RESUMEN

Bioorthogonal cycloaddition reactions between tetrazines and strained dienophiles are widely used for protein, lipid and glycan labelling because of their extremely rapid kinetics. However, controlling this chemistry in the presence of living mammalian cells with a high degree of spatial and temporal precision remains a challenge. Here we demonstrate a versatile approach to light-activated formation of tetrazines from photocaged dihydrotetrazines. Photouncaging, followed by spontaneous transformation to reactive tetrazine, enables live-cell spatiotemporal control of rapid bioorthogonal cycloaddition with dienophiles such as trans-cyclooctenes. Photocaged dihydrotetrazines are stable in conditions that normally degrade tetrazines, enabling efficient early-stage incorporation of bioorthogonal handles into biomolecules such as peptides. Photocaged dihydrotetrazines allow the use of non-toxic light to trigger tetrazine ligations on living mammalian cells. By tagging reactive phospholipids with fluorophores, we demonstrate modification of HeLa cell membranes with single-cell spatial resolution. Finally, we show that photo-triggered therapy is possible by coupling tetrazine photoactivation with strategies that release prodrugs in response to tetrazine ligation.

Asunto(s)

Compuestos Heterocíclicos , Animales , Reacción de Cicloadición , Colorantes Fluorescentes , Células HeLa , Humanos , Mamíferos , Proteínas

RESUMEN

Activating mutations in the small GTPase NRAS are responsible for driving tumor growth in several cancers. Unfortunately, the development of NRAS inhibitors has proven difficult due to the lack of hydrophobic binding pockets on the protein's surface. To overcome this limitation, we chose to target the post-translational S-palmitoyl modification of NRAS, which is required for its signaling activity. Utilizing an amphiphile-mediated depalmitoylation (AMD) strategy, we demonstrate the ability to directly cleave S-palmitoyl groups from NRAS and inhibit its function. C8 alkyl cysteine causes a dose-dependent decrease in NRAS palmitoylation and inhibits downstream signaling in melanoma cells with an activating mutation in NRAS. This compound reduces cell growth in NRAS-driven versus non-NRAS-driven melanoma lines and inhibits tumor progression in an NRAS-mutated melanoma xenograft mouse model. Our work demonstrates that AMD can effectively suppress NRAS activity and could represent a promising new avenue for discovering lead compounds for treatment of NRAS-driven cancers.

Asunto(s)

GTP Fosfohidrolasas/antagonistas & inhibidores , Lipoilación , Melanoma/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , GTP Fosfohidrolasas/metabolismo , Células HeLa , Humanos , Melanoma/patología , Proteínas de la Membrana/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Neoplasias Cutáneas/patología

RESUMEN

Amphiphilic molecules self-assemble into supramolecular structures of various sizes and morphologies depending on their molecular packing and external factors. Transformations between various self-assembled morphologies are a matter of great fundamental interest. Recently, we reported the discovery of a novel class of single-chain galactopyranosylamide amphiphiles that self-assemble to form vesicles in water. Here, we describe how the vesicles composed of the amphiphile N-oleoyl ß-d-galactopyranosylamine (GOA) undergo a morphological transition to fibers consisting of mainly flat sheet-like structures. Moreover, we show that this transformation is reversible in a temperature-dependent manner. We used several optical microscopy and electron microscopy techniques, circular dichroism spectroscopy, small-angle X-ray scattering, and differential scanning calorimetry, to fully investigate and characterize the morphological transformations of GOA and provide a structural basis for such phenomena. These studies provide significant molecular insight into the structural polymorphism of sugar-based amphiphiles and foresee future applications in rational design of self-assembled materials.

