Gene Therapy and Molecular Biology最新文献

High grade malignant gliomas are genetically unstable, heterogeneous and highly infiltrative; all characteristics that lend glioma cells superior advantages in resisting conventional therapies. Unfortunately, the median survival time for patients with glioblastoma multiforme remains discouraging at 12-15 months from diagnosis. Neuroimmunologists/oncologists have focused their research efforts to harness the power of the immune system to improve brain tumor patient survival. In the past 30 years, small numbers of patients have been enrolled in a plethora of experimental immunotherapy Phase I and II trials. Some remarkable anecdotal responses to immune therapy are evident. Yet, the reasons for the mixed responses remain an enigma. The inability of the devised immunotherapies to consistently increase survival may be due, in part, to intrinsically-resistant glioma cells. It is also probable that the tumor compartment of the tumor-bearing host has mechanisms or produces factors that promote tumor tolerance and immune suppression. Finally, with adoptive immunotherapy of ex vivo activated effector cell preparations, the existence of suppressor T cells within them theoretically may contribute to immunotherapeutic failure. In this review, we will summarize our own studies with immunotherapy resistant glioma cell models, as well as cover other examined immunosuppressive factors in the tumor microenvironment and immune effector cell suppressor populations that may contribute to the overall immune suppression. An in-depth understanding of the obstacles will be necessary to appropriately develop strategies to overcome the resistance and improve survival in this select population of cancer patients.

Lentivirus vector systems have been developed for the safe delivery of foreign genes to target tissues. However, the use of these systems for delivering specific proteins to target cells has been largely unexplored. To test this concept, the lentivirus expression plasmid pLenti was utilized to overexpress in producer cells a YFP-fusion protein that is specifically targeted to glycolipid-enriched membrane rafts, which is the site of virus assembly. Our data show that virus generated in producer cells that expressed the YFP fusion protein were able to effectively label target cells by a 2-3 hr incubation with the virus. Labeling of the target cells was specific to the lentivirus, as it was blocked by pre-incubating the virus with antibody to the surface protein, and it was not affected by pre-treating the target cells with cyclohexamide. T cells that were labeled using the lentivirus underwent a robust stimulation following crosslinking the T cell receptor, thus showing that T cells labeled using lentivirus remained responsive to extracellular cues. Altogether, these results show that overexpression of foreign proteins in lentivirus producer cells can yield protein-loaded viruses, which can then function to deliver the protein to target cells. Thus, our findings suggest an avenue for targeting specific proteins to cells where foreign gene expression is not feasible.

The eukaryotic nucleus houses a significant amount of information that is carefully ordered to ensure that genes can be transcribed as needed throughout development and differentiation. The genome is partitioned into regions containing functional transcription units, providing the means for the cell to selectively activate some, while keeping other regions of the genome silent. Over the last quarter of a century the structure of chromatin and how it is influenced by epigenetics has come into the forefront of modern biology. However, it has thus far failed to identify the mechanism by which individual genes or domains are selected for expression. Through covalent and structural modification of the DNA and chromatin proteins, epigenetics maintains both active and silent chromatin states. This is the "other" genetic code, often superseding that dictated by the nucleotide sequence. The nuclear matrix is rich in many of the factors that govern nuclear processes. It includes a host of unknown factors that may provide our first insight into the structural mechanism responsible for the genetic selectivity of a differentiating cell. This review will consider the nuclear matrix as an integral component of the epigenetic mechanism.

Nucleic acid enzymes have been used with great success for studying natural processes in the central nervous system (CNS). We first provide information on the structural and enzymatic differences of various ribozymes and DNAzymes. We then discuss how they have been used to explore new therapeutic approaches for treating diseases of the CNS. They have been tested in various systems modeling retinitis pigmentosum, proliferative vitreoretinopathy, Alzheimer's disease, and malignant brain tumors. For these models, effective targets for nucleic acid enzymes have been readily identified and the rules for selecting cleavage sites have been well established. The bulk of studies, including those from our laboratory, have emphasized their use for gliomas. With the availability of multiple excellent animal models to test glioma treatments, good progress has been made in the initial testing of nucleic acid enzymes for brain tumor therapy. However, opportunities still exist to significantly improve the delivery and efficacy of ribozymes to achieve effective treatment. The future holds significant potential for the molecular targeting and therapy of eye diseases, neurodegenerative disorders, and brain tumors with these unique treatment agents.

Gene therapy strategies requiring long-term high-level expression from integrated genes are currently limited by inconsistent levels of expression. This may be observed as variegated, silenced or position-dependent gene expression. Each of these phenomena involve suppressive chromatin structures. We hypothesized that by actively conferring an open chromatin structure on integrated vectors would increase transgene expression. To test this idea we used a 100bp element from the β-globin locus control region (LCR) which is able to independently open local chromatin structure in erythroid tissues. This element includes binding sites for GATA-1, NF-E2, EKLF and Sp-1 and is evolutionarily conserved. We constructed a series of MSCV-based vectors containing the β-globin gene driven by a minimal β-globin promoter with combinations of the HSFE and LCR derived enhancer elements. Pools of MEL clones containing integrated vectors were analyzed for chromatin structure and β-globin gene expression. The HSFE increased the extent of nuclease sensitive chromatin over the promoters of the constructs. The most effective vector included tandem copies of the HSFE and produced a 5-fold increase in expression compared to the promoter alone. These results indicate that the HSFE is able to augment the opening of β-globin promoter chromatin structure and significantly increase gene expression in the context of an integrated retroviral vector.