Vectors that repress heterologous promoter activity

Details Vectors that repress heterologous promoter activity Vectors that repress heterologous promoter activity

1997-09-08

2004-05-04

Housel, James (Department: 1648)

Chemistry: molecular biology and microbiology

Vector, per se

C514S04400A, C424S199100, C424S207100

Reexamination Certificate

active

06730511

ABSTRACT:

BACKGROUND OF THE INVENTION
Mouse mammary tumour virus (MMTV) is a retrovirus that is associated with mammary tumorigenesis in susceptible mice (Salmons, B. and Güzburg, W. H.,
Virus Res.,
8:81-102, 1987). The virus is transmitted from the mother mouse to the suckling offspring via the milk. In addition to the usual retroviral genes gag, pol and env, the Long Terminal Repeat (LTR) of Mouse Mammary Tumour Virus (MMTV) contains an open reading frame (ORF) (Donehower, L. A. et al.,
J. Virol.,
37:226-238, (1981); Kennedy, N. et al.,
Nature,
295:622-624 (1982)) which is highly conserved between different MMTV isolates (Brandt-Carlson, C. et al.,
Virology,
193:171-185 (1993)). Although ORF specific transcripts have yet to be cloned, in part due to their low abundance, a splice acceptor site has been mapped immediately upstream of the 3′ LTR which is presumed to generate putative 1.7 kb ORF transcripts (Wheeler, D. A., et al.,
J. Virol.,
46:42-49 (1983); van Ooyen, A. J. et al.,
J. Virol.,
46:362-370 (1983)). Recently, a novel promoter has been identified in the MMTV 5′LTR and transcripts initiating from this promoter also splice to the ORF acceptor site (Güzburg, W. H. et al.,
Nature,
364:154-158 (1993)), increasing the potential for diversity of ORF related products.
Two biological activities, defined by functional assays, have been ascribed to products of the ORF. One of these activities is a transcriptional repressor, Naf, which downregulates in trans expression from MMTV based constructs (Salmons, B., et al.,
J. Virol.,
64:6355-6359, (1990); Güzburg, W. H. and Salmons, B.,
Biochem. J.,
283:625-632 (1992)). The second activity displayed by the MMTV ORF is a superantigen. (Sag) activity (Choi, Y., et al.,
Nature,
350:203-207 (1991); Acha-Orbea, H., et al.,
Nature,
350:207-211 (1991)). Expression of Sag in vivo results in the stimulation and growth, followed by deletion, of reactive T cells (reviewed in Acha-Orbea, H. and MacDonald, H. R.,
Trends in Microbiology,
1:32-34 (1993)). This effect is specific in that the Sag of a given MMTV variant interacts with specific classes of the twenty described V13 chains of the T cell receptor (Pullen, A. M., et al.,
J. Exp. Med.,
175:41-47 (1992), Huber, B. T.,
Trends in Genetics,
8:399-402 (1992)).
The viral Sag has been shown to be a type II membrane anchored glycoprotein of 45 KDa by in vitro translation studies (Korman, A. J., et al.,
The EMBO J.,
11:1901-1905 (1992), Knight, A. M., et al.,
Eur. J. Immunol.,
175:879-882 (1992)). Further, Sag proteins of 45/47 kDa have also been synthesized in baculovirus (Brandt-Carlson, C. and Butel, J. S.,
J. Virol.,
65:6051-6060 (1991); Mohan, N. et al.,
J. Exp. Med.,
177:351-358 (1993)) and vaccinia virus (Krummenacher, C. and Diggelmann, H.,
Mol. Immunol.,
30:1151-1157 (1993)) expression systems. This 45/47 kDa glycoprotein may require processing to a 18 kDa cleavage product (Winslow, G. M. et al.,
Cell,
71:719-730 (1992)). A Sag specific monoclonal antibody detects Sag expression on LPS-activated, but not nonstimulated, B cells even though the latter cells express a functional Sag. Thus undetectable levels of Sag are sufficient for superantigen activity ((Winslow, G. M. et al.,
Cell,
71:719-730 (1992); Winslow, G. M. et al.,
Immunity,
1:23-33 (1994)).
The use of retroviral vectors (RV) for gene therapy has received much attention and currently is the method of choice for the transferral of therapeutic genes in a variety of approved protocols both in the USA and in Europe (Kotani, H., et al.,
Human Gene Therapy,
5:19-28 (1994)). However, most of these protocols require that the infection of target cells with the RV carrying the therapeutic gene occurs in vitro, and successfully infected cells are then returned to the affected individual (Rosenberg, S. A., et. al.,
Human Gene Therapy,
3:75-90 (1992), Anderson, W. F.,
