timidina Quinase/genética , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Técnicas de Cocultura , Cães , Ganciclovir/farmacologia , Genes Transgênicos Suicidas , Terapia Genética , Glioblastoma/genética , Herpes Simples/genética , Humanos , Lentivirus/genética , Células-Tronco Mesenquimais/metabolismo , Camundongos , Timidina Quinase/metabolismo , Transdução Genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Biochem Pharmacol ; 197: 114893, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34968484

RESUMO

Suicide Gene Therapy (SGT) aims to introduce a gene encoding either a toxin or an enzyme making the targeted cell more sensitive to chemotherapy. SGT represents an alternative approach to combat pathologies where conventional treatments fail such as pancreatic cancer or the high-grade glioblastoma which are still desperately lethal. We review the possibility to use SGT to treat these cancers which have shown promising results in vitro and in preclinical trials. However, SGT has so far failed in phase III clinical trials thus further improvements are awaited. We can now take advantages of the many advances made in SGT for treating cancer to combat other pathologies such as HIV-1 infection. In the review we also discuss the feasibility to add SGT to the therapeutic arsenal used to cure HIV-1-infected patients. Indeed, preliminary results suggest that both productive and latently infected cells are targeted by the SGT. In the last section, we address the limitations of this approach and how we might improve it.


Assuntos
Terapias Complementares/métodos , Genes Transgênicos Suicidas/genética , Terapia Genética/métodos , Infecções por HIV/genética , HIV-1/genética , Neoplasias/genética , Animais , Terapias Complementares/tendências , Terapia Genética/tendências , Infecções por HIV/terapia , Humanos , Neoplasias/terapia
7.
Curr Gene Ther ; 22(1): 23-39, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34238158

RESUMO

Recently, genetic engineering by various strategies to stimulate gene expression in a specific and controllable mode is a speedily growing therapeutic approach. Genetic modification of human stem or progenitor cells, such as Embryonic Stem Cells (ESCs), Neural Progenitor Cells (NPCs), Mesenchymal Stem/Stromal Cells (MSCs), and Hematopoietic Stem Cells (HSCs) for direct delivery of specific therapeutic molecules or genes has been evidenced as an opportune plan in the context of regenerative medicine due to their supported viability, proliferative features, and metabolic qualities. On the other hand, a large number of studies have investigated the efficacy of modified stem cells in cancer therapy using cells from various sources, disparate transfection means for gene delivery, different transfected yields, and wide variability of tumor models. Accordingly, cell-based gene therapy holds substantial aptitude for the treatment of human malignancy as it could relieve signs or even cure cancer succeeding expression of therapeutic or suicide transgene products; however, there exist inconsistent results in this regard. Herein, we deliver a brief overview of stem cell potential to use in cancer therapy and regenerative medicine and importantly discuss stem cells based gene delivery competencies to stimulate tissue repair and replacement in concomitant with their potential to use as an anti-cancer therapeutic strategy, focusing on the last two decades' in vivo studies.


Assuntos
Células-Tronco Mesenquimais , Neoplasias , Terapia Baseada em Transplante de Células e Tecidos , Genes Transgênicos Suicidas , Humanos , Células-Tronco Mesenquimais/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Medicina Regenerativa/métodos
8.
Front Immunol ; 12: 755639, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34737753

