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Retroviral vectors that are able to sustain multiple rounds of replication may find many applications. However, one critical feature of such vectors is the ability to maintain an intact transgene cassette during repeated rounds of replication. We here report on the stability of a translational cassette consisting of an internal ribosome entry site followed by the enhanced green fluorescent protein coding sequence inserted in different configurations into murine leukemia virus genomes. In two of the constructs, the insert was located in the upstream part of the U3 region while in the third construct it was inserted in the 3' untranslated region of the viral genome. Furthermore, in two of the constructs, the translational cassette was flanked by a direct repeat, while no such structure flanked the third construct. Our results show that deletion of the heterologous translational cassette is observed for all constructs upon extended cell culture and that the number of replication rounds before revertants are detected can be postponed by decreasing the length of the repeat flanking the translational cassette.

Background: Human gene therapy vectors based on primate lentiviruses harbour in contrast to oncoretroviruses the risk of vector mobilization by human immunodeficiency viruses (HIV). Infection of cells transduced with a lentiviral vector by HIV could lead to packaging of the lentiviral vector RNA into HIV particles and transfer of the vector., Methods: A new approach based on primer complementation was developed to reduce the risk of vector mobilization. The primer binding site (PBS) of an SIV-based vector was mutated abolishing tRNA primer binding and thus blocking reverse transcription. This block was efficiently by-passed during vector production by providing an artificial tRNA matching the mutated PBS with titers reaching 10(6) infectious units/ml., Results: Primer-complemented SIV vectors were mobilized from transduced cells by HIV-1 >150-fold less efficiently than vectors with wild-type PBS. Mobilization of the primer-complemented SIV vector by SIV was inhibited to a lesser extent indicating reduced efficacy of the primer complementation approach for preventing mobilization of lentiviral vectors by homologous virus. The analysis of the PBS of the vector DNA in target cells transduced with vectors containing mutated PBS in the absence of a matched tRNA suggests that formation of heterozygous particles followed by priming on the helper RNA and strand switch during reverse transcription can lead to mobilization of the primer-complemented vector by SIV, but not HIV-1. Although self-inactivating vectors were more efficient in preventing vector mobilization by HIV-1 than primer-complemented vectors, mobilization remained undectable only if both approaches were combined., Conclusions: The primer complementation approach should further reduce the risk of mobilization of self-inactivating SIV-based vectors by HIV-1 and thus increase their safety., (Copyright 2004 John Wiley & Sons, Ltd.)

The envelope protein of retroviruses is responsible for viral entry into host cells. Here, we describe a mutational library approach to dissect functional domains of the envelope protein involving a retroviral vector, which expresses both the envelope protein of Akv murine leukemia virus (MLV) and the neomycin phosphotransferase II (Neo) selection marker from the same transcript. Envelope expression was achieved by inserting an internal ribosome entry site (IRES) between the neo and the env genes. We found the structure of the linker between the IRES element and env to be critical for sufficient envelope expression. This vector functions as a replication competent mini-virus in a culture of NIH 3T3 derived semi-packaging cells that express the viral Gag and Pol proteins. Titers comparable to those of wild type virus were achieved by this system. To test this vector system, we created a random mutational library of Arg 85 and Asp 86 in the first variable region of Akv envelope protein. Homologous amino acids to Asp 86 in Moloney and Friend murine leukemia viruses are thought to be directly involved in receptor binding. Subsequent selection of mutants capable of infecting murine NIH 3T3 cells indicated that the wild type aspartic acid or another hydrophilic residue at position 86 is an important determinant for envelope function.

Although retroviral vectors based upon the murine leukemia virus have good safety records from clinical trials, attention to safety issues is crucial for the advancement of retroviral gene therapy. Key issues are to reduce uncontrolled transfer of viral or non-viral genetic information and to prevent the formation of replicating retroviruses. Safety features are also being incorporated into the novel attenuated lentiviral vectors that open new prospects for gene delivery. Here, we highlight features developed to restrict the transfer of viral genes, immobilize transfer vectors in target cells, or control interactions with other retroviruses in producer or target cells, as well as new developments in tissue-targeted and regulated vectors.

