A transient three-plasmid expression system for the production of high titer retroviral vectors.

We have constructed a series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and carried on plasmids containing SV40 origins of replication. These two features greatly enhanced retroviral gene expression when introduced into cell lines carrying the SV40 large T antigen. The two packaging components, gag-pol and env, were placed on separate plasmids to reduce helper virus formation. Using a highly transfectable human cell line and sodium butyrate to further increase expression of each component, we achieved helper-free viral stocks of approximately 10(7) infectious units/ml by 48 h after transient co-transfection with the three plasmid components. This system can be used both for the generation of high titer retroviral stocks for transduction and for the rapid screening of a large number of MLV gag-pol or env mutants.     

Targeted vectors for gene therapy of cancer and retroviral infections

Gene therapy has developed to a technology which rapidly moved from the laboratory bench to the bedside in the clinic. This implies safe, efficient and targeted gene transfer systems for suitable application to the patient. Beside the development of such gene transfer vectors of viral or nonviral origin, improvement of cell type specific and inducible gene expression is pivotal for successful gene therapy leading to targeted gene action. Numerous gene therapy approaches for treatment of cancer and retroviral infections utilize cell type specific and/or regulatable promoter and enhancer sequences for the selective expression of therapeutic genes in the desired cell populations and tissues. In this article the recent developments and the potential of expression targeting are reviewed for gene therapy approaches of cancer and retroviral infections.     

An essential role for the His-8 residue of the SDF-1alpha-chimeric, tropism-redirected Env protein of the Moloney murine leukemia virus in regulating postbinding fusion events

BACKGROUND: To use retroviral vectors for the cell-specific delivery of genes, it is necessary to redirect their receptor tropism to cell-specific receptors. Previously, we reported that a Moloney murine leukemia virus (MLV) retroviral vector containing a human stromal-derived factor-1alpha (SDF-1alpha)-chimeric envelope protein (Env) (S3) acquired the ability to transduce human cells via CXCR4, the cognate receptor for SDF-1alpha, while retaining the ability to transduce mouse cells via mCAT1. METHODS: We constructed expression plasmids for derivatives of the S3 Env protein; S3-D84K containing an Asp-84-to-Lys (D84K) substitution, S3-H8R-D84K containing D84K and an additional His-8-to-Arg substitution, and S3-D84K-RY containing D84K and additional Gln-227-to-Arg plus Asp-243-to-Tyr substitutions which have been suggested to suppress the loss of function of His-8. Cellular expression, virion incorporation, and entry functions of these derivatives were investigated. RESULTS: All three derivatives were incorporated into virions. The S3-D84K vector lost its ecotropism, but could transduce CXCR4-expressing human and mouse cells at titers of 10(3) to 10(4) colony-forming units (cfu)/ml. The S3-H8R-D84K vector did not show transduction, although its Env protein could bind to CXCR4. The transduction titer of the S3-D84K-RY vector via CXCR4 was slightly lower than that of the S3-D84K vector. These results indicate that the His-8 residue of the S3-D84K Env protein is indispensable and may be fully functional in postbinding membrane fusion. CONCLUSIONS: Insertion of a ligand at Pro-79 of the Moloney MLV Env protein has proved to be a valuable strategy for constructing direct targeting retroviral vectors, since it permits the formation of a redirected Env protein without ecotropism, and it does not disrupt the function of the essential His-8 residue.     

A series of vectors that simplify mammalian gene targeting

In order to facilitate the procedure of mammalian gene targeting, we have produced and functionally tested a series of generic vectors. Homologous recombination has been achieved with each vector. The vectors are designed for both replacement and insertional recombination, are suitable for hit and run strategies and contain all necessary genetic elements for both positive-negative and promoterless/gene fusion enrichment of homologous integrations. Multiple unique restriction sites are included to simplify the incorporation of genomic targeting sequences.     

Effect of genetic background and culture conditions on the production of herpesvirus-based gene therapy vectors.

Herpes simplex virus type-1 (HSV-1) represents a unique vehicle for the introduction of foreign DNA to cells of a variety of tissues. The nature of the vector, the cell line used for propagation of the vector, and the culture conditions will significantly impact vector yield. An ideal vector should be deficient in genes essential for replication as well as those that contribute to its cytotoxicity. Advances in the engineering of replication-defective HSV-1 vectors to reduce vector-associated cytotoxicity and attain sustained expression of target genes make HSV-1 an attractive gene-delivery vehicle. However, the yield of the less-cytotoxic vectors produced in standard tissue-culture systems is at least three order of magnitudes lower than that achieved with wild-type virus. The low overall yield and the complexity involved in the preparation of HSV vectors at high concentrations represent major obstacles in use of replication-defective HSV-derived vectors in gene therapy applications. In this work, the dependence of the vector yield on the genetic background of the virus is examined. In addition, we investigated the production of the least toxic, lowest-yield vector in a CellCube bioreactor system. After initial optimization of the operational parameters of the cellcube system, we were able to attain virus yields similar to or better than those values attained using the tissue culture flask system for vector production with significant savings with respect to time, labor, and cost.     