RESUMO

T cells engineered with chimeric antigen receptor (CAR-T cells) are an effective treatment in patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia or B-cell non-Hodgkin lymphoma. Despite the reported exciting clinical results, the CAR-T cell approach needs efforts to improve the safety profile, limiting the occurrence of adverse events in patients given this treatment. Besides the most common side effects, such as cytokine release syndrome and CAR-T cell-related encephalopathy syndrome, another potential issue involves the inadvertent transduction of leukemia B cells with the CAR construct during the manufacturing process, thus leading to the possibility of a peculiar mechanism of antigen masking and treatment resistance. In this study, we investigated whether the inclusion of the inducible caspase 9 (iC9) suicide gene in the CAR construct design could be an effective safety switch to control malignant CAR+ B cells, ultimately counteracting this serious adverse event. iC9 is a suicide gene able to be activated through binding with an otherwise inert small biomolecule, known as AP1903. The exposure of iC9.CAR.CD19-DAUDI lymphoma and iC9.CAR.CD19-NALM-6 leukemia cells in vitro to 20 nM of AP1903 resulted into the prompt elimination of CAR+ B-leukemia/lymphoma cell lines. The results obtained in the animal model corroborate in vitro data, since iC9.CAR.CD19+ tumor cells were controlled in vivo by the activation of the suicide gene through administration of AP1903. Altogether, our data indicate that the inclusion of the iC9 suicide gene may result in a safe CAR-T cell product, even when manufacturing starts from biological materials characterized by heavy leukemia blast contamination.


Assuntos
Caspase 9 , Genes Transgênicos Suicidas , Imunoterapia Adotiva/métodos , Leucemia de Células B , Linfoma de Células B , Receptores de Antígenos Quiméricos/uso terapêutico , Animais , Linhagem Celular Tumoral , Humanos , Camundongos
9.
J Neuroendocrinol ; 33(12): e13057, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34748241

RESUMO

Vasopressin-synthesizing neurons are located in several brain regions, including the hypothalamic paraventricular nucleus (PVN), supraoptic nucleus (SON) and suprachiasmatic nucleus (SCN). Vasopressin has been shown to have various functions in the brain, including social recognition memory, stress responses, emotional behaviors and circadian rhythms. The precise physiological functions of vasopressin-synthesizing neurons in specific brain regions remain to be clarified. Conditional ablation of local vasopressin-synthesizing neurons may be a useful tool for investigation of the functions of vasopressin neurons in the regions. In the present study, we characterized a transgenic rat line that expresses a mutated human diphtheria toxin receptor under control of the vasopressin gene promoter. Under a condition of salt loading, which activates the vasopressin gene in the hypothalamic PVN and SON, transgenic rats were i.c.v. injected with diphtheria toxin. Intracerebroventricular administration of diphtheria toxin after salt loading depleted vasopressin-immunoreactive cells in the hypothalamic PVN and SON, but not in the SCN. The number of oxytocin-immunoreactive cells in the hypothalamus was not significantly changed. The rats that received i.c.v. diphtheria toxin after salt loading showed polydipsia and polyuria, which were rescued by peripheral administration of 1-deamino-8-d-arginine vasopressin via an osmotic mini-pump. Intrahypothalamic administration of diphtheria toxin in transgenic rats under a normal hydration condition reduced the number of vasopressin-immunoreactive neurons, but not the number of oxytocin-immunoreactive neurons. The transgenic rat model can be used for selective ablation of vasopressin-synthesizing neurons and may be useful for clarifying roles of vasopressin neurons at least in the hypothalamic PVN and SON in the rat.


Assuntos
Técnicas de Transferência de Genes , Genes Transgênicos Suicidas , Neurônios/metabolismo , Vasopressinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Toxina Diftérica/farmacologia , Deleção de Genes , Genes Transgênicos Suicidas/efeitos dos fármacos , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Masculino , Neurônios/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/metabolismo , Ratos , Ratos Endogâmicos Lew , Ratos Transgênicos , Núcleo Supraóptico/efeitos dos fármacos , Núcleo Supraóptico/metabolismo , Vasopressinas/genética
10.
Int J Biol Sci ; 17(15): 4122-4139, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34803487

