Vaccinia viral/retroviral chimeric vectors.

Retroviral vectors have become important tools in gene therapy due to a number of desirable properties, including efficient gene delivery and stable genomic integration. Some shortcomings, however, still remain to be solved. Retroviral vectors cannot be grown to as high titers as for example adenoviruses or vaccinia viruses, they tend to be unstable and are sensitive to lysis by complement when transfused into patients. The search for more robust retroviral delivery systems has led to the development of hybrid viral vectors trying to combine the broadly estimated features of retroviral vectors with advantageous properties of a second viral vector system. Chimeric systems with retroviruses and adeno-alphavirus and herpesviruses have been reported. This review is dedicated to vaccinia virus, a widely used vector in molecular and cell biology, as a chimeric carrier for retroviral vector units. In the first poxviral/retroviral constructs, retroviral vector units integrated into a defective vaccinia vector, gave rise to transduction competent particles. Due to the high insertion capacity of the vaccinia system, also the packaging components could be inserted into the carrier virus resulting in a system that is independent of retroviral packaging cell lines. Moreover, since vaccinia is a cytoplasmic virus that does not recognize nuclear transcription and processing signals, retroviral vectors with introns and internal transcription stops could be constructed that transduce complex gene cassettes. The topic of this review is the vaccinia viral / retroviral vector system and possible applications to gene therapy.

Retroviral vectors produced in the cytoplasmic vaccinia virus system transduce intron-containing genes.

Introns and polyadenylation (pA) sites are known to improve transcript stability and nuclear-cytoplasmic transport and are normally present in efficient gene expression vectors. Standard retroviral vectors, however, do not allow the inclusion of such sequence elements, as mRNA processing at internal splice and pA sites interferes with the production of functional full-length vector genomes. In this report we examined the capability of hybrid vaccinia/retroviral vectors to transduce complex gene cassettes with nuclear RNA processing signals within the retroviral genome. A retroviral vector was constructed that contains a gene of interest (the human coagulation factor IX [FIX] cDNA), including an intron and an internal pA site. The modified proviral vector genome was cloned downstream of a vaccinia virus promoter and was inserted into the vaccinia virus genome. Infection of a packaging cell line with the recombinant vaccinia virus vector resulted in secretion of retroviral particles at average titers of 10(5) CFU per ml of cell culture supernatant. Due to the cytoplasmic transcription and the nonrecognition of nuclear transcription signals in the vaccinia virus system, full-length transcripts were obtained that still contained the intron. In the retrovirally transduced cell lines the FIX transcripts were terminated at the internal pA site. The transcripts were quantitatively spliced, and FIX was secreted. Recombinant cell lines with stable single-copy inserts containing sequence elements necessary for efficient gene function could be generated. Thus, a relatively simple cytoplasmic system for the generation of complex retroviral vectors is described. Retroviral vectors transducing intron-containing gene cassettes may play a further role in gene therapy applications.

Highly efficient induction of protective immunity by a vaccinia virus vector defective in late gene expression.

Vaccinia viruses defective in the essential gene coding for the enzyme uracil DNA glycosylase (UDG) do not undergo DNA replication and do not express late genes in wild-type cells. A UDG-deficient vaccinia virus vector carrying the tick-borne encephalitis (TBE) virus prM/E gene, termed vD4-prME, was constructed, and its potential as a vaccine vector was evaluated. High-level expression of the prM/E antigens could be demonstrated in infected complementing cells, and moderate levels were found under noncomplementing conditions. The vD4-prME vector was used to vaccinate mice; animals receiving single vaccination doses as low as 10(4) PFU were fully protected against challenge with high doses of virulent TBE virus. Single vaccination doses of 10(3) PFU were sufficient to induce significant neutralizing antibody titers. With the corresponding replicating virus, doses at least 10-fold higher were needed to achieve protection. The data indicate that late gene expression of the vaccine vector is not required for successful vaccination; early vaccinia virus gene expression induces a potent protective immune response. The new vaccinia virus-based defective vectors are therefore promising live vaccines for prophylaxis and cancer immunotherapy.

Poxviral/retroviral chimeric vectors allow cytoplasmic production of transducing defective retroviral particles.

Defective vaccinia viruses were constructed that express functional Moloney murine leukemia virus-based vector genomes, giving rise to substantial titers of transduction-competent retrovirus particles after infection of a retroviral packaging cell line. For this purpose, the proviral retrovirus genome, engineered into the vaccinia virus mutant, was subjected to several modifications, including the replacement of retroviral promoter sequences by vaccinia virus sequences and the precise fusion of the transcription stop signal downstream of and the removal of such signals within the transcription unit, allowing cytoplasmic transcription of distinct full-length retroviral transcripts. Vaccinia-mediated expression of retroviral vector particles could be observed as early as 3 h postinfection and resulted in stable transduction of NIH/3T3 target cells at higher titers than the control performed by conventional plasmid transfections. Thus at least part of the vaccinia life cycle and retroviral assembly can occur concomitantly. Due to the favorable properties of vaccinia vectors, including high coding capacity, stability, and wide host range, defective vaccinia viral/retroviral chimeric vectors are promising tools for gene therapy applications.

Dominant host range selection of vaccinia recombinants by rescue of an essential gene.

We report the rescue of a defective vaccinia virus, forming the basis for a stringent selection protocol to generate replicating recombinant virus without the need for marker cassettes and selection agents. Plaques of recombinant virus could be isolated solely by their ability to grow in wild-type cells normally supporting the growth of vaccinia virus. All growth-competent clones analyzed contained the gene of interest in the intended genomic locus and displayed foreign gene expression to the same levels as was seen with classical recombinants obtained by insertion into the vaccinia virus thymidine kinase locus. The system is based on a defective vaccinia virus, expressing exclusively early genes, termed eVAC-1, and an insertion plasmid vector providing the essential function, the uracil DNA glycosylase gene. In addition, the defective virus is free of selection and color marker genes, thus also representing a basic vector for the generation of defective recombinants.

Construction of a vaccinia virus deficient in the essential DNA repair enzyme uracil DNA glycosylase by a complementing cell line.

The vaccinia virus D4R open reading frame, encoding the essential DNA repair enzyme uracil DNA glycosylase, was expressed in two permanent cell lines, the rabbit kidney cell line RK13 and the human fibroblast cell line 293. The temperature-sensitive vaccinia virus mutant ts4149, which maps within D4R, was able to grow under restrictive conditions in both of these transformed cell lines. Cell clones complemented D4R function to various degrees, demonstrating complementation of an essential vaccinia virus gene by a cell line constitutively expressing the essential function. Thus, the complementing host cells allowed the rescue of a virus defective in the D4R gene, demonstrating that this system may be used for the propagation of defective cytoplasmic DNA viruses. The defective virus grew to high yields only in the engineered cell lines. The data support the hypothesis that early gene products, such as uracil DNA glycosylase, supplied in trans can fully complement essential viral functions.