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Design of 5' untranslated sequences in retroviral vectors developed for medical use.

ABSTRACT: Utilizing genetic modules of simple retroviruses, we have developed a novel generation of gene transfer vectors with improved therapeutic potential. In the 5' untranslated "leader" sequences, all AUG codons which may aberrantly initiate translation and all viral coding sequences were removed. Thus, the probability of expressing unwanted peptides and the potential for homologous recombination with retroviral genes were largely reduced, and the cloning capacity was increased. The transgene was inserted to replace the viral gag sequences, and a new minimal splice acceptor was introduced, resulting in increased expression with all genes tested (those coding for human multidrug resistance 1 and enhanced green fluorescent protein, as well as the lacZ gene). These vectors may represent attractive tools for human gene therapy, because they increase the efficiency of transgene expression and may also increase safety in medical applications.

SUBMITTER: Hildinger M

PROVIDER: S-EPMC104187 | biostudies-literature | 1999 May

REPOSITORIES: biostudies-literature

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Design of 5' untranslated sequences in retroviral vectors developed for medical use.

Hildinger M M Abel K L KL Ostertag W W Baum C C

Journal of virology 19990501 5

Utilizing genetic modules of simple retroviruses, we have developed a novel generation of gene transfer vectors with improved therapeutic potential. In the 5' untranslated "leader" sequences, all AUG codons which may aberrantly initiate translation and all viral coding sequences were removed. Thus, the probability of expressing unwanted peptides and the potential for homologous recombination with retroviral genes were largely reduced, and the cloning capacity was increased. The transgene was ins ...[more]

PMID: 10196304

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Project description:BACKGROUND: Endogenous retroviral sequences (ERVs) are integral parts of most eukaryotic genomes and vastly outnumber exogenous retroviruses (XRVs). ERVs with a relatively complete structure were retrieved from the genetic archives of humans and chickens, diametrically opposite representatives of vertebrate retroviruses (over 3300 proviruses), and analyzed, using a bioinformatic program, RetroTector, developed by us. This rich source of proviral information, accumulated in a local database, and a collection of XRV sequences from the literature, allowed the reconstruction of a Pol based phylogenetic tree, more extensive than previously possible. The aim was to find traits useful for classification and evolutionary studies of retroviruses. Some of these traits have been used by others, but they are here tested in a wider context than before. RESULTS: In the ERV collection we found sequences similar to the XRV-based genera: alpha-, beta-, gamma-, epsilon- and spumaretroviruses. However, the occurrence of intermediates between them indicated an evolutionary continuum and suggested that taxonomic changes eventually will be necessary. No delta or lentivirus representatives were found among ERVs. Classification based on Pol similarity is congruent with a number of structural traits. Acquisition of dUTPase occurred three times in retroviral evolution. Loss of one or two NC zinc fingers appears to have occurred several times during evolution. Nucleotide biases have been described earlier for lenti-, delta- and betaretroviruses and were here confirmed in a larger context. CONCLUSION: Pol similarities and other structural traits contribute to a better understanding of retroviral phylogeny. "Global" genomic properties useful in phylogenies are i.) translational strategy, ii.) number of Gag NC zinc finger motifs, iii.) presence of Pro N-terminal dUTPase (dUTPasePro), iv.) presence of Pro C-terminal G-patch and v.) presence of a GPY/F motif in the Pol integrase (IN) C-terminal domain. "Local" retroviral genomic properties useful for delineation of lower level taxa are i.) host species range, ii.) nucleotide compositional bias and iii.) LTR lengths.

Dataset Information

Design of 5' untranslated sequences in retroviral vectors developed for medical use.

Publications

Design of 5' untranslated sequences in retroviral vectors developed for medical use.

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