RESUMO

Objective: Angiogenesis is involved in multiple biological processes, including atherosclerosis (AS) and cancer. Dickkopf1 (DKK1) plays many roles in both tumors and AS and has emerged as a potential biomarker of cancer progression and prognosis. Targeting DKK1 is a good choice for oncological treatments. Many anticancer therapies are associated with specific cardiovascular toxicity. However, the effects of DKK1 neutralizing therapy on AS are unclear. We focused on how DKK1 affected angiogenesis in AS and ox-LDL-induced human umbilical vein endothelial cells (HUVECs). Methods: ApoE-/- mice were fed a high-fat diet and then injected with DKK1i or DKK1 lentivirus to study the effects of DKK1. In vitro, promoter assays, protein analysis, database mining, dual-luciferase reporter assay (DLR), electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and coimmunoprecipitation (co-IP) were used to study the mechanism of DKK1 biogenesis. Cell migration and angiogenesis assays were performed to investigate the function and regulatory mechanisms of DKK1. Results: DKK1 participated in angiogenesis both in the plaques of ApoE-/- mice by knockdown or overexpression of DKK1 and ox-LDL-induced HUVECs. DKK1 induced angiogenesis (increasing migration and capillary formation, inducing expression of VEGFR-2/VEGF-A/MMP) via the CKAP4/PI3K pathway, independent of Wnt/ß-catenin. ox-LDL increased the expression and nuclear transfer of Ets-1 and c-jun, and induced the transcriptional activity of DKK1 in HUVECs. Ets-1, along with c-jun and CBP, could bind to the promoter of DKK1 and enhance DKK1 transcription. MiR33a-5p was downregulated in ox-LDL induced HUVECs and aortic artery of high-fat diet ApoE-/- mice. Ets-1 was a direct target of miR33a-5p. MiR33a-5p/Ets-1/ DKK1 axis contributed to angiogenesis. Conclusions: MiR33a-5p/Ets-1/DKK1 signaling participated in ox-LDL-induced angiogenesis of HUVECs via the CKAP4/PI3K pathway. These new findings provide a rationale and notable method for tumor therapy and cardiovascular protection.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , MicroRNAs/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/induzido quimicamente , Dieta Hiperlipídica/efeitos adversos , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica/fisiologia , Genes Transgênicos Suicidas , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lipoproteínas LDL , Masculino , Camundongos , Camundongos Knockout para ApoE , MicroRNAs/genética , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Interferência de RNA , Transdução de Sinais
11.
Eur Rev Med Pharmacol Sci ; 25(18): 5701-5724, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34604962

RESUMO

OBJECTIVE: Bipolar disorder (BD) is a severe disorder, and it is associated with an increased risk of mortality. About 25% of patients with BD have attempted and 11% have died by suicide. All these characteristics suggest that the disorders within the bipolar spectrum are a crucial public health problem. With the development of molecular genetics in recent decades, it was possible to more easily detect risk genes associated with this disorder. This study aimed at summarizing the findings of systematic reviews and meta-analyses on the topic and assessing the quality of the available evidence. MATERIALS AND METHODS: PubMed/Medline and Web of Science were searched to identify systematic reviews and meta-analyses published during 2013-2019. Standard methodology was applied to synthesize and assess the retrieved literature. RESULTS: This systematic review identifies a number of potential risk genes associated with bipolar disorder whose mechanism of action has yet to be confirmed. They are divided into several groups: 1) a list of the most significant susceptibility genetic factors associated with BD; 2) the implication of the ZNF804A gene in BD; 3) the role of genes involved in calcium signaling in BD; 4) DNA methylation in BD; 5) BD and risk suicide genes; 6) susceptibility genes for early-onset BD; 7) candidate genes common to both BD and schizophrenia; 8) genes involved in cognitive status in BD cases; 9) genes involved in structural alteration in BD brain tissue; 10) genes involved in lithium response in BD. CONCLUSIONS: Future research should concentrate on molecular mechanisms by which genetic variants play a major role in BD. Supplemental research is needed to replicate the applicable results.