Asunto(s)

Agua , Rastreo Diferencial de Calorimetría , Temperatura

RESUMEN

Tumor-specific adenoviral vectors comprise a fruitful gene-based diagnostic imaging and therapy research area for advanced stage of cancer, including metastatic disease. However, clinical translation of viral vectors has encountered considerable obstacles, largely due to host immune responses against the virus. Here, we explored the utilization of an immunosuppressant, rapamycin, to circumvent the anti-adenovirus immunity in immunocompetent murine prostate cancer models. Rapamycin diminished adenoviral-induced acute immune response by inhibiting NF-κB activation; it also reduced the scale and delayed the onset of inflammatory cytokine secretion. Further, we found that rapamycin abrogated anti-adenovirus antibody production and retarded the function of myeloid cells and lymphocytes that were activated upon viral administration in pre-immunized hosts. Thus, the co-administration of rapamycin prolonged and enhanced adenovirus-delivered transgene expression in vivo, and thereby augmented the imaging capability of adenoviral vectors in both bioluminescent and positron emission tomography modalities. Furthermore, we showed that despite an excellent response of cancer cells to a cytotoxic gene therapeutic vector in vitro, only minimal therapeutic effects were observed in vivo in pre-immunized mice. However, when we combined gene therapy with transient immunosuppression, complete tumor growth arrest was achieved. Overall, transient immunosuppression by rapamycin was able to boost the diagnostic utility and therapeutic potentials of adenoviral vectors.

Asunto(s)

Adenoviridae/genética , Terapia de Inmunosupresión/métodos , Inmunosupresores/farmacología , Imagen Molecular , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/terapia , Sirolimus/farmacología , Inmunidad Adaptativa/efectos de los fármacos , Animales , Línea Celular Tumoral , Ganciclovir/farmacología , Terapia Genética/efectos adversos , Vectores Genéticos/genética , Inmunidad Innata/efectos de los fármacos , Inmunización , Masculino , Ratones , Imagen Molecular/efectos adversos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/inmunología , Seguridad , Timidina Quinasa/genética , Transgenes/genética

RESUMEN

We sought to modify adenoviral (Ad) particles by incorporating the advantageous characteristics of non-viral gene delivery vehicles to complement the viral vectors. α-Amino acid-N-carboxyanhydride chemistry was used to synthesize homopolypeptides and diblock copolypeptides that possess well-defined secondary structures. Using cryo-electron and fluorescence microscopy, we showed that these polypeptides can coat the surfaces of Ad particles in a non-covalent manner to modify their transduction properties. The coated Ad particles were found to bind to and be internalized by cells. In contrast to reports using covalently PEGylated Ad particles, we found that our physically coated Ad hybrid complexes facilitate gene transfer both in vitro and in vivo. We showed that our polypeptide coating was able to shield the Ad particles from the neutralizing effect of antibodies and mitigate the binding of blood coagulation factor (Factor X) in vitro. The coating also reduced the antigenicity of Ad in immunocompetent mice. The biodistribution of the systemically administered hybrid complexes mirrored the behavior of both viral and non-viral vectors, exhibiting liver tropism as well as enhanced lung transduction. These data demonstrated that our non-covalent modification was able to alter Ad's interactions with cells and organs with retention of transduction efficiency. Advantages such as facile coating of the Ad vector, design flexibility and ease of attaching ligands to the polypeptides make this system potentially useful as a platform for adding functionalities to Ad to target cancer metastasis.

Asunto(s)

Adenoviridae/genética , Portadores de Fármacos/química , Técnicas de Transferencia de Gen , Vectores Genéticos , Péptidos/química , Transducción Genética , Animales , Anticuerpos Antivirales/sangre , Línea Celular , Microscopía por Crioelectrón , Estabilidad de Medicamentos , Proteínas Fluorescentes Verdes/genética , Humanos , Luciferasas de Luciérnaga/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Microscopía Fluorescente , Tamaño de la Partícula , Dispersión de Radiación , Propiedades de Superficie

