Cap-independent multicistronic retroviral vectors

Details Cap-independent multicistronic retroviral vectors Cap-independent multicistronic retroviral vectors

1993-01-26

2001-11-20

LeGuyader, John L. (Department: 1635)

Chemistry: molecular biology and microbiology

Vector, per se

C435S325000, C536S023100, C536S024100

Reexamination Certificate

active

06319707

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to genetic engineering involving recombinant DNA technology, and particularly to retroviral vectors for producing coordinately expressed polycistronic mRNA in transfected host cells.
BACKGROUND OF THE INVENTION
Translation of typical eukaryotic mRNAs begins with the binding of initiation factors and the 40S small ribosomal subunit to the capped 5′ end of an mRNA, followed by the migration of this complex to the first AUG codon in a suitable context for initiation of translation, where the complete ribosomal initiation complex is formed and protein synthesis begins. Picornavirus infection of cells causes a reduction in the translation of cellular RNA. In the case of poliovirus and several other members of the picornavirus family, the reduction is at the step of cap-dependent binding of ribosomal components to cellular mRNAs, and is due to inactivation of cap binding protein eIF-4F by a viral protease (25,26; see the appended Citations). Uncapped viral RNA continues to be translated in a cap-independent manner, and is dependent on the presence of unusually long 5′ nontranslated regions (NTRs), ranging from 650 to 1300 nucleotides (nt), in the viral mRNA. These NTRs contain multiple AUG codons that appear not to initiate translation. In the case of poliovirus, mutation of 6 of the 7 upstream AUG codons had no effect on virus replication in cultured cells, and mutations in the seventh AUG only reduced the replication rate of the virus (20).
Several lines of evidence suggest that picornavirus NTRs provide sites for direct binding of ribosomes and thus allow internal initiation of protein translation from downstream AUG codons. First, although downstream coding regions in multicistronic RNAs are usually expressed poorly in mammalian cells or in in vitro mammalian translation systems, insertion of the 5′ NTR from either poliovirus or encephalomyocarditis virus (EMCV) promotes efficient translation of downstream cistrons in in vitro and transient in vivo assays (3,4,22,28). Second, insertion of a 5′ NTR upstream of a heterologous protein coding region renders translation of that cistron independent of poliovirus infection in cultured cells and in cellular extracts from infected and uninfected cells (3,21,22,28). Lastly, internal binding of ribosomes to 5′ NTR regions has been demonstrated in vitro, and involves the binding of additional cellular but not viral proteins (5,8,23). A limitation of these experiments using plasmid vectors is the use of in vitro or transient in vivo assays for measurement of translation initiation. Furthermore, it was not apparent that such picornavirus 5′ NTR systems could operate in a retroviral context.
A general limitation of prior retroviral vector techniques is that, if more than one gene (i.e., more than one cistron) is to be included in the retroviral construct, an internal promoter (e.g., an SV40 promoter) or mRNA alternative splicing may be required to obtain independent expression of the second gene. Otherwise (in the absence of splicing) transcription of the retroviral DNA will result in a polycistronic mRNA that encodes a polyprotein. Unfortunately, using multiple internal promoters or alternative splicing is only a partial solution to this problem and frequently leads to other difficulties because, for example, (a) each gene is independently expressed and selecting for one gene does not insure that expression of the other gene will be optimal; (b) the level of alternative splicing is highly variable in different types of cells; and (c) multiple promoters may interfere with one another. Although it is routine in the retroviral vector art to engineer such prior polycistronic vectors, many technical problems are commonly encountered. For example, selectable markers are routinely used to select transformed cells having integrated retroviral DNA, and while the growth of cells expressing high levels of the marker are favored, the selection of cells expressing the other gene, e.g., of therapeutic interest, may not be favored. It would therefore be highly desirable to be able to select simultaneously for expression of the marker gene and the gene of interest, but without encoding a polyprotein or relying on an alternatively spliced mRNA. Thus, it would be highly desirable to provide a retroviral construct in which the expression of two or more genes could be linked such that selection for optimal expression of one gene will ensure the coordinate expression of the other gene(s). For example, it would be desirable to be able to select simultaneously for expression of a selectable marker, a second gene of interest encoding a cytosolic protein, and a third gene of interest encoding a secreted protein.
SUMMARY OF THE INVENTION
To overcome the aforementioned limitations of the prior art, we have examined internal initiation from 5′ NTRs by using bicistronic retroviral vectors for the generation of stable cell lines. We find that insertion of encephalomyocarditis virus (EMCV) or poliovirus 5′ NTRs between the two coding regions markedly stimulates translation of the downstream cistron. Because both proteins are coordinately expressed from a single mRNA, this approach to the synthesis of multiple proteins by retroviral vectors has advantages over other methods that employ alternate splicing or internal promoters. Hence selection for expression of one of the genes should ensure expression of the other, which is not the case when using other strategies.
The invention provides, in one embodiment, retroviral constructs capable of forming a proviral genome in a host cell including a first nucleotide coding sequence, a second nucleotide coding sequence, and a third nucleotide sequence capable of hybridizing under stringent conditions to a 5′ nontranslated region (NTR) of a picornavirus RNA or its complementary RNA strand. The first, second, and third nucleotide sequences are operably linked in the retroviral construct such that transcription of the proviral genome gives rise to a messenger RNA molecule containing transcripts of the first, second, and third nucleotide sequences. In a representative embodiment, one of the first and second nucleotide sequences encodes a detectable marker and the other of the nucleotide coding sequences encodes a therapeutic gene product. The retroviral construct will typically also contain retroviral elements for a 5′ long terminal repeat sequence, a 3′ long terminal repeat sequence having a poly-A tail, a psi-packaging signal, and a 5′ segment of gag capable of promoting encapsidation of the retroviral construct into a retroviral vector particle.
Coordinate expression of the first and second coding sequence is ultimately achieved because the transcript of the third nucleotide sequence in the aforesaid messenger RNA molecule contains a nucleic acid capable of forming a regulatory stem-loop nucleic acid structure followed by at least one operable AUG start codon. This regulatory stem-loop nucleic acid structure is capable of operably binding a translation initiation complex in a host cell such that the transcripts of the first and second nucleotide sequences in the messenger RNA molecule are coordinately expressed in the host cell.
In a related embodiment, the invention provides packaging host cells transfected with the subject retroviral construct. The transfected cells are capable of encapsidating infective retroviral vector particles having a virion RNA complementary to the first, second, and third nucleotide sequences in the aforesaid proviral genome. Infective retroviral vector particles encapsidated by the packaging host cells are thereby provided.
In another related embodiment, the invention provides producer host cells transduced with the subject retroviral vector particles. The genome of the producer host cell contains a first proviral genome corresponding to the subject retroviral construct in combination with a second proviral genome containing a gag and pol gene and a third proviral genome containing an env

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