Assuntos
Transtorno Bipolar/genética , Sinalização do Cálcio/genética , Metilação de DNA/genética , Genes Transgênicos Suicidas/genética , Estudos de Associação Genética , Predisposição Genética para Doença/genética , Fatores de Transcrição Kruppel-Like/genética , Humanos , Esquizofrenia/genética
12.
Bioengineered ; 12(1): 6572-6578, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34506254

RESUMO

Differentiated thyroid cancer (DTC), such as papillary thyroid cancer, has a good prognosis after routine treatment. However, in the course of treatment, 5% to 20% of cases may dedifferentiate and can be transformed into dedifferentiated DTC (deDTC) or anaplastic thyroid cancer, leading to treatment failure. To date, several drugs have been used effectively for dedifferentiated thyroid cancer, whereas gene therapy may be a potential method. Literature reported that double suicide genes driven by human telomerase reverse transcriptase promoter (hTERTp) can specifically express in cancer cells and kill them. However, the weak activity of hTERTp limits its further research. To overcome this weakness, we constructed a novel chitosan nanocarrier containing double suicide genes driven by a 'gene switch' (a cascade of radiation enhancer E9 and a hTERTp). The vector was labeled with iodine-131 (131I). On one hand, E9 can significantly enhance the activity of hTERTp under the weak radiation of 131I, thereby increasing the expression of double suicide genes in deDTC cells. On the other hand, 131I also plays a certain killing role when it enters host cells. The proposed nanocarrier has good specificity for deDTC cells and thus deserves further study.


Assuntos
Nanopartículas , Regiões Promotoras Genéticas/genética , Telomerase/genética , Neoplasias da Glândula Tireoide , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Genes Transgênicos Suicidas/genética , Terapia Genética , Humanos , Nanopartículas/química , Nanopartículas/toxicidade , Transfecção
13.
Int J Mol Sci ; 22(17)2021 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-34502287

RESUMO

Gene-directed enzyme prodrug gene therapy (GDEPT) theoretically represents a useful method to carry out chemotherapy for cancer with minimal side effects through the formation of a chemotherapeutic agent inside cancer cells. However, despite great efforts, promising preliminary results, and a long period of time (over 25 years) since the first mention of this method, GDEPT has not yet reached the clinic. There is a growing consensus that optimal cancer therapies should generate robust tumor-specific immune responses. The advent of checkpoint immunotherapy has yielded new highly promising avenues of study in cancer therapy. For such therapy, it seems reasonable to use combinations of different immunomodulators alongside traditional methods, such as chemotherapy and radiotherapy, as well as GDEPT. In this review, we focused on non-viral gene immunotherapy systems combining the intratumoral production of toxins diffused by GDEPT and immunomodulatory molecules. Special attention was paid to the applications and mechanisms of action of the granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that is widely used but shows contradictory effects. Another method to enhance the formation of stable immune responses in a tumor, the use of danger signals, is also discussed. The process of dying from GDEPT cancer cells initiates danger signaling by releasing damage-associated molecular patterns (DAMPs) that exert immature dendritic cells by increasing antigen uptake, maturation, and antigen presentation to cytotoxic T-lymphocytes. We hypothesized that the combined action of this danger signal and GM-CSF issued from the same dying cancer cell within a limited space would focus on a limited pool of immature dendritic cells, thus acting synergistically and enhancing their maturation and cytotoxic T-lymphocyte attraction potential. We also discuss the problem of enhancing the cancer specificity of the combined GDEPT-GM-CSF-danger signal system by means of artificial cancer specific promoters or a modified delivery system.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Imunoterapia/métodos , Neoplasias/terapia , Animais , Vacinas Anticâncer/farmacologia , Genes Transgênicos Suicidas , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Neoplasias/imunologia , Terapia Viral Oncolítica/métodos , Pró-Fármacos/farmacologia , Timidina Quinase/genética , Timidina Quinase/farmacologia
14.
Theranostics ; 11(17): 8254-8269, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34373740

