Modular retro-vectors for transgenic and therapeutic use.

Retroviruses have been used for many years as vectors for human gene therapy as well as for making transgenic animals. However, the efficient insertion of genes by retroviruses is often complicated by transcriptional inactivation of the retroviral long terminal repeats (LTRs) and by the production of replication-competent retroviruses (RCR). Solutions to these and other difficulties are being found in modular vectors, in which the desirable features of different vector systems are combined. Examples of synergistic vectors include virosomes (liposome/virus delivery), adeno-retro vectors, and MLV/VL30 chimeras. As gene delivery systems become increasingly complex, methodology is also needed for precise assembly of modular vectors. Gene self-assembly (GENSA) technology permits seamless vector construction and simultaneous, multifragment assembly.

Fusogenic effects of murine retroviruses and cationic enhancers of transduction.

The maturation of retrovirus particles involves proteolytic cleavage of the envelope glycoprotein transmembrane component, resulting in conversion of the virus particle to a fusogenic or infectious state. Susceptible murine cells exposed to virus-containing supernatants from ecotropic retroviral helper cells occasionally fused to neighboring cells, resulting in syncytia (giant cells with multiple nuclei). Polycationic molecules dramatically enhanced the effect, leading to widespread cell death. The degree of cell fusion was dependent upon the retroviral envelope subtype (ecotropic-->amphotropic, gibbon ape leukemia virus was negative) as well as on the polycationic reagent used (G9 dendrimer-->Lipofectamine-->polybrene). Cell fusion effects were not mediated by the retroviral vector backbone, because virus-containing supernatants from helper cells (without vector) and vector producer cells had a similar effect. Human target cells were not fused by any type of murine retrovirus; in addition, amphotropic virus from human helper cells was not fusogenic toward murine cells. Thus, fusogenic effects were important during the propagation of vectors using murine helper cells but were not a significant factor during the transduction of human cells.

Biosynthetic retrovectoring systems for gene therapy.

Chemical and physical methods of introducing genes into cells (transfection) are being combined with viral transduction as one possible approach toward overcoming the shortcomings of current gene transfer methods. Although several different strategies are being developed worldwide, this article focuses on modification of retroviral vectoring systems. One goal of this work is to combine the safety and ease of transfection methods with the permanency that is presently achieved only by integrating viruses. Work with retrotransposon pseudotypes, synthetic retrovectors, and liposomal delivery of retrovirus vectors is discussed.

Expression of VL30 vectors in human cells that are targets for gene therapy.

Novel vectors made from mouse VL30 retrotransposons were tested in cell types that are targets for gene therapy, including normal human cells (skeletal muscle epithelium, bronchial epithelium, mammary epithelium), Epstein-Barr virus-transformed peripheral blood lymphocytes (PBLs), and various tumor cell lines. The long terminal repeat (LTR) transcriptional promoter, derived from the retroelement NVL-3, expressed abundant mRNA containing the bacterial neomycin resistance gene (neo) in all cell types tested. The amounts of neo RNA detected on RNA blots from normal vs. transformed cells were similar, although relatively less RNA was expressed in PBLs than in other cell types. Vector RNA expression in PBLs persisted during six months of continuous culture. Transcription was regulated by fibroblast growth factor (bFGF) and insulin, with the effects of each being cell-type-dependent. Thus, VL30 vectors introduced and expressed transgenes at significant levels in a number of cell types that are of interest for human gene therapy of metabolic and neoplastic diseases.

Transmission of endogenous VL30 retrotransposons by helper cells used in gene therapy.

Retrovirus-derived vectors and packaging cell lines are basic components used for gene transfer in human gene therapy. To eliminate recombinational and transcriptional problems associated with retroviral vectors, synthetic retrotransposon VL30 vectors were devised. During experimentation with these new vectors, extensive cotransmission of endogenous VL30 retrotransposon sequences was observed, originating within helper cell lines used in gene therapy experiments. The RNA was efficiently packaged into helper virus and was transmitted to recipient human cells, where it was again expressed as RNA. Transmission occurred regardless of whether the vector was retrovirus-derived or VL30, or if no vector was used. Endogenous VL30 RNA was readily detected in unselected recipient cells after a single exposure to helper virus, demonstrating a high efficiency of transmission compared with a cotransmitted VL30 vector, which contained an extensive deletion of internal sequences. These studies indicated that VL30 retrotransposons were ubiquitously transmitted by murine helper cells. Furthermore, the data strongly suggested that improvements in gene transfer may be obtained, both by using nonmurine helper cells (to reduce competitive inhibition by endogenous VL30) and by using VL30-derived vectors with intact packaging sequences.

Synthetic retrotransposon vectors for gene therapy.

New gene therapy methods are rapidly being developed to permit the expression of tumor suppressor genes, cytotoxins, anticancer antigens, and immunoregulatory proteins in the treatment of cancer. Large-scale testing in humans has been delayed by questions concerning the safety and effectiveness of preferred retroviral vectors and helper cells. These vector systems are limited by their ability to undergo homologous recombination with endogenous retroviruses or helper-viral sequences, resulting in release of replication-competent retrovirus (RCR). In addition, transcriptional inactivation of the retroviral promoter often occurs, caused in part by methylation of CpG islands in the retroviral long terminal repeats (LTRs). We report the production of highly specific retrovectors using gene amplification together with oligonucleotide building blocks. The synthetic vectors were based on mouse VL30 retrotransposon NVL3, and lacked homology to retroviral helper gene sequences. Three of four constructs made by gene amplification yielded biologically active vectors. These constructs efficiently transmitted and stably inserted a neomycin resistance marker gene into the genome of recipient cells, expressing an abundant RNA species of the expected size in the absence of detectable replication competent retrovirus. The vectors and techniques described enable widely applicable expression modes using generic helper cells, and require only approximately 1.3 kb of cis-acting vector RNA sequences for faithful transfer and expression of genetic material.