RESUMO
High-level systemic delivery of viral vectors to tumors has proved problematic as a result of immune neutralization, nonspecific adhesion, and clearance of circulating viral particles. Some cell types localize to tumors in response to particular biological properties associated with tumor growth. Their use to deliver viral vectors to tumors would allow precious viral stocks to be protected until they can be released at high local concentrations. Here, we describe a mechanism by which retroviral vector production by T cells can be regulated by a tumor-specific trigger through engagement of a chimeric immune receptor (CIR) with its target antigen. The virus that is released from the T cells can also be transcriptionally targeted. Finally, we show that it is possible to use vector-loaded, antigen-triggered human T cells as therapeutic, tumor-specific vector delivery cells in models of both local intratumoral and systemic delivery to both lung and liver metastases. This strategy incorporates multiple levels of targeting into the delivery system at the stages of surface targeting, viral production, and gene expression.
Assuntos
Terapia Genética/métodos , Retroviridae/genética , Transcrição Gênica , Adenoviridae/genética , Animais , Adesão Celular , Citometria de Fluxo , Vetores Genéticos , Proteínas de Fluorescência Verde , Humanos , Células Jurkat , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Nus , Modelos Genéticos , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Linfócitos T/metabolismo , Linfócitos T/virologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
We aimed to use cell-based carriers to direct vector production to target sites for systemic therapy. We used T cells engineered to express a chimeric T cell receptor that can specifically recognize target cells expressing the tumor-associated carcinoembryonic antigen (CEA). These T cells were modified to produce a retrovirus under tight pharmacological control using the rapamycin-inducible transcriptional regulatory system. The retroviral vectors produced were transcriptionally targeted to CEA-expressing target cells. We found that vector production and transgene expression from these T cells in vitro was dependent on pharmacological induction and expression of CEA in target cells, respectively. Mice bearing metastatic tumors that received cell carriers delivering the HSVtk gene demonstrated a significant increase in survival, but only in response to pharmacological induction of vector production. Interestingly, the therapeutic effect required the presence of the tumor-specific chimeric receptor on T cells. Further studies demonstrated that systemic delivery of tumor-specific T cells to mice bearing metastatic tumors caused recruitment of nonspecific T cells to the tumor site. We hypothesize that this enhanced targeting to tumor sites is responsible for the efficiency of T cell-mediated retroviral gene transfer and that this principle can be used to enhance systemic therapies using immune-cell carriers.
Assuntos
Antígenos de Neoplasias/imunologia , Antígeno Carcinoembrionário/imunologia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Células Jurkat/virologia , Vírus da Leucemia Murina de Moloney/genética , Proteínas Recombinantes de Fusão/imunologia , Sirolimo/farmacologia , Transcrição Gênica/efeitos dos fármacos , Adenocarcinoma/patologia , Adenocarcinoma/secundário , Animais , Neoplasias Colorretais/patologia , Sistemas de Liberação de Medicamentos , Corantes Fluorescentes , Regulação Viral da Expressão Gênica , Genes Sintéticos , Vetores Genéticos/uso terapêutico , Humanos , Células Jurkat/transplante , Neoplasias Hepáticas Experimentais/secundário , Neoplasias Hepáticas Experimentais/terapia , Melanoma/patologia , Melanoma/secundário , Camundongos , Vírus da Leucemia Murina de Moloney/fisiologia , Especificidade de Órgãos , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Simplexvirus/enzimologia , Simplexvirus/genética , Sequências Repetidas Terminais , Timidina Quinase/genética , Transdução Genética , Transfecção , Proteínas Virais/genética , Replicação Viral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Replication-competent adenoviral vectors are potentially far more efficient than replication-defective vectors. However, for reasons of safety, there is a need to restrict viral replication both spatially, by limiting replication to certain cell types, and temporally. To control replication temporally, we have developed a system, based on the small-molecule dimerizer rapamycin, for regulating the replication of adenoviral vectors. In this system, one adenoviral vector, AdC4, expresses transcription factors whose activity is regulated by the non-immunosuppressive rapamycin analog AP21967. A second vector, Ad(Z12-I-E1aE1b19k), contains E1 genes placed downstream of binding sites for the regulated transcription factor. Co-infection of several cell lines by the vector pair leads to dimerizer-dependent E1 expression and an increase in viral replication, as shown by Southern blots and replication assays. Furthermore, expression of a reporter gene from a replication-defective vector, Ad-GM-CSF, can be augmented by up to 18-fold by co-infection with the pair of conditionally replicating vectors in the presence of dimerizer. Similar results are obtained when the vectors are directly injected into subcutaneous HT1080 xenograft tumors in nude mice. We believe that vectors based on this principle will be a useful additional tool to enhance efficiency and safety of gene delivery for anti-cancer therapy.