Anti-viral vectors

Details Anti-viral vectors Anti-viral vectors

2001-12-11

2004-08-31

Park, Hankyel T. (Department: 1648)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S005000, C435S069100, C435S091400, C435S320100, C435S455000, C536S023720

Reexamination Certificate

active

06783981

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to novel viral vectors capable of delivering anti-viral inhibitory RNA molecules to target cells.
BACKGROUND TO THE INVENTION
The application of gene therapy to the treatment of AIDS and HIV infection has been discussed widely (Lever, 1995). The types of therapeutic gene proposed usually fall into one of two broad categories. In the first the gene encodes protein products that inhibit the virus in a number of possible ways. One example of such a protein is the RevM10 derivative of the HIV Rev protein. The RevM10 protein acts as a transdominant negative mutant and so competitively inhibits Rev function in the virus. Like many of the protein-based strategies, the RevM10 protein is a derivative of a native HIV protein. While this provides the basis for the anti-HIV effect, it also has serious disadvantages. In particular, this type of strategy demands that in the absence of the virus there is little or no expression of the gene. Otherwise, healthy cells harbouring the gene become a target for the host cytotoxic T lymphocyte (CTL) system, which recognises the foreign protein. The second broad category of therapeutic gene circumvents these CTL problems. The therapeutic gene encodes inhibitory RNA molecules; RNA is not a target for CTL recognition.
There are several types of inhibitory RNA molecules known: anti-sense RNA, ribozyrnes, competitive decoys and external guide sequences (EGSs).
External guide sequences, first identified by Forster and Altman (1990), are RNA sequences that are capable of directing the cellular protein RNase P to cleave a particular RNA sequence. In vivo, they are found as part of precursor tRNAs where they function to direct cleavage by the cellular riboprotein RNase P in vivo of the tRNA precursor to form mature tRNA. However, in principle, any RNA can be targeted by a custom-designed EGS RNA for specific cleavage by RNase P in vitro or in viva. For example, Yuan et al. (1992) demonstrate a reduction in the levels of chloramphenicol activity in cells in tissue culture as a result of introducing an appropriately designed EGS.
In recent years a number of laboratories have developed retroviral vector systems based on HIV. In the context of anti-HIV gene therapy these vectors have a number of advantages over the more conventional murine based vectors such as murine leukaemia virus (MLV) vectors. Firstly, HIV vectors would target precisely those cells that are susceptible to HIV infection. Secondly, the HIV-based vector would transduce cells such as macrophages that are normally refractory to transduction by murine vectors. Thirdly, the anti-HIV vector genome would be propagated through the CD4+ cell population by any virus (HIV) that escaped the therapeutic strategy. This is because the vector genome has the packaging signal that will be recognised by the viral particle packaging system. These various attributes make HIV-vectors a powerful tool in the field of anti-HIV gene therapy.
A combination of inhibitory RNA molecules and an HIV-based vector would be attractive as a therapeutic strategy. However, until now this has not been possible. Vector particle production takes place in producer cells which express the packaging components of the particles and package the vector genome. The inhibitory RNA sequences that are designed to destroy the viral RNA would therefore also interrupt the expression of the components of the HIV-based vector system during vector production. The present invention aims to overcome this problem.
SUMMARY OF THE INVENTION
It is therefore an object of the invention to provide a system and method for producing viral particles, in particular HIV particles, which carry nucleotide constructs encoding inhibitory RNA molecules such as external guide sequences, optionally together with other classes of inhibitory RNA molecules such as ribozyrnes and/or antisense RNAs directed against a corresponding virus, such as HIV, within a target cell, that overcomes the above-mentioned problems. The system includes both a viral genome encoding the inhibitory RNA molcules and nucleotide constructs encoding the components required for packaging the viral genome in a producer cell. However, in contrast to the prior art, although the packaging components have substantially the same amino acid sequence as the corresponding components of the target virus, the inhibitory RNA molecules do not affect production of the viral particles in the producer cells because the nucleotide sequence of the packaging components used in the viral system have been modified to prevent the inhibitory RNA molecules from effecting cleavage or degradation of the RNA transcripts produced from the constructs. Such a viral particle may be used to treat viral infections, in particular HIV infections.
Accordingly the present invention provides a viral vector system comprising:
(i) a first nucleotide sequence encoding an external guide sequence capable of binding to and effecting the cleavage by RNase P of a second nucleotide sequence, or transcription product thereof, encoding a viral polypeptide required for the assembly of viral particles; and
(ii) a third nucleotide sequence encoding said viral polypeptide required for the assembly of viral particles, which third nucleotide sequence has a different nucleotide sequence to the second nucleotide sequence such that the third nucleotide sequence, or transcription product thereof, is resistant to cleavage directed by the external guide sequence.
Preferably, said system further comprises at least one further first nucleotide sequence encoding a gene product capable of binding to and effecting the cleavage, directly or indirectly, of a second nucleotide sequence, or transcription product thereof, encoding a viral polypeptide required for the assembly of viral particles, wherein the gene product is selected from an external guide sequence, a ribozyme and an anti-sense ribonucleic acid.
In another aspect, the present invention provides a viral vector production system comprising:
(i) a viral genome comprising at least one first nucleotide sequence encoding a gene product capable of binding to and effecting the cleavage, directly or indirectly, of a second nucleotide sequence, or transcription product thereof, encoding a viral polypeptide required for the assembly of viral particles;
(ii) a third nucleotide sequence encoding said viral polypeptide required for the assembly of the viral genome into viral particles, which third nucleotide sequence has a different nucleotide sequence to the second nucleotide sequence such that said third nucleotide sequence, or transcription product thereof, is resistant to cleavage directed by said gene product;
wherein at least one of the gene products is an external guide sequence capable of binding to and effecting the cleavage by RNase P of the second nucleotide sequence.
Preferably, in addition to an external guide sequence, at least one gene product is selected from a ribozyme and an anti-sense ribonucleic acid, preferably a ribozyme.
Preferably, the viral vector is a retroviral vector, more preferably a lentiviral vector, such as an HIV vector. The second nucleotide sequence and the third nucleotide sequences are typically from the same viral species, more preferably from the same viral strain. Generally, the viral genome is also from the same viral species, more preferably from the same viral strain.
In the case of retroviral vectors, the polypeptide required for the assembly of viral particles is selected from gag, pol and env proteins. Preferably at least the gag and pol sequences are lentiviral sequences, more preferably HIV sequences. Alternatively, or in addition, the env sequence is a lentiviral sequence, more preferably an HIV sequence.
In a preferred embodiment, the third nucleotide sequence is resistant to cleavage directed by the gene product as a result of one or more conservative alterations in the nucleotide sequence which remove cleavage sites recognised by the at least one gene product and/or binding sites for the at least one gene p

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