Anti-viral vectors

Details Anti-viral vectors Anti-viral vectors

2000-04-20

2003-04-01

Nguyen, Dave Trong (Department: 1632)

Chemistry: molecular biology and microbiology

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

C435S069100, C435S091400, C435S320100, C435S455000

Reexamination Certificate

active

06541248

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 (14). 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 (16). 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 (17, 25). 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. The RNA molecules may be anti-sense RNA (15, 31), ribozymes (5) or competitive decoys (1).
Ribozymes are enzymatic RNA molecules which catalyse sequence-specific RNA processing. The design and structure of ribozymes has been described extensively in the literature in recent years (3, 7, 31). Amongst the most powerful systems are those that deliver multitarget ribozymes that cleave RNA of the target virus at multiple sites (5, 21).
In recent years a number of laboratories have developed retroviral vector systems based on HIV (2, 4, 18, 19, 22-24, 27, 32, 35, 39, 43). 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 (22, 23). Secondly, the HIV-based vector would transduce cells such as macrophages that are normally refractory to transduction by murine vectors (19, 20). Thirdly, the anti-HIV vector genome would be propagated through the CD4+ cell population by any virus (HIV) that escaped the therapeutic strategy (7). 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 the multitarget ribozyme 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 ribozymes 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 ribozymes 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 molecules 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 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; 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 said gene product.
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.
The gene product is typically an RNA inhibitory sequence 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 product. For example, where the gene product is a ribozyme, the third nucleotide sequence is adapted to be resistant to cleavage by the ribozyme.
Preferably the third nucleotide sequence is codon optimised for expression in host cells. The host cells, which term includes producer cells and packaging cells, are typically mammalian cells.
In a particularly preferred embodiment, (i) the viral genome is an HIV genome comprising nucleotide sequences encoding anti-HIV ribozymes and/or anti-HIV antisense sequences directed against HIV packaging component sequences (such as gag-pol) in a target HIV and (ii) the viral system for producing packaged HIV particles further comprises nucleotide constructs encoding the same packaging components (such as gag-pol proteins) as in the target HIV wherein the sequence of the nucleotide constructs is different from that found in the target HIV so that the anti-HIV ribozyme and/or antisense HIV sequences cannot effect cleavage or degradation of the gag-pol transcripts during production of the HIV particles in producer cells.
The present

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