Science,
256:808-813 (1992)). Such ex vivo gene therapy protocols are ideal for correction of medical conditions in which the target cell population can be easily isolated (e.g., lymphocytes). Additionally the ex vivo infection of target cells allows the administration of large quantities of concentrated virus which can be rigorously safety tested before use.
Unfortunately, only a fraction of the possible applications for gene therapy involve target cells that can be easily isolated, cultured and then reintroduced. Additionally, the complex technology and associated high costs of ex vivo gene therapy effectively preclude its disseminated use world-wide. Future facile and cost-effective gene therapy will require an in vivo approach in which the viral vector, or cells producing the viral vector, are directly administered to the patient in the form of an injection or simple implantation of RV producing cells.
This kind of-in vivo approach-, of course, introduces a variety of new problems. First of-all, and above all, safety consideration have to be addressed. Virus will be produced, possibly from an-implantation of virus producing cells, and there will be-no opportunity to precheck the produced virus. It is important to be aware of the finite risk involved in the use of such systems, as well as trying to produce new systems that minimize this risk. The essentially random integration of the proviral form of the retroviral genome into the genome of the infected cell led to the identification of a number of cellular proto-oncogenes by virtue of their insertional activation (Varmus, H. “Retroviruses”,
Science,
240:1427-1435 (1988)). The possibility that a similar mechanism may cause cancers in patients treated with RVs carrying therapeutic genes intended to treat other preexistent medical conditions, has posed a recurring ethical problem. Most researchers would agree that the probability of the replication defective RV, such as all those currently used, integrating. into or near a cellular gene involved in controlling cell proliferation is vanishingly small. However, it is generally also assumed that the explosive expansion of a population of replication competent retrovirus from a single infection event, will eventually provide enough integration events to make such a phenotypic integration a very real possibility.
Retroviral vector systems are optimized to minimize the chance of replication competent virus being present. However, it has been well documented that recombination events between components of the RV system can lead to the generation of potentially pathogenic replication competent virus and a number of generations of vector systems have been constructed to minimize the risk of recombination (Salmons, B. and Günzburg, W. H.,
Human Gene Therapy,
4:129-141 (1993)). However, little is known about the finite probability of these events. Since it will never be possible to reduce the risk associated with this or other viral vector systems to zero, an informed risk-benefit decision will always have to be taken. Thus it becomes very important to empirically: determine the chance of (Donehower, L. A. et al.,
J. Virol.,
37:226-238, (1981)) insertional disruption or activation of single genes by retrovirus integration and (Kennedy, N. et al.,
Nature,
295:622-624 (1982)) the risk of generation of replication competent virus by recombination in current generations of packaging cell lines. A detailed examination of the mechanism by which these events occur will also allow the construction of new types of systems designed to limit these events.
A further consideration for practical in vivo gene therapy, both from safety considerations as well as from an efficiency and from a purely practical point of view, is the targeting of RVs. It is clear that therapeutic genes carried by vectors should not be indiscriminately expressed in all tissues and cells, but rather only in the requisite target cell. This is especially important if the genes to be transferred are toxin genes aimed at ablating specific tumour cells. Ablation of other, nontarget cells would obviously be very undesirable. Targeting of the expression of carried therapeutic

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