RESUMO

Background: Mesenchymal stem cells (MSCs) have been applied as a promising vehicle for tumour-targeted delivery of suicide genes in the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) suicide gene therapy against malignant gliomas. The efficiency of this strategy is largely dependent on the bystander effect, which relies on high suicide gene expression levels and efficient transportation of activated GCV towards glioma cells. However, up to now, the methods to enhance the bystander effect of this strategy in an efficient and safe way are still lacking and new approaches to improve this therapeutic strategy are required. Methods: In this study, MSCs were gene transfected using magnetosome-like ferrimagnetic iron oxide nanochains (MFIONs) to highly express HSV-tk. Both the suicide and bystander effects of HSV-tk expressed MSCs (MSCs-tk) were quantitatively evaluated. Connexin 43 (Cx43) expression by MSCs and glioma cells was measured under different treatments. Intercellular communication between MSCs and C6 glioma cells was examined using a dye transfer assay. Glioma tropism and the bio-distribution of MSCs-tk were observed. Anti-tumour activity was investigated in the orthotopic glioma of rats after intravenous administration of MSCs-tk followed by intraperitoneal injection of GCV. Results: Gene transfection using MFIONs achieved sufficient expression of HSV-tk and triggered Cx43 overexpression in MSCs. These Cx43 overexpressing MSCs promoted gap junction intercellular communication (GJIC) between MSCs and glioma cells, resulting in significantly inhibited growth of glioma through an improved bystander effect. Outstanding tumour targeting and significantly prolonged survival with decreased tumour size were observed after the treatment using MFION-transfected MSCs in glioma model rats. Conclusion: Our results show that iron oxide nanoparticles have the potential to improve the suicide gene expression levels of transfected MSCs, while promoting the GJIC formation between MSCs and tumour cells, which enhances the sensitivity of glioma cells to HSV-tk/GCV suicide gene therapy.


Assuntos
Terapia Genética/métodos , Glioma , Nanopartículas Magnéticas de Óxido de Ferro/administração & dosagem , Células-Tronco Mesenquimais/metabolismo , Animais , Antivirais/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Conexina 43/genética , Conexina 43/metabolismo , Ganciclovir/farmacologia , Expressão Gênica/efeitos dos fármacos , Genes Transgênicos Suicidas , Glioma/tratamento farmacológico , Glioma/genética , Humanos , Ratos , Simplexvirus/genética , Timidina Quinase/genética , Timidina Quinase/farmacologia , Transfecção/métodos , Carga Tumoral/efeitos dos fármacos
15.
Virol J ; 18(1): 107, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059075

RESUMO

Reducing the pool of HIV-1 reservoirs in patients is a must to achieve functional cure. The most prominent HIV-1 cell reservoirs are resting CD4 + T cells and brain derived microglial cells. Infected microglial cells are believed to be the source of peripheral tissues reseedings and the emergence of drug resistance. Clearing infected cells from the brain is therefore crucial. However, many characteristics of microglial cells and the central nervous system make extremely difficult their eradication from brain reservoirs. Current methods, such as the "shock and kill", the "block and lock" and gene editing strategies cannot override these difficulties. Therefore, new strategies have to be designed when considering the elimination of brain reservoirs. We set up an original gene suicide strategy using latently infected microglial cells as model cells. In this paper we provide proof of concept of this strategy.


Assuntos
Encéfalo/virologia , Genes Transgênicos Suicidas , Infecções por HIV , HIV-1 , Latência Viral , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Edição de Genes , Humanos , Microglia/virologia
16.
Nat Commun ; 12(1): 3275, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34045451

RESUMO

Despite advancements in human pluripotent stem cells (hPSCs) differentiation protocols to generate appropriate neuronal progenitors suitable for transplantation in Parkinson's disease, resultant grafts contain low proportions of dopamine neurons. Added to this is the tumorigenic risk associated with the potential presence of incompletely patterned, proliferative cells within grafts. Here, we utilised a hPSC line carrying a FailSafeTM suicide gene (thymidine kinase linked to cyclinD1) to selectively ablate proliferative cells in order to improve safety and purity of neural transplantation in a Parkinsonian model. The engineered FailSafeTM hPSCs demonstrated robust ventral midbrain specification in vitro, capable of forming neural grafts upon transplantation. Activation of the suicide gene within weeks after transplantation, by ganciclovir administration, resulted in significantly smaller grafts without affecting the total yield of dopamine neurons, their capacity to innervate the host brain or reverse motor deficits at six months in a rat Parkinsonian model. Within ganciclovir-treated grafts, other neuronal, glial and non-neural populations (including proliferative cells), were significantly reduced-cell types that may pose adverse or unknown influences on graft and host function. These findings demonstrate the capacity of a suicide gene-based system to improve both the standardisation and safety of hPSC-derived grafts in a rat model of Parkinsonism.