RESUMEN

UNLABELLED: Better intraprostatic cancer imaging techniques are needed to guide clinicians in prostate cancer treatment decisions. Because many genes are specifically overexpressed in cancer cells, one strategy to improve prostate cancer detection is to image intraprostatic cancer-specific transcriptional activity. Because of the obstacles of weak cancer- or tissue-specific promoter activity and bladder clearance of many PET tracers, intraprostatic PET of gene transcriptional activity has not been previously reported. METHODS: The two-step transcriptional amplification (TSTA) system that amplifies the prostate-specific antigen promoter activity was used for PET imaging of the reporter gene herpes simplex virus type-1 sr39 thymidine kinase (HSV1-sr39tk). The TSTA-sr39tk system was injected directly into prostates or prostatic tumors as a replication-incompetent adenovirus (AdTSTA-sr39tk) and imaged using PET. RESULTS: AdTSTA-sr39tk was able to image prostate-specific antigen promoter transcriptional activity by 9-(4-(18)F-fluoro-3-[hydroxymethyl]butyl)guanine PET, in both mouse and canine prostates in vivo. Ex vivo small-animal PET images, scintigraphic counts, and sr39tk expression analysis confirmed the specificity of the observed signal. CONCLUSION: Here, by combining the TSTA-amplified signal with a protocol for tracer administration, we show that in vivo PET detection of transcriptional activity is possible in both mouse and immunocompetent canine prostates. These results suggest that imaging applications using transcription-based tumor-specific promoters should be pursued to better visualize cancer foci that escape detection by conventional biopsies.

Asunto(s)

Tomografía de Emisión de Positrones , Próstata/metabolismo , Transcripción Genética , Adenoviridae/genética , Animales , Perros , Estudios de Factibilidad , Genes Reporteros/genética , Células HEK293 , Herpesvirus Humano 1/enzimología , Humanos , Inmunocompetencia/genética , Masculino , Ratones , Estadificación de Neoplasias , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , Antígeno Prostático Específico/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Timidina Quinasa/genética

RESUMEN

Tumor-infiltrating myeloid cells (TIMs) support tumor growth by promoting angiogenesis and suppressing antitumor immune responses. CSF-1 receptor (CSF1R) signaling is important for the recruitment of CD11b(+)F4/80(+) tumor-associated macrophages (TAMs) and contributes to myeloid cell-mediated angiogenesis. However, the impact of the CSF1R signaling pathway on other TIM subsets, including CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs), is unknown. Tumor-infiltrating MDSCs have also been shown to contribute to tumor angiogenesis and have recently been implicated in tumor resistance to antiangiogenic therapy, yet their precise involvement in these processes is not well understood. Here, we use the selective pharmacologic inhibitor of CSF1R signaling, GW2580, to demonstrate that CSF-1 regulates the tumor recruitment of CD11b(+)Gr-1(lo)Ly6C(hi) mononuclear MDSCs. Targeting these TIM subsets inhibits tumor angiogenesis associated with reduced expression of proangiogenic and immunosuppressive genes. Combination therapy using GW2580 with an anti-VEGFR-2 antibody synergistically suppresses tumor growth and severely impairs tumor angiogenesis along with reverting at least one TIM-mediated antiangiogenic compensatory mechanism involving MMP-9. These data highlight the importance of CSF1R signaling in the recruitment and function of distinct TIM subsets, including MDSCs, and validate the benefits of targeting CSF1R signaling in combination with antiangiogenic drugs for the treatment of solid cancers.

Asunto(s)

Anisoles/farmacología , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Movimiento Celular/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Pirimidinas/farmacología , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Animales , Carcinoma Pulmonar de Lewis/patología , Línea Celular Tumoral , Neoplasias Pulmonares/patología , Macrófagos/citología , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Células Mieloides/efectos de los fármacos , Células Mieloides/patología , Trasplante de Neoplasias , Neovascularización Patológica/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Ratas , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

Imaging in medicine has been classically based on the anatomical description of organs. In the past 15 years, new imaging techniques based on gene expression that characterize a pathological process have been developed. Molecular imaging is the use of such molecules to image cell-specific characteristics. Here, we review recent advances in molecular imaging, taking as our prime example lymph node (LN) metastasis in prostate cancer. We describe the new techniques and compare their accuracy in detecting LN metastasis in prostate cancer. We also present new molecular strategies for improving tumor detection using adenoviruses, molecular promoters and amplification systems. Finally, we present the concept of 'in vivo pathology', which envisages using molecular imaging to accurately localize metastatic lesions based on the molecular signature of the disease.

