Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2002-09-18
2004-11-23
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S005000, C435S006120, C435S070100, C435S069700, C435S173300, C435S325000
Reexamination Certificate
active
06821753
ABSTRACT:
This application is a national phase application of International Patent Appln. No. PCT/GB00/04981 filed Dec. 22, 2000, which designated the United States and was published in English.
FIELD OF THE INVENTION
The present invention relates to replication incompetent herpes simplex viruses capable of efficiently transferring genes to multiple sites within the nervous system. It also relates to the use of such viruses in the study and treatment of diseases and conditions of the nervous system.
BACKGROUND TO THE INVENTION
Herpes simplex viruses (HSV) 1 and 2 have often been suggested as a vector for gene delivery to the nervous system and also other cell types (for reviews see Coffin and Latchman 1995, Fink et al. 1996). As a vector HSV has a number of potential advantages in that it naturally enters latency in neurons, providing the possibility of long term gene expression, does not integrate into the host genome, preventing insertional mutagenesis (for example the activation of oncogenes or inactivation of tumour suppressor genes), can accept very large DNA insertions allowing the delivery of multiple genes, is easy to propagate, and can infect a wide variety of other cell types as well as neurons. HSV also has the unique ability among viruses currently under development as vectors in that it can be efficiently transported along nerves to the cell body (usually in the spine) by retrograde axonal transport following an initial peripheral infection. However, while this property of retrograde axonal transport of HSV vectors has been observed with replication competent vectors in the peripheral nervous system (PNS), it has not previously been exploited in vectors used in the central nervous system (CNS), probably due to limitations in the vectors which have previously been available.
While HSVI is highly prevalent in the human population, in the vast majority of cases giving no obvious signs of disease, for use as a vector the virus must be disabled for safety and so as to minimise toxicity to target cells. Various strategies for disablement have been reported including the removal of genes which are unnecessary for growth in vitro but necessary for pathogenesis in vivo. Such genes include those encoding thymidine kinase (TK; Ho and Mokarski 1988), ribonucleotide reductase (Goldstein and Weller 1988) and ICP34.5 (Coffin et al. 1995). However for minimal toxicity it has become apparent that expression of the regulatory immediate early genes ICPO, ICP4, ICP22 and ICP27, which are themselves cytotoxic, must be minimised (Johnson et al. 1994, Johnson et al. 1992, Wu et al. 1996, Samaniego et al. 1998, Krisky et al. 1998). Such reductions in IE gene expression minimise transcription from the vast majority of the 80 or so other genes in the HSV genome. Removal of ICP4 or ICP27 completely prevents virus growth and so such deletions must be complemented in the cells used for virus propagation (e.g. Deluca et al. 1985). Deletion of ICP22 and/or ICPO, while these genes are not absolutely essential for virus growth (Sacks and Shaffer 1987, Stow and Stow 1986, Post and Roizman 1981, Sears et al. 1985), reduces virus titre. Thus for the growth of HSV mutants with multiple IE gene deficiencies, cell lines must be produced which effectively complement deletions from the virus, and for effective growth of viruses with deletions in ICP4, ICP27, ICP22 and ICPO, all these deficiencies would optimally need to be complemented. However as the IE proteins are highly cytotoxic (Johnson et al. 1994), IE gene expression in cell lines must be tightly regulated. This is usually achieved by the use of the homologous IE gene promoters which are relatively inactive in the absence of virus infection (e.g. E5 cells [ICP4], B130/2 cells [ICP27], E26 cells [ICP4+ICP27], F06 cells [ICP4+ICP27+ICPO]; Deluca and Schaffer 1987, Howard et al. 1998, Samaniego et al. 1995, Samaniego et al. 1997). This reduces the problem of IE protein cytotoxicity but still leaves an inherent problem in the generation of cells which are highly effective at complementing multiple IE gene deficiencies.
A second strategy to reduce IE gene expression, rather than deletion of the IE genes themselves, is to include mutations in the gene encoding vmw65. vmw65 is a virion protein which transactivates IE promoters after virus infection (Batterson and Roizman 1983, Pellet et al. 1985), and while an essential structural protein, specific mutations abolish the trans-activating capability of the protein without affecting the structural integrity of the virus (Ace et al. 1989, Smiley and Duncan 1997). These mutations vastly reduce IE gene expression although at high multiplicity or with the inclusion of hexamethylene bisacetamide (HMBA) in the media still allow efficient virus growth in culture (McFarlane et al. 1992).
Thus, for the construction of vector viruses the approach could also be taken of combining mutations in vmw65, which should reduce expression of all the IE genes, with deletion of ICP27 and/or ICP4, the two essential IE genes, giving viruses as above in which overall IE gene expression is minimised. This is the approach we have taken to generate non-toxic HSV vectors in which IE gene expression has been minimised but which can still be grown in culture using cell lines containing the genes encoding ICP27, ICP4 and the equine herpes virus (EHV) homologue of vmw65 (encoded by EHV gene 12; Thomas et al. 1999).
The development of HSV vectors has also required a second problem to be overcome before they can be used to take advantage of the natural lifecycle of the virus in which a latent state is maintained in neurons. This problem results from the finding that in most cases promoters driving genes inserted into an HSV vector genome are rapidly transcriptionally inactivated as the virus enters latency, including the promoter which usually drives the expression of the only HSV transcripts present during latency, the latency associated transcripts (LATs). To solve this problem, a number of approaches have been taken:
First, it was found that a Moloney murine leukaemia virus (MMLV) promoter linked to a fragment of the LAT promoter (‘LAP 1’; Goins et al. 1994) and inserted into the gene encoding glycoprotein C (gC) was able to drive expression during latency, although neither LAPI or the MMLV promoter alone, or a number of other promoters linked to LAPI or alone allowed this to occur (Lokensgard et al. 1994). MMLV alone inserted in ICP4 (Dobson et al. 1990, Bloom et al. 1995) or in LAPI (Carpenter and Stevens 1996) however was active, which may be speculated to be due to the proximity of these regions to the endogenous LATs rather than when distant to the LAT region (in gC) as before. In other approaches it was found that LAP2 alone could give expression during latency when inserted in gC (Goins et al. 1994), but this expression was very weak, and that LAP2 linked to LAPI, like MMLV linked to LAPI, could also maintain latent gene expression when inserted in gC (Lokensgard 1997). Finally it was found that insertion of an internal ribosome entry site (IRES) into the 2 Kb LAT allowed expression of a downstream marker gene (with poly A site) during latency (Lachmann and Efstathiou 1997). However while the above approaches have demonstrated latent gene expression in the PNS, as yet latent gene expression in the brain has only been demonstrated in a very small number of transduced cells (Dobson et al. 1990, Bloom et al. 1995, Carpenter and Stevens 1996) and effective replication defective disabled HSV vectors for the long term gene transfer to the brain have not previously been available.
We have taken an alternative approach to the development of promoters allowing latent gene expression from within the context of an HSV vector where we have found that elements of the LAT region can be used to confer a long term activity onto promoters which are not usually active during latency by placing them downstream of a region designated LAT P2 (promoters inserted after HSVI nt 120,219; Genbank file HEICG: W0
Biovex Limited
Nixon & Vanderhye PC
Park Hankyel T.
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