Reliable evaluation of tumor growth in animal models depends upon accurate identification of all malignant cells in affected organs. An ideal tumor cell label is non-toxic, labels the cells in a population uniformly and does not affect their biological behavior. A good candidate for such a cell label is enhanced green fluorescence protein (EGFP). However, the stability of EGFP expression and the characteristics of EGFP-marked tumors cells in vivo have not yet been elucidated in detail. We here report that a B16 murine melanoma subline stably transduced with an EGFP/Neo encoding retroviral vector display the same growth patterns in vitro and in vivo as the parental cell line. Furthermore, the transduced cells were found to maintain the level of EGFP expression in vivo for at least 15 days. Thus, B16 malignant melanoma cells stably transduced with the gene for EGFP seem well suited for studies on tumor growth in mouse models.

A murine leukemia virus-derived replication-competent retroviral vector with a translational cassette for the enhanced green fluorescence protein (EGFP) was previously found to function efficiently in cell culture (Jespersen et al., 1999, Gene 239, 227-235). We here report that infection of newborn NIH Swiss mice gives rise to EGFP expression in a majority of spleen cells within the first days after infection. Among the nonadherent spleen cells, B, T, and NK lymphocytes were found to be efficiently marked by EGFP by flow cytometry analysis, whereas the adherent spleen cells were negative in most animals. Analysis at time points up to 60 days after infection reveals a decline in EGFP-positive spleen cells over time. Viremia analysis and PCR analysis of spleen DNA indicate that viruses that have lost the translation cassette predominate at later stages. The results provide a model for efficient gene delivery to spleen cells in a time window of 1 to 2 weeks after infection of newborns. Although this type of vector will not in itself be applicable in the clinic, we envision that efficient in vivo gene delivery will be valuable by analysis of differentiation and proliferation of hematopoietic cells in animal models and by development and evaluation of effectors interfering with these processes.

Co-encapsidation of retroviral RNAs into virus particles allows for the generation of recombinant proviruses through events of template switching during reverse transcription. By use of a forced recombination system based on recombinational rescue of replication- defective primer binding site-impaired Akv-MLV-derived vectors, we here examine putative genetic interactions between vector RNAs and copackaged endogenous retroviral RNAs of the murine leukaemia virus (MLV) and VL30 retroelement families. We show (i) that MLV recombination is not blocked by nonhomology within the 5' untranslated region harbouring the supposed RNA dimer-forming cis -elements and (ii) that copackaged retroviral RNAs can recombine despite pronounced sequence dissimilarity at the cross-over site(s) and within parts of the genome involved in RNA dimerization, encapsidation and strand transferring during reverse transcription. We note that recombination-based rescue of primer binding site knock-out retroviral vectors may constitute a sensitive assay to register putative genetic interactions involving endogenous retroviral RNAs present in cells of various species.

The cis-elements that direct packaging and dimerization of retroviral RNAs overlap, and it has been suggested that dimerization is required for RNA packaging. This also implies that heterodimerization would be necessary for co-packaging and recombination. Moreover, co-packaging of distinct RNAs may be reduced if incapable of heterodimerizing. In this study, we have designed a novel two-vector rescue system in which co-packaging and interstrand transfer are necessary for transduction. Thus, the rescue titer is a measure of the ability of a given vector combination to co-package and subsequently generate a provirus. In the current MLV-based set-up, we explored Akv- and MLV-like-endogenous virus (MLEV)-derived vectors with modulated dimerization signals. Results show that rescue is influenced by competition at the level of RNA packaging, as well as complementarity between dimerization elements. Altogether, the results support the hypothesis that complementarity between dimerization elements may favor co-packaging of distinct retroviral RNAs. [Copyright &y& Elsevier]