Asunto(s)

Diagnóstico por Imagen/métodos , Metástasis Linfática/diagnóstico por imagen , Neoplasias de la Próstata/patología , Animales , Humanos , Metástasis Linfática/diagnóstico , Metástasis Linfática/patología , Imagen por Resonancia Magnética , Masculino , Tomografía de Emisión de Positrones , Neoplasias de la Próstata/diagnóstico , Radiografía

RESUMEN

UNLABELLED: Because of its high selectivity and specificity for the imaging reporter probe 9-(4-(18)F-fluoro-3-[hydroxymethyl]butyl)guanine ((18)F-FHBG), the herpes simplex virus type 1 thymidine kinase (HSV1-tk) variant sr39tk is actively being studied as a PET reporter gene. We recently demonstrated the capability of using a prostate-specific transcriptional amplification PET reporter vector, AdTSTA-sr39tk, to target prostate cancer lymph node metastasis. However, one area that warrants further study is the examination of the sensitivity of PET by determining the minimum percentage of cells expressing the sr39tk transgene needed for detection. Addressing this question could determine the sensitivity of vector-mediated sr39tk PET in cancer-targeting strategies. METHODS: DU-145, PC-3, and CWR22Rv.1 prostate cancer cell lines (a total of 1 x 10(6) cells) were studied, of which 7%, 10%, 25%, 50%, or 70% were transduced with the lentiviral vector constitutively expressing HSV1-sr39tk-IRES-enhanced green fluorescent protein (EGFP). Cells were subcutaneously implanted into the left shoulder of severe combined immunodeficient mice and evaluated. Tumor cells comparably transduced with an EGFP control vector were implanted on the right shoulder. Mice were imaged using PET with (18)F-FHBG at 8, 15, and 22 d after tumor implant. On day 23, tumors were isolated and analyzed for sr39tk transgene expression by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR), Western blotting, immunohistochemistry, and flow cytometry for EGFP expression. RESULTS: Results showed a linear relationship between the level of sr39tk expression and the quantity of tracer accrual in DU-145, with the minimal value for PET detection at 10%. The magnitude of tracer retention in sr39tk-expressing cells was amplified over time as the tumor grew. Protein levels in the stepwise titration increased with the percentage of sr39tk-transduced cells. CONCLUSION: The stepwise titration of prostate cancer cells transduced with the lenti-CMV-sr39tk-IRES-EGFP determined the minimum number of sr39tk-expressing tumor cells necessary to be detected by PET using the (18)F-FHBG reporter probe. Furthermore, PET signal correlated well with traditional methods of protein evaluation such as flow cytometry, quantitative RT-PCR, Western blotting, and immunohistochemistry. Unlike the traditional methods, however, the use of PET is noninvasive and will be more advantageous in clinical situations.

Asunto(s)

Guanina/análogos & derivados , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/genética , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/metabolismo , Timidina Quinasa/farmacocinética , Animales , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos/métodos , Expresión Génica , Guanina/farmacocinética , Lentivirus/genética , Masculino , Ratones , Técnicas de Sonda Molecular , Tomografía de Emisión de Positrones/métodos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Radiofármacos/farmacocinética , Timidina Quinasa/genética , Transducción Genética/métodos

RESUMEN

The accurate assessment of nodal involvement in prostate cancer is crucial to planning treatment, yet there is a shortage of noninvasive imaging techniques capable of visualizing nodal lesions directly. This study demonstrates the feasibility of using recombinant human adenoviral vectors to detect nodal metastases in a human prostate cancer model. This was achieved by the prostate-restricted expression of optical and positron emission tomography (PET) imaging reporter genes by the viral vector coupled with the innate lymphotropic properties of adenovirus. We show that peritumoral administration of these vectors results in the direct detection of reporter gene expression in metastatic lesions within sentinel lymph nodes. Notably, this approach parallels the current lymphoscintigraphy method but enables the direct PET visualization of sentinel lymph node metastases, eliminating the need for invasive lymphadenectomy. These findings may lead to more effective diagnostic and therapeutic strategies for individuals with advanced-stage prostate cancer.