Assuntos
Engenharia Celular/métodos , Genes Transgênicos Suicidas , Doença de Parkinson Secundária/terapia , Transplante de Células-Tronco/métodos , Animais , Apoptose/genética , Diferenciação Celular , Linhagem Celular , Proliferação de Células/genética , Modelos Animais de Doenças , Neurônios Dopaminérgicos/fisiologia , Feminino , Genes bcl-1/genética , Xenoenxertos/citologia , Xenoenxertos/patologia , Células-Tronco Embrionárias Humanas/fisiologia , Humanos , Masculino , Mesencéfalo/citologia , Mesencéfalo/patologia , Oxidopamina/administração & dosagem , Oxidopamina/toxicidade , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/patologia , Ratos , Transplante de Células-Tronco/efeitos adversos , Transplante de Células-Tronco/normas , Timidina Quinase/genética
17.
Cancer Med ; 10(9): 3085-3100, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33826244

RESUMO

Lung cancer is the most common cause of cancer-related death in developed countries; therefore, the generation of effective targeted therapeutic regimens is essential. Recently, gene therapy approaches toward malignant cells have emerged as attractive molecular therapeutics. Previous studies have indicated that stanniocalcin-1 (STC-1), a hormone involved in calcium and phosphate homeostasis, positively regulates proliferation, apoptosis resistance, and glucose metabolism in lung cancer cell lines. In this study, we investigated if targeting STC-1 in tumor cells could be a promising strategy for lung cancer gene therapy. We confirmed that STC-1 levels in peripheral blood were higher in lung cancer patients than in healthy donors and that STC-1 expression was observed in five out of eight lung cancer cell lines. A vector expressing a suicide gene, uracil phosphoribosyltransferase (UPRT), under the control of the STC-1 promoter, was constructed (pPSTC-1 -UPRT) and transfected into three STC-1-positive cell lines, PC-9, A549, and H1299. When stably transfected, we observed significant cell growth inhibition using 5-fluorouracil (5-FU) treatment. Furthermore, growth of the STC-1-negative lung cancer cell line, LK-2 was significantly arrested when combined with STC-1-positive cells transfected with pPSTC-1 -UPRT. We believe that conferring cytotoxicity in STC-1-positive lung cancer cells using a suicide gene may be a useful therapeutic strategy for lung cancer.


Assuntos
Terapia Genética/métodos , Glicoproteínas/metabolismo , Neoplasias Pulmonares/terapia , Terapia de Alvo Molecular/métodos , Pentosiltransferases/metabolismo , Células A549 , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Feminino , Fluoruracila/uso terapêutico , Genes Reporter , Genes Transgênicos Suicidas , Glucose/metabolismo , Glicoproteínas/sangue , Glicoproteínas/genética , Humanos , Luciferases/genética , Luciferases/metabolismo , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pentosiltransferases/genética , Plasmídeos , Regiões Promotoras Genéticas/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/genética , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Commun Biol ; 4(1): 165, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547399

RESUMO

Cataplexy is triggered by laughter in humans and palatable food in mice. To further evaluate mice's cataplexy, we examined courtship behavior in orexin neuron-ablated mice (ORX-AB), one of the animal models of narcolepsy/cataplexy. Wild-type female mice were placed into the home cage of male ORX-AB and cataplexy-like behavior was observed along with ultrasonic vocalizations (USVs), also known as the "love song". ORX-AB with a female encounter showed cataplexy-like behavior both during the dark and light periods, whereas ORX-AB with chocolate predominantly showed it during the dark period. During the light period observation, more than 85% of cataplexy-like bouts were preceded by USVs. A strong positive correlation was observed between the number of USVs and cataplexy-like bouts. Cataplexy-like behavior in narcoleptic mice is a good behavioral measure to study the brain mechanisms behind positive emotion because they can be induced by different kinds of positive stimuli, including chocolate and female courtship.