Isolation of targeted AAV2 vectors from novel virus display libraries

Random peptide ligands displayed on viral capsids are emerging tools for selection of targeted gene transfer vectors even without prior knowledge of the potential target cell receptor. We have previously introduced adeno-associated viral (AAV)-displayed peptide libraries that ensure encoding of displayed peptides by the packaged AAV genome. A major limitation of these libraries is their contamination with wild-type (wt) AAV. Here we describe a novel and improved library production system that reliably avoids generation of wt AAV by use of a synthetic cap gene. Selection of targeted AAV vectors from wt-containing and the novel wt-free libraries on cell types with different permissivity for wt AAV2 replication suggested the superiority of the wt-free library. However, from both libraries highly specific peptide sequence motifs were selected which improved transduction of cells with moderate or low permissivity for AAV2 replication. Strong reduction of HeLa cell transduction compared to wt AAV2 and only low level transduction of non-target cells by some selected clones showed that not only the efficiency but also the specificity of gene transfer was improved. In conclusion, our study validates and improves the unique potential of virus display libraries for the development of targeted gene transfer vectors.     

pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants

Agro-infiltration of leaf tissue with binary vectors harbouring a sequence of interest is a rapid method of expressing proteins in plants. It has recently been shown that flanking the sequence to be expressed with a modified 5'-untranslated region (UTR) and the 3'-UTR from Cowpea mosaic virus (CPMV) RNA-2 (CPMV- HT ) within the binary vector pBINPLUS greatly enhances the level of expression that can be achieved [ Sainsbury, F. and Lomonossoff, G.P. (2008) Plant Physiol . 148 , 1212–1218]. To exploit this finding, a series of small binary vectors tailored for transient expression (termed the pEAQ vectors) has been created. In these, more than 7 kb of non-essential sequence was removed from the pBINPLUS backbone and T-DNA region, and unique restriction sites were introduced to allow for accommodation of multiple expression cassettes, including that for a suppressor of silencing, on the same plasmid. These vectors allow the high-level simultaneous expression of multiple polypeptides from a single plasmid within a few days. Furthermore, vectors have been developed which allow the direct cloning of genes into the binary plasmid by both restriction enzyme-based cloning and GATEWAY recombination. In both cases, N- or C-terminal histidine tags may be fused to the target sequence as required. These vectors provide an easy and quick tool for the production of milligram quantities of recombinant proteins from plants with standard plant research techniques at a bench-top scale.     

Lack of specificity of cell-surface protease targeting of a cytotoxic hyperfusogenic gibbon ape leukaemia virus envelope glycoprotein

Background

In a strategy termed “Protease Targeting”, retroviral vectors carrying an EGF infectivity‐blocking domain fused to the N‐terminus of the envelope SU via a MMP (matrix metalloproteinase)‐cleavable linker were successfully used to target gene delivery to EGF receptor‐(EGF‐R‐)positive tumour cells over‐expressing MMPs. In the current study, we aimed to investigate whether this strategy could be applied to (a) limit the cytotoxic activity of a hyperfusogenic GALV therapeutic gene, and (b) enhance the immune‐stimulatory properties of GALV via local, MMP‐mediated release human granulocyte‐macrophage colony stimulating factor (GM‐CSF).

Methods

We generated GALV envelope expression constructs displaying EGF or GM‐CSF blocking ligands at the N‐terminus of GALV envelope SU via a non‐cleavable, Factor Xa protease or MMP‐cleavable linker and investigated their cytotoxicity on MMP‐positive and negative cell lines.

Results

The unmodified hyperfusogenic GALV envelope was cytotoxic to all cell lines tested. The non‐cleavable linker GALV envelope constructs caused no cytotoxicity, demonstrating efficient inhibition by the displayed domains. Moderate activation of fusion of the protease‐cleavable linker constructs was observed in all cell lines, regardless of their level of MMP expression and of the specificity of the linker. High levels of the ‘blocking domain’ were detected in the cell supernatants due to dissociation of the surface unit (SU) from the transmembrane (TM) component of the GALV envelope glycoprotein TM.

Conclusions

Unlike protease targeting in the context of retroviral vectors, protease activation of the cytotoxicity of GALV envelope by cleavage of a fusion blocking ligand at the cell surface does not appear to be specifically mediated by cell‐surface MMPs. In addition, shedding of the SU‐fusion protein from the TM limits the general applicability of this strategy for cancer gene therapy. Specificity of cell‐cell fusion mediated by GALV envelope cannot be manipulated in the same fashion as virus‐cell fusion. Copyright © 2002 John Wiley & Sons, Ltd.

     

microRNA调控的靶向性病毒载体在基因治疗中的应用

安全、有效、具有靶向性的病毒载体是基因治疗药物在临床上得以应用的关键。microRNA是一类单链、内源性的转录后调控小分子,它的发现为开发具有靶向性调控能力的病毒载体提供了新的研究方法。以下在介绍microRNA调节病毒载体靶向性原理的基础上,着重介绍microRNA在清除复制能力病毒的污染、消除转基因特异性免疫、增强肿瘤靶向性基因治疗、开发活体疫苗等领域的应用。