Asunto(s)

Adenoviridae/genética , Adenoviridae/metabolismo , Regulación Viral de la Expresión Génica , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Animales , Humanos , Indoles , Ganglios Linfáticos/patología , Metástasis Linfática , Masculino , Ratones , Ratones Transgénicos , Metástasis de la Neoplasia , Trasplante de Neoplasias , Tomografía de Emisión de Positrones/métodos , Tomografía Computarizada por Rayos X/métodos

RESUMEN

Prostate cancer dissemination is difficult to detect in the clinic, and few treatment options exist for patients with advanced-stage disease. Our aim was to investigate the role of tumor lymphangiogenesis during metastasis. Further, we implemented a noninvasive molecular imaging technique to facilitate the assessment of the metastatic process. The metastatic potentials of several human prostate cancer xenograft models, LAPC-4, LAPC-9, PC3 and CWR22Rv-1 were compared. The cells were labeled with luciferase, a bioluminescence imaging reporter gene, to enable optical imaging. After tumor implantation the animals were examined weekly during several months for the appearance of metastases. Metastatic lesions were confirmed by immunohistochemistry. Additionally, the angiogenic and lymphangiogenic profiles of the tumors were characterized. To confirm the role of lymphangiogenesis in mediating metastasis, the low-metastatic LAPC-9 tumor cells were engineered to overexpress VEGF-C, and the development of metastases was evaluated. Our results show CWR22Rv-1 and PC3 tumor cell lines to be more metastatic than LAPC-4, which in turn disseminates more readily than LAPC-9. The difference in metastatic potential correlated with the endogenous production levels of lymphangiogenic growth factor VEGF-C and the presence of tumor lymphatics. In agreement, induced overexpression of VEGF-C in LAPC-9 enhanced tumor lymphangiogenesis leading to the development of metastatic lesions. Taken together, our studies, based on a molecular imaging approach for semiquantitative detection of micrometastases, point to an important role of tumor lymphatics in the metastatic process of human prostate cancer. In particular, VEGF-C seems to play a key role in prostate cancer metastasis.

Asunto(s)

Vasos Linfáticos/fisiología , Neoplasias de la Próstata/patología , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Metástasis de la Neoplasia , Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/metabolismo , Factor C de Crecimiento Endotelial Vascular/genética , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

A better understanding of the in vivo biodistribution of adenoviral vectors would enable the researcher to anticipate potential side effects due to off-targeted site of transduction, and aid in the strategic design of gene therapy. We combined real-time polymerase chain reaction with in vivo optical imaging to examine viral transduction in liver, lung, spleen, kidney, prostate, and lymph nodes. A replication-deficient serotype 5 adenoviral vector expressing the firefly luciferase gene under the control of a constitutive cytomegalovirus promoter was administered in vivo via different routes. Intravenous and intraperitoneal injections resulted in greatest gene expression and viral DNA in the liver, whereas intraperitoneal injections led to a greater extent of gene delivery to the prostate. Although prostate-directed injection resulted in dominant gene expression in the targeted site, leakage of the vector to other organs was also observed. Vector injection into the lymphatic-rich paw tissue or the subcutaneous tissue of shoulder or chest followed the expected lymphatic drainage pattern, resulting in the accumulation of viral vector in ipsilateral brachial and axillary lymph nodes. Collectively, this study demonstrates that each tissue retains various amounts of adenoviral vector, depending on the route of administration. This knowledge is useful in the strategic design and implementation of adenovirus-mediated gene therapies.