Assuntos
Cataplexia/patologia , Corte , Neurônios/patologia , Excitação Sexual , Vocalização Animal/fisiologia , Animais , Comportamento Animal/fisiologia , Cataplexia/genética , Cataplexia/fisiopatologia , Cataplexia/psicologia , Corte/psicologia , Genes Transgênicos Suicidas , Masculino , Camundongos , Camundongos Transgênicos , Narcolepsia/genética , Narcolepsia/patologia , Narcolepsia/fisiopatologia , Narcolepsia/psicologia , Neurônios/metabolismo , Orexinas/deficiência , Orexinas/genética , Orexinas/metabolismo
19.
Virol J ; 18(1): 31, 2021 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-33516234

RESUMO

BACKGROUND: Gene therapy approaches using hematopoietic stem cells to generate an HIV resistant immune system have been shown to be successful. The deletion of HIV co-receptor CCR5 remains a viable strategy although co-receptor switching to CXCR4 remains a major pitfall. To overcome this, we designed a dual gene therapy strategy that incorporates a conditional suicide gene and CCR5 knockout (KO) to overcome the limitations of CCR5 KO alone. METHODS: A two-vector system was designed that included an integrating lentiviral vector that expresses a HIV Tat dependent Thymidine Kinase mutant SR39 (TK-SR39) and GFP reporter gene. The second non-integrating lentiviral (NIL) vector expresses a CCR5gRNA-CRISPR/Cas9 cassette and HIV Tat protein. RESULTS: Transduction of cells sequentially with the integrating followed by the NIL vector allows for insertion of the conditional suicide gene, KO of CCR5 and transient expression of GFP to enrich the modified cells. We used this strategy to modify TZM cells and generate a cell line that was resistant to CCR5 tropic viruses while permitting infection of CXCR4 tropic viruses which could be controlled via treatment with Ganciclovir. CONCLUSIONS: Our study demonstrates proof of principle that a combination gene therapy for HIV is a viable strategy and can overcome the limitation of editing CCR5 gene alone.


Assuntos
Técnicas de Inativação de Genes , Genes Transgênicos Suicidas , Terapia Genética/métodos , Infecções por HIV/terapia , Receptores CCR5/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Células HEK293 , Humanos , Transdução Genética
20.
Biomolecules ; 11(1)2021 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-33477716

RESUMO

Herein we report the first proof for the application of type II 2'-deoxyribosyltransferase from Lactobacillus delbrueckii (LdNDT) in suicide gene therapy for cancer treatment. To this end, we first confirm the hydrolytic ability of LdNDT over the nucleoside-based prodrugs 2'-deoxy-5-fluorouridine (dFUrd), 2'-deoxy-2-fluoroadenosine (dFAdo), and 2'-deoxy-6-methylpurine riboside (d6MetPRib). Such activity was significantly increased (up to 30-fold) in the presence of an acceptor nucleobase. To shed light on the strong nucleobase dependence for enzymatic activity, different molecular dynamics simulations were carried out. Finally, as a proof of concept, we tested the LdNDT/dFAdo system in human cervical cancer (HeLa) cells. Interestingly, LdNDT/dFAdo showed a pronounced reduction in cellular viability with inhibitory concentrations in the low micromolar range. These results open up future opportunities for the clinical implementation of nucleoside 2'-deoxyribosyltransferases (NDTs) in cancer treatment.


Assuntos
Genes Transgênicos Suicidas , Nucleosídeos/farmacologia , Pentosiltransferases/metabolismo , Pró-Fármacos/farmacologia , Desoxiadenosinas/metabolismo , Fluoruracila/química , Fluoruracila/farmacologia , Glicosídeo Hidrolases/metabolismo , Glicosilação/efeitos dos fármacos , Células HeLa , Humanos , Lactobacillus/enzimologia , Simulação de Dinâmica Molecular , Nucleosídeos/química , Pró-Fármacos/química
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