Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
1997-09-17
2001-02-27
Salimi, Ali R. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant virus encoding one or more heterologous proteins...
C424S204100, C424S229100, C435S005000, C435S235100, C435S320100, C435S173300, C514S04400A
Reexamination Certificate
active
06193980
ABSTRACT:
The present invention relates to constructs for delivery of sequences of interest to cells of an individual, for instance using recombinant viruses. This can have a therapeutic aim: and examples of the constructs and cells containing them can be useful also, for example, in production of a polypeptide which can then be used as desired (e.g. as an immunogen). By employing a latency active promoter of a latency associated transcript (LAT) region of a herpes simplex virus, long-term, high-level expression of a reporter sequence can be achieved.
It is often desirable to deliver exogenous DNA to cells in order to provide a missing gene or to help correct abnormal cellular behaviour. The present invention is not generally concerned with any difficulties that may be associated with delivery of nucleic acid to cells.
Many viruses have evolved to deliver nucleic acid into the nucleus of the cell, where it can be expressed. Certain viruses have been genetically engineered to carry a gene to be delivered, and can deliver it to host cells such-as those the virus normally infects. Gene delivery vectors have also been based on attenuated or genetically disabled virus.
A number of genetically engineered viruses have been used to deliver foreign genes to cells both in vitro and in vivo. For certain purposes it is desirable for the gene to be stably expressed, producing biologically active amounts of its product on a long term basis. This remains a problem in a number of contexts.
Herpes Simplex Virus (HSV) is a ubiquitous pathogen of man which is capable of acutely infecting many cell types, and can persist long term in the sensory neurons of the host's dorsal root ganglia. This state, described as viral latency, is characterised by the persistence of the viral genome in the nucleus of the neuron without any detectable production of viral proteins, or interference with normal cellular metabolism. HSV's ability to establish latency in neurons makes it an attractive-candidate as a gene delivery vector for the nervous system.
Though the virus does not produce any detectable protein product during latency, there is continuing RNA transcription. This latency associated transcription comes from a single region of the viral genome (the latency associated transcript or LAT region) and is driven by the latency active promoter (LAP). The TATA box and basal transcriptional regulatory sequences, which constitute the core LAT promoter, reside approximately 700 bp upstream of the 2 kb major LAT.
HSV-1 is considered a good candidate vector for CNS gene therapy because it is able to establish life-long latent infections in human sensory neurons. During latency, the viral genome appears to be maintained episomally, and hence there appears to be no danger of insertional mutagenesis or inactivation of host genes. Furthermore, there is no detectable production of viral proteins during latency, and no evidence that the latent state interferes with the normal metabolism of the host cell.
Viral gene expression during latency appears to be limited to 2 or 3 nuclear RNA species which accumulate to high levels in sensory neurons harbouring latent virus (the LATs) (3). These transcripts are driven by a complex promoter region (LAP) (4). A functional-LAT region is not essential for the establishment of latency, and viruses whose LAT region has been deleted can still establish latency (5), and in some cases, express LATs (6). In fact it seems that the LATs may be involved in reactivation, as LAT negative viruses do not reactivate efficiently (7). Latency can be established in the absence of any viral gene expression, and seems to be a default pathway for the virus when it enters a cell where productive infection is not possible.
Studies using diverse promoters to drive expression of a reporter gene (usually &bgr;-galactosidase) in animal models, have shown transient reporter gene expression, but that this is not long-lived. This has led to much work trying to define what elements of the LAT region and promoter (LAP) are involved in maintaining its long-term transcriptional activity. Further work has been done to try to utilise the LAP and other sequence elements in the LAT region to facilitate long-term expression of reporter genes.
A rabbit &bgr;-globin gene inserted downstream of the TATA box of the LAP made &bgr;-globin RNA during latent infection, but at lower levels than LAT in wild type infection (8). When the experiments were repeated using the endogenous LAT promoters to drive &bgr;-galactosidase or nerve growth factor genes, no RNA could be detected by in situ hybridisation (9).
The same group have since used a recombinant defective virus with a Moloney Murine Leukaemia Virus (MMLV) LTR, Lac Z construct inserted into ICP4, and a deletion of the 5′ part of LAT, and demonstrated &bgr;-galactosidase expression in sensory ganglia (10). Gene expression was also assessed in motor neurons of the hypoglossal nucleus, where there was abundant transient expression. The results were taken to indicate that the MMLV LTR did not remain active during latency in motor neurons. When the MMLV LTR is moved away from the viral repeats (where it is near to endogenous LAT sequences) and inserted into the gC locus, it was not longer able to produce long-term gene expression. If the region upstream of the LAP is also inserted into the gC locus upstream of the LTR, this virus is capable of producing gene expression in sensory neurons. The LAT sequences did not have similar facilitating effects when inserted upstream of the murine metallothionein promoter (11).
Other groups have also used the LAT promoter in vectors. Wolfe et al used a recombinant virus with the &bgr;-glucoronidsase (GUSB) gene inserted into a LAT deletion downstream of the LAP in an attempt to correct the deficiency in GUSB deficient mice (12). A corneal infection route was used, and, although there was no phenotypic improvement in the condition of the mice, they were able to detect some GUSB positive cells as much as 18 weeks post inoculation. Miyanohara et al have used a variety of HSV-1 vectors to attempt to deliver genes to the liver of mice (13). Using a LAT promoter to drive HbsAg or canine factor IX, they saw low levels of protein in the serum for about 3 weeks after direct injection of the vector into the liver.
These studies suggest that the LAT promoter may be able to produce prolonged, albeit low level, expression of foreign genes in the CNS and even in non-neuronal cells.
Goins et al.
J. Virol
. 68: 2239-2252 (1994) and WO96/27672 postulated the presence of a second latency-active promoter, tested for by experiments involving transient gene expression, located in the HSV1 U
L
flanking repeats.
Specification WO 96/27672 (published later than the priority date claimed for the present application) (Glorioso & Fink) concerns the structure of a herpesvirus promoter for transcription of a non-herpes gene in a cell latently infected with a herpes virus, e.g. peripheral neurons and cranial nerve ganglia.
HSV-1 based vectors have also been constructed using lytic cycle HSV promoters (gC (14), IE110 (15)), strong non-specific promoters (CMV IE MMLV LTR) and neuron specific promoters (NSE (16)). On the whole, these studies show only transient gene expression in both peripheral and CNS neurons. As discussed above, the MMLV LTR can give long-term expression, but only when inserted close to LAT elements.
The present inventors now show experimentally that by inserting a reporter gene, preceded by an internal ribosomal entry site (to allow efficient translation), downstream of the LAP in the LAT region, a vector that gives stable, high level reporter gene expression during viral latency can be produced.
In initial attempts to construct HSV vectors which would give long-term gene expression the present inventors decided to use a murine RNA polymerase I (RNA pol I) promoter reporter gene construct inserted into the LAT region. RNA pol I is responsible for the transcription of ribosomal RNAs, and is active in all cell types. Native RNA pol I transcripts are
Efstathiou Stacey
Lachmann Robin H.
Cambridge University Technical Services Limited
Klarquist Sparkman Campbell & Leigh & Whinston, LLP
Salimi Ali R.
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