Asunto(s)

Adenoviridae/genética , Vectores Genéticos , Animales , Secuencia de Bases , Cartilla de ADN/genética , Expresión Génica , Terapia Genética/métodos , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Hígado/virología , Luciferasas de Luciérnaga/genética , Mediciones Luminiscentes , Ganglios Linfáticos/virología , Masculino , Ratones , Ratones SCID , Reacción en Cadena de la Polimerasa , Próstata/virología , Proteínas Recombinantes/genética , Distribución Tisular

RESUMEN

Significant advances in gene therapy have been made as a result of the improvement of gene delivery systems, discovery of new therapeutic genes, better understanding of mechanisms of disease progression, exploration and improvement of tissue-specific gene regulatory sequences, and development of better prodrug/enzyme systems. This review discusses adenoviral-based and prostate-specific cancer gene therapy--emphasizing tissue-specific promoter choices to increase gene therapy safety and specificity--and the development of prostate-targeted vectors, with a focus on the two-step transactivation system for amplifying gene expression, specifically in prostate cancer cells. Several examples will be discussed for the scientific basis and therapeutic applications. In addition, prostate cancer gene therapy clinical trials and future directions in this field will also be described briefly.

Asunto(s)

Terapia Genética , Neoplasias de la Próstata/terapia , Animales , Técnicas de Transferencia de Gen , Humanos , Masculino , Neoplasias de la Próstata/genética

RESUMEN

Cancer gene therapy based on tissue-restricted expression of cytotoxic gene should achieve superior therapeutic index over an unrestricted method. This study compared the therapeutic effects of a highly augmented, prostate-specific gene expression method to a strong constitutive promoter-driven approach. Molecular imaging was coupled to gene therapy to ascertain real-time therapeutic activity. The imaging reporter gene (luciferase) and the cytotoxic gene (herpes simplex thymidine kinase) were delivered by adenoviral vectors injected directly into human prostate tumors grafted in SCID mice. Serial bioluminescence imaging, positron emission tomography, and computed tomography revealed restriction of gene expression to the tumors when prostate-specific vector was employed. In contrast, administration of constitutive active vector resulted in strong signals in the liver. Liver serology, tissue histology, and frail condition of animals confirmed liver toxicity suffered by the constitutive active cohorts, whereas the prostate-targeted group was unaffected. The extent of tumor killing was analyzed by apoptotic staining and human prostate marker (prostate-specific antigen). Overall, the augmented prostate-specific expression system was superior to the constitutive approach in safeguarding against systemic toxicity, while achieving effective tumor killing. Integrating noninvasive imaging into cytotoxic gene therapy will provide a useful strategy to monitor gene expression and therapeutic efficacy in future clinical protocols.

Asunto(s)

Genes Transgénicos Suicidas , Terapia Genética , Tomografía de Emisión de Positrones , Neoplasias de la Próstata/diagnóstico por imagen , Timidina Quinasa/genética , Animales , Masculino , Ratones , Ratones SCID , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/terapia , Timidina Quinasa/metabolismo , Tomografía Computarizada por Rayos X , Trasplante Heterólogo

RESUMEN

PURPOSE: A highly augmented, prostate-specific two-step transcriptional amplification (TSTA) method was developed with the ultimate goal of delivering an effective and safe gene-based treatment to prostate cancer patients. Because very limited treatment options are available for recurrent hormone refractory prostate cancer (HRPC), it is imperative to assess whether the prostate-specific antigen (PSA) promoter-based TSTA gene therapy will be functional in HRPC. EXPERIMENTAL DESIGN: We tested the TSTA-driven adenovirus vector on three androgen-dependent and six HRPC models. Real-time gene expression was monitored by both optical imaging and the combined modality of positron emission tomography (PET) and computed tomography. RESULTS: The TSTA-driven firefly luciferase expressing adenoviral vector was active in all androgen receptor (AR)-expressing HRPC models, but inactive in AR- and PSA-negative lines. Interestingly, the TSTA-mediated gene expression was induced by hydrocortisone in MDA PCa 2b, a cell line with mutated AR that possesses altered ligand specificity. In animal models, the TSTA-mediated optical signal was more robust in the HRPC than androgen-dependent tumors. In a parallel trend, a TSTA vector that expresses the herpes simplex virus thymidine kinase PET reporter gene also displayed more robust PET signal in the HRPC tumor. CONCLUSIONS: The activity of TSTA system is AR dependent and it recapitulates the functional status of endogenous AR. These data support the conclusion that AR function is activated in HRPC despite castrated levels of androgen. Together with the fact that majority of recurrent prostate cancers express AR and PSA, we foresee that the TSTA approach can be a promising gene therapy strategy for the advanced stages of prostate cancer.

