Retroviral gene transfer vectors

Details Retroviral gene transfer vectors Retroviral gene transfer vectors

1999-05-06

2003-04-15

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Process of mutation, cell fusion, or genetic modification

Introduction of a polynucleotide molecule into or...

C435S320100, C435S235100, C514S04400A

Reexamination Certificate

active

06548301

ABSTRACT:

The invention relates to novel retroviral gene transfer vectors, preferably expression vectors, which, because of a reduced content of viral genes, are distinguished by a higher safety standard and expression of non-viral nucleotide sequences in higher amounts.
The expression of foreign proteins in pro- and eukaryotic cells, i.e. of proteins which are usually expressed only in very small amounts, or not at all, in these cells, plays an essential role in research into the function of proteins, production of proteins and treatment of diseases. A prerequisite for expression of a foreign gene is that the genetic material which codes for the protein is introduced into the target cell. This is as a rule achieved with the aid of so-called vectors. Vectors are DNA molecules into which the DNA which codes for the protein to be expressed has been cloned and which contain DNA sequences which are of importance for expression of the protein.
For stable gene transfer into mammalian cells, retroviral vectors based on mouse leukaemia viruses (MLV) are currently the most frequently used and best characterized system (Miller, A. D. (1993) Methods Enzymol, 217; 581-599). Fields of use of retroviral vectors are, for example, over-expression of proteins with the aim of obtaining pure proteins, expression cloning with the aim of identifying new proteins, and stable expression of a protein in a body cell with the aim of a therapeutic action of the expressed protein. The level of transgene expression in the cell system relevant to the disease is decisive for the therapeutic potential of gene transfer here. For clinical use of gene transfer vectors, the safety of the vectors is furthermore of decisive importance. On the one hand, expression of viral gene products must be excluded here, since these may display a pathogenic action in the body. On the other hand, recombination with naturally occurring viruses should be ruled out, to prevent viral proteins being expressed under the control of the regulatory element introduced into the vector, or novel viruses with unknown properties being formed.
Retroviral vectors can exist in two forms, as proviral DNA or as vector RNA. Proviral double-stranded DNA is integrated in a stable manner in the genome of the target cell. From this, the so-called genomic vector transcript is read, which is built up like a cell mRNA (messenger RNA), and is packed in viral particles and transmits the genetic information of the retrovirus. After retroviral infection of a target cell, the genomic vector transcript is transcribed by reverse transcription into a new provirus and integrated in a stable manner into the genome of the cell (Miller, A. D. (1993) Methods Enzymol. 217: 581-599).
The provirus is flanked at the 5′- and at the 3′-end by “long terminal repeats” (LTR), which include the regions U3, R and U5 (FIG.
1
). The U3 region contains enhancer/promoter sequences which control transcription of the vector. The R region carries the polyadenylation signal. The U5 region contains sequences which are necessary for integration of the retrovirus. The coding sequences of the exogenous protein are usually between 5′- and 3′-LTR in the vector and are flanked by control sequences of regulatory importance which are essential for the progress of the retroviral life cycle and at the same time influence the half-life of the RNA and the translation efficiency of the exogenous protein. The transcription start of the RNA lies at the boundary of the R region of the 5′-LTR. The transcription end is determined by polyadenylation and termination signals in the R region of the 3′-LTR. A polyadenosine tail is attached at the end of the R region of the 3′-LTR. Splicing signals of the retrovirus/vector can lead to transcripts with internal deletions.
The 5′-untranslated region of the retroviral vector is composed of (A. D. Miller (1993) loc. cit.):
R region and U5 region of the 5′-LTR (approx. 150 nucleotides, necessary for reverse transcription and integration)
Primer binding site (18 nucleotides, necessary for reverse transcription)
Leader region (in current retroviral vectors at least 800 nucleotides long). This follows the primer binding site and extends to the start of the coding sequence. The leader region contains the packing and dimerization signal, which is necessary for incorporation of the RNA into retroviral particles. At the start of the leader lies the retroviral splicing donor, and at the end of the leader there can be a cryptic (i.e. recognized only in a portion of the transcripts) splicing acceptor. The splicing donor and splicing acceptor determine the start and end of the RNA sequences which can be removed from the primary transcript by the splicing operation even before export into the cytoplasm. The efficiency of the splicing reaction depends on the suitability of the splicing signals. In addition to the sequences of the splicing donor and splicing acceptor, the polypyrimidine tract lying before the splicing acceptor and the subsequent so-called branch site (see below) determine the efficiency of the splicing (Zhuang, Y. A. et al. (1989) Proc. Natl. Acad. Sci. USA, 86: 2752-2756).
The efficiency of the translation is impaired by the long length of the 5′-untranslated region (5′-UTR). The splicing signals of the leader can give rise to shortened transcripts with significantly shorter 5′-UTR, which have an increased translation efficiency (Armentano, D. et al. (1987) J. Virol. 61: 1647-1620; Bender, M. A. et al. (1987) J. Virol. 61: 1639-1646; Krall, W. J. et al. (1996) Gene Ther. 3: 37-48).
The 3′-untranslated region of the RNA contains the polypurine tract, which is necessary for the reverse transcription, and the U3 and R region of the 3′-LTR.
After the retroviral life cycle, the U3 region of the 3′-UTR is copied into both LTRs. If the U3 region contains enhancer/promoter regions, these control the transcription of the vector-RNA in the infected cell (Baum, C. et al. (1995) J. Virol. 69: 7541-7547). The end of the RNA is the polyadenosine tail of approx. 200 nucleotides, which co-determines the stability in the cytoplasm.
In addition to the control elements, retroviruses contain nucleotide sequences which code for viral proteins. These include the gag gene, which codes for structure proteins of the virus, the pro gene, which codes for the virion protease, the pol gene, which codes for the reverse transcriptase, and the env gene, which codes for virus envelope glycoproteins. If the retroviral vector does not contain one of these genes, it is replication-incompetent. To produce infectious virus particles from a replication-incompetent retroviral vector, a helper virus and/or a packing-competent cell line which provide the properties lacking from the retroviral vector are required.
The viral sequences contained in the retroviral vectors can give rise to recombination with complementary retroviruses in the packing cell, as a result of which replication-competent retroviruses, which can induce leukaemias and encephalopathies in animal studies, can form (Anderson, W. F. (1993) Hum. Gene Ther. 4; 311-321; Münk, C. (1997) PNAS 94: 5837-5842). The residual gag and pol genes furthermore contain a large number of cryptic reading frames, i.e. nucleotide sequences which code for an amino acid sequence in a reading frame other than for the actual gene, but in the normal case are not read, since the control elements and/or the start codon are missing. Nevertheless, it cannot be ruled out that immunogenic or toxic peptides can be generated in the target cell by the open reading frame.
The target cell of the retroviral expression vector is transduced with infectious virus particles. These particles are produced with the aid of a packing-competent cell line. It is important here that as many infectious virus particles as possible per ml of cell culture supernatant are produced by the packing-competent cells, that is to say that the virus titre, stated in infectious units per ml of culture medium, is as hi

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