Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-11-03
2001-02-06
Priebe, Scott D. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S008000, C800S003000, C800S008000, C514S04400A
Reexamination Certificate
active
06184436
ABSTRACT:
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to a transgenic (Tg) mice expressing at least HIV-1 nef or the entire HIV-1 coding sequences under the control of the human CD4 gene promoter flanked by the enhancer of the mouse CD4 gene to serve as a small animal model of AIDS.
(b) Description of Prior Art
The human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of AIDS. Although the biology of HIV-1 has been intensively studied, the pathogenic mechanisms by which the depletion of CD4
+
T lymphocytes occurs and how the severe immune disease characteristic of advanced AIDS is induced are not completely understood. A major obstacle in studying the pathogenesis of HIV-1 infection in vivo and the development of effective vaccines has been the lack of suitable animal models. A small, readily available animal model that could duplicate, in whole or in part, the pathological changes observed in AIDS patients would be a great asset for AIDS research.
Cell tropism for infection by HIV-1 is largely determined by the cell-surface expression of the CD4 antigen and the newly discovered co-receptors (CXCR4/fusin, CCR5). Immature T-cell precursors and mature CD4
+
T cells and cells of the dendritic/macrophage lineage (monocytes, dendritic cells, Langerhans cells, macrophages and microglial cells) are those subsets of cells mainly targeted by HIV-1 infection. Ideally, an animal model for HIV-1 infection and AIDS should have the same subsets of cells infectable by or expressing HIV-1. To date, no animal model has been generated that fulfills all these criteria. The best model currently used still remains SAIDS, an AIDS-like syndrome induced in primates by SIV, a simian lentivirus having a structure very similar to that of HIV-1. However, this model is not widely available to all scientists and a virus different from HIV-1 is used for infection. Severe combined immunodeficient (SCID) mice reconstituted with human lymphoid cells have also been used for infection with HIV-1. This biological system offers the advantage of active HIV-1 replication in small animals with a severe depletion of the engrafted CD4
+
T cells. However, these mice are relatively difficult to generate and other important manifestations of AIDS are not observed. The third type of models for HIV-1-induced disease currently available are transgenic (Tg) mice expressing all or some HIV-1 gene products. Although the initial interaction of HIV-1 with its cellular receptors and the early replication steps and reinfection cycles are bypassed and cannot be studied in these Tg models, they nevertheless allow investigation of post-integration events in the virus life cycle, such as HIV-1 protein-mediated cytopathic effects and the subsequent host responses.
Several HIV-1 Tg animal models have been developed (Klotman PE et al., AIDS, 1995, 9:313-324). The use of the HIV-1 LTR to express the whole HIV-1 genome in Tg mice led to the development of an AIDS-like syndrome, but this phenotype was observed in a single line (no 13) of Tg mice (Leonard J M et al., Science, 1988, 242:1665-1670). LTR-dependent expression of the 3′ half of the HIV-1 genome in Tg mice induced a severe nephropathy. The HIV-1 LTR-driven expression of either nef or tat in Tg mice was found to induce epidermal hyperplasia, while expression of gag or protease in lens fiber cells appears to be responsible for the development of cataracts. Attempts to express HIV-1 nef more specifically in lymphoid cells of Tg mice were made by using the T-cell specific CD3 &dgr; promoter/enhancer element, the TCR&bgr; chain-enhancer promoter element or the CD2 regulatory elements. These Tg animals showed varying degrees of depletion of CD4
+
thymocytes and of peripheral T cells, depending on the promoter used. However, additional important characteristics of AIDS were not observed in these mice while other features, such as a large increase of the B cell population, not seen in human AIDS, was observed (Lindemann D et al., J. of Experimental Medicine, 1994, 179:797-807).
It would be highly desirable to be provided with a small animal model of AIDS such as a transgenic mice which would develop a severe AIDS-like disease which would dependent on the levels of HIV-1 expression.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide a small animal model of AIDS such as a transgenic mice which would develop a severe AIDS-like disease which would dependent on the levels of HIV-1 expression.
In an attempt to develop more relevant model of HIV-1 infection, it was an aim of the present invention to express HIV-1 gene products in the same cells of Tg mice as those usually found infected in HIV-1 positive individuals, i.e. CD4 T
+
cells and cells of the dendritic/macrophage lineage. The human CD4 gene promoter sequences flanked by the enhancer of the mouse CD4 gene were used to express the whole HIV-1 coding sequences in Tg mice. These Tg mice should represent a model of a relatively high steady-state viral RNA load since all relevant cell subsets should express the transgene in Tg mice. In accordance with the present invention, evidence is presented here that indeed these Tg mice develop a severe AIDS-like disease which is dependent on the levels of HIV-1 expression.
In accordance with the present invention there is provided a transgenic mouse to serve as a small animal model of AIDS which comprises a HIV-1 DNA sequence coding for at least nef under the control of the human CD4 gene promoter flanked by the enhancer of the mouse CD4 gene for expression in T cells and in cells of monocyte/microphage lineage.
In accordance with the present invention there is provided a transgenic mouse to serve as a small animal model of AIDS which comprises a HIV-1 DNA genome essentially consisting in entire HIV-1 coding sequences under the control of the human CD4 gene promoter flanked by the enhancer of the mouse CD4 gene for expression in T cells and in cells of monocyte/microphage lineage.
In accordance with the present invention there is provided a transgenic mouse to serve as a small animal model of AIDS in which the germ cells and somatic cells carry at least one copy of a single transgene that comprises:
a) a HIV-1 DNA sequence coding for at least nef; and
b) a human CD4 gene promoter operatively linked to the HIV-1 DNA genome, wherein the promoter is flanked by the enhancer of the mouse CD4 gene for expression in T cells and in cells of monocyte/microphage lineage;
wherein the transgene is introduced into the mouse or an ancestor thereof as a single transgene.
In accordance with the present invention there is provided a transgenic mouse to serve as a small animal model of AIDS in which the germ cells and somatic cells carry at least one copy of a single transgene that comprises:
a) a HIV-1 DNA genome essentially consisting in entire HIV-1 coding sequences; and
b) a human CD4 gene promoter operatively linked to the HIV-1 DNA genome, wherein the promoter is flanked by the enhancer of the mouse CD4 gene for expression in T cells and in cells of monocyte/microphage lineage;
wherein the transgene is introduced into the mouse or an ancestor thereof as a single transgene.
The preferred transgenic mouse of the present invention has the human CD4 gene promoter linked at the is 3′ end of the HIV-1 DNA genome.
In accordance with the present invention there is provided a method to screen for therapeutic agents for the treatment of AIDS, which comprises the steps of: a) administering the therapeutic agent to the mouse of the present invention; and b) determining the effects of the therapeutic agent.
In accordance with the present invention there is provided a method for producing a transgenic mouse to serve as a small animal model of AIDS, which comprises the steps of:
a) transferring a transgene into a mouse fertilized oocyte, which transgene comprises:
i) a HIV-1 DNA sequence coding for at least nef or the entire HIV-1 genome; and
ii) a human CD4 gene promoter operatively linked to the HIV-1 DNA sequence, wherein the p
Hanna Zaher
Jolicoeur Paul
Kay Denis G.
Côté France
Institut de Recherches Cliniques de Montreal
Priebe Scott D.
Shabey Ogilvy Renault
Woitach Joseph T.
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