Asunto(s)

Perfilación de la Expresión Génica , Terapia Genética , Antígeno Prostático Específico/biosíntesis , Neoplasias de la Próstata/terapia , Receptores Androgénicos/genética , Adenoviridae , Antiinflamatorios/farmacología , Resistencia a Antineoplásicos , Femenino , Amplificación de Genes , Vectores Genéticos , Humanos , Hidrocortisona/farmacología , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Masculino , Tomografía de Emisión de Positrones , Regiones Promotoras Genéticas , Antígeno Prostático Específico/análisis , Neoplasias de la Próstata/genética , Receptores Androgénicos/fisiología , Tomografía Computarizada por Rayos X , Transcripción Genética , Células Tumorales Cultivadas

RESUMEN

Targeted gene transduction to specific tissues and organs through intravenous injection would be the ultimate preferred method of gene delivery. Here, we report successful targeting in a living animal through intravenous injection of a lentiviral vector pseudotyped with a modified chimeric Sindbis virus envelope (termed m168). m168 pseudotypes have high titer and high targeting specificity and, unlike other retroviral pseudotypes, have low nonspecific infectivity in liver and spleen. A mouse cancer model of metastatic melanoma was used to test intravenous targeting with m168. Human P-glycoprotein was ectopically expressed on the surface of melanoma cells and targeted by the m168 pseudotyped lentiviral vector conjugated with antibody specific for P-glycoprotein. m168 pseudotypes successfully targeted metastatic melanoma cells growing in the lung after systemic administration by tail vein injection. Further development of this targeting technology should result in applications not only for cancers but also for genetic, infectious and immune diseases.

Asunto(s)

Marcación de Gen/métodos , Melanoma Experimental/terapia , Virus Sindbis/genética , Animales , Terapia Genética/métodos , Vectores Genéticos , Lentivirus/genética , Luciferasas/biosíntesis , Melanoma Experimental/secundario , Ratones , Proteínas del Envoltorio Viral/genética

RESUMEN

Gene-based therapy is a promising and flexible therapeutic approach to manage diverse types of cancer. The lack of convincing therapeutic success of current gene therapy protocols in part, can be attributed to the inability to monitor gene expression at the targeted site in the living subject. Linking molecular imaging to gene therapy will enable real-time assessment of the therapeutic process and the refinement of treatment protocols. This review will cover two common imaging modalities, positron emission tomography (PET) and bioluminescence imaging (BLI), used in pre-clinical and clinical gene therapy applications. Strategies to develop more specific and robust cancer gene therapy and imaging approaches will be discussed. Coupling PET to gene therapy of cancer has already been implemented in several clinical studies. This approach would help to improve the efficacy and safety of future gene therapy clinical trials.

Asunto(s)

Perfilación de la Expresión Génica/métodos , Terapia Genética/métodos , Mediciones Luminiscentes , Neoplasias/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Expresión Génica , Genes Reporteros , Marcadores Genéticos , Humanos , Luciferasas , Sustancias Luminiscentes , Modelos Biológicos , Neoplasias/genética , Transcripción Genética , Transducción Genética

RESUMEN

Gene expression-based imaging coupled to gene therapy will permit the prediction of therapeutic outcome. A significant challenge for successful gene therapy is to achieve a high-level of specific gene expression; however, tissue-specific promoters are weak. We postulate that if the weak activity of tissue-specific promoters can be amplified to the levels of strong viral promoters, which have been successful in preclinical scenarios, while retaining specificity, the therapeutic index of gene therapy can be greatly augmented. With this in mind, we developed a two-step transcriptional activation (TSTA) system. In this two-tiered system, a modified prostate-specific antigen promoter was employed to drive a potent synthetic transcriptional activator, GAL4-VP2. This, in turn, activated the expression of a GAL4-dependent reporter or therapeutic gene. Here we demonstrate that recombinant adenoviral vectors (Ads) in which we have incorporated prostate-targeted TSTA expression cassettes retain cell specificity and androgen responsiveness in cell culture and in animal models, as measured by noninvasive optical bioluminescence imaging. We investigated the mechanism of TSTA in different adenoviral configurations. In one configuration, both the activator and the reporter components are inserted into a single Ad (AdTSTA-FL). The activity of AdTSTA-FL exceeds that of a cytomegalovirus promoter-driven vector (AdCMV-FL), while maintaining tissue specificity. When the activator and reporter components are placed in two separate Ads, androgen induction is more robust than for the single AdTSTA-FL. Based on these findings, we hope to refine the TSTA Ads further to improve the efficacy and safety of prostate cancer gene therapy.

Asunto(s)

Adenoviridae/genética , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/terapia , Andrógenos/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Femenino , Genes Reporteros , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Ratones , Ratones SCID , Microscopía por Video , Modelos Genéticos , Trasplante de Neoplasias , Regiones Promotoras Genéticas , Factores de Tiempo , Transcripción Genética , Activación Transcripcional

RESUMEN

The greatest challenge in cancer treatment is to achieve the highest levels of specificity and efficacy. Cancer gene therapy could be designed specifically to express therapeutic genes to induce cancer cell destruction. Cancer-specific promoters are useful tools to accomplish targeted expression; however, high levels of gene expression are needed to achieve therapeutic efficacy. Incorporating an imaging reporter gene in tandem with the therapeutic gene will allow tangible proof of principle that gene expression occurs at the correct location and at a sufficient level. Gene-based imaging can advance cancer detection and diagnosis. By combining the cancer-targeted imaging and therapeutic strategies, the exciting prospect of a 'one-two punch' to find hidden, disseminated cancer cells and destroy them simultaneously can potentially be realized.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Terapia Genética/métodos , Neoplasias/genética , Neoplasias/terapia , Transcripción Genética/genética , Animales , Humanos , Neoplasias/diagnóstico , Neoplasias/patología , Especificidad de Órganos

RESUMEN

The early androgen-dependent (AD) phase of prostate cancer is dependent on the androgen receptor (AR). However, it is unclear whether AR is fully functional in recurrent prostate cancer after androgen withdrawal. To address this issue we interrogated AR signaling in AD and recurrent prostate cancer xenografts using molecular imaging, chromatin immunoprecipitation, and immunohistochemistry. In the imaging experiments, an adenovirus bearing a two-step transcriptional activation cassette, which amplifies AR-dependent firefly luciferase reporter gene activity, was injected into tumors implanted into severe combined immunodeficiency mice. A charge-coupled device optical imaging system detected the initial loss and then resumption of AR transcriptional activity in D-luciferin-injected mice as tumors transitioned from AD to recurrent growth. The results of chromatin immunoprecipitation and immunohistochemical localization experiments correlated with the Ad two-step transcriptional activation imaging signal. AR localized to the nucleus and bound to the endogenous prostate-specific antigen enhancer in AD tumors but exited the nucleus and dissociated from the enhancer upon castration. However, AR reentered the nucleus and rebound the prostate-specific antigen enhancer as the cancer transitioned into the recurrent phase. Surprisingly, RNA polymerase II and the general factor TFIIB remained bound to the gene throughout the transition. Our data support the concept that AR is fully functional in recurrent cancer and suggest a model by which a poised but largely inactive transcription complex facilitates reactivation by AR at castrate levels of ligand.

Asunto(s)

Neoplasias Hormono-Dependientes/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/fisiología , Adenoviridae/genética , Animales , Cromatina/genética , Cromatina/metabolismo , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones SCID , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Trasplante de Neoplasias , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/patología , Pruebas de Precipitina , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Transducción de Señal , Activación Transcripcional , Trasplante Heterólogo , Células Tumorales Cultivadas