Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1997-11-03
2001-02-13
Priebe, Scott D. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S021000, C800S022000, C800S025000, C435S325000
Reexamination Certificate
active
06187993
ABSTRACT:
The present invention relates to transgenic animals which can act as a model for a human disease state. In particular, the invention relates to transgenic mammals which can serve as a non-human model for psoriasis.
Psoriasis is a skin condition that affects around 2% of the world's population and is characterized clinically by the presence of rounded, circumscribed, erythematous, dry, scaling patches of various sizes, covered by greyish white or silvery white, umbilicated and lamellar scales, which have a predilection for the exterior surfaces, nails, scalp, genitalia and lumbosacral region.
Although in its mildest form it may merely be a nuisance which it is desirable to control for cosmetic reasons, in more severe forms it can lead to severe pustule and scab formation and may be associated with arthritis.
In the earliest stages of psoriasis mild epidermal hyperplasia occurs, including mis-expression of keratins 6 and 16, and increased proliferation in the basal layer. Capillaries in the dermis become enlarged due to immune infiltrate, and mild inflammation is noticed in the epidermis as well as the dermis. The numbers and types of localised T-cell present are increased in both these tissues. In epidermis, regions of parakeratosis alternate with regions of hyperkeratosis. Mitotic activity in the dermis also has been found to increase.
In the later stages of psoriasis inflammation increases in the epidermis to the point where pustules or cysts often develop. This increased inflammation results in flaking, reddened skin. In the most generalised forms of psoriasis, marked acanthosis occurs in the epidermis, that is bell-shaped fingers of dermis invaginate the overlaying epidermis to the point where the epidermis becomes very thin focally. Also, focal necrosis of epidermal tissue may result in more severe pustule and scab formation.
Human epidermal keratinocytes express several adhesive receptors that belong to the integrin family of &agr;/&bgr; heterodimers. Several of the keratinocyte integrins share a common &bgr;
1
subunit: &agr;
2
&bgr;
1
mediates binding to collagen and laminin; &agr;
3
&bgr;
1
is a receptor for laminin and epiligrin; and &agr;
5
&bgr;
1
is the keratinocyte fibronectin receptor. The cells also express &agr;
v
&bgr;
5
, which is a vitronectin receptor, and &agr;
6
&bgr;
4
, which is a component of hemidesmosomes and &agr;
8
&bgr;
1
and &agr;
9
&bgr;
1
. &bgr;
1
integrins not only mediate keratinocyte adhesion to extracellular matrix proteins, but also play a role in intercellular adhesion, lateral migration, stratification, proliferation and the regulation of terminal differentiation.
Integrin expression is largely confined to the basal, proliferative, layer of keratinocytes both in adult skin and during embryonic development (Hertle et al (1992)
J. Clin. Invest
. 89, 1892-1901). The absence of integrins from the surface of suprabasal, terminally differentiating cells reflects a two-stage downregulation of receptor function and expression (Hotchin and Watt (1992)
J. Biol. Chem
. 267, 14852-14858). The first stage occurs in basal keratinocytes that become committed to terminal differentiation: there is no reduction in the level of &bgr;
1
integrins on the cell surface, but the ability of the receptors to bind extracellular matrix proteins is substantially decreased (Hotchin and Watt (1992)
J. Biol. Chem
. 267, 14852-14858) probably reflecting a change in receptor conformation (O'Toole et al (1990)
Cell Reg
. 1, 883-893). The second stage of downregulation is the loss of integrins from the surface of cells that have left the basal layer (Hotchin and Watt (1992)
J. Biol. Chem
. 267, 14852-14858). Although integrin expression is normally confined to basal keratinocytes, there are situations in which suprabasal keratinocytes co-express integrins (reviewed by Watt and Hertle (1994) in
The Keratinocyte Handbook
, eds. I. M. Leigh, E. B. Lane and F. M. Watt, Cambridge University Press), notably during wound healing (Jones et al (1993)
J. Pathol
. 169, 235-244), in psoriatic lesions (Hertle et al (1992)
J. Clin. Invest
. 89, 1892-1901) and in some squamous cell carcinomas (Jones et al (1993)
J. Pathol
. 169, 235-244. Suprabasal integrin expression can be transient: in the healing of small suction blister wounds it occurs at the-time of wound closure, when the keratinocytes are hyperproliferative, but returns to normal one week later (Hertle et al (1992)
J. Clin. Invest
. 89, 1892-1901). Suprabasal expression is not a direct response to inflammation since it is not induced by intradermal injections of TNF&agr; or IFN&ggr; (Hertle et al (1995)
J. Invest. Dermatol
. 104, 260-265). Suprabasal integrin expression has also been noted in eczema and lichen planus.
Genetic factors have been implicated in the epidemiology of psoriasis (Elder et al (1994)
J. Invest. Dermatol
. 24S-27S) and the immune system plays a role in the pathogenesis of psoriasis (Baadsgaard et al (1990)
J. Invest. Dermatol
. 95, 32S-34S.
At present, no-one knows what causes psoriasis.
Mild psoriasis is presently treated using an emollient. In more troublesome cases, local application of salicylic acid, coal tar, dithranol or calcipotriol can be used. Photochemotherapy using psoralens with long-wave ultraviolet irradiation is sometimes used and etretinate, a retinoid, may be given in severe or complicated psoriasis. Acitretin, a metabolite of etretinate, may also be used in severe cases, as can methotrexate and cyclosporin.
There is a need for improved treatments for psoriasis. However, no adequate model system exists for testing potential treatments. Although suprabasal expression of integrins has been found on some occasions to be associated with psoriasis (and other conditions, as noted above), there was no reason to believe that such expression could cause psoriasis, especially since the disease had been thought to have such a strong immune system involvement.
Objects of the present invention are to provide an animal model of psoriasis, means of producing such an animal model and uses for the animal model.
A first aspect of the invention provides a nucleic acid construct comprising a promoter capable of directing expression in the suprabasal cells of the epidermis and means to cause expression of an integrin subunit in the suprabasal cells.
By “means to cause expression of an integrin subunit in the suprabasal cells” we include an integrin subunit coding region and other coding regions whose expression in the suprabasal cells causes expression of an integrin subunit in the suprabasal cells. It is particularly preferred if the means to cause expression of an integrin subunit in the suprabasal cells is an integrin subunit coding region.
It is preferred if the nucleic acid construct is a DNA construct although RNA constructs, such as retroviral constructs, are included in which case the promoter is active when it is copied into DNA within a host cell.
By “a promoter capable of directing expression in the suprabasal cells of the epidermis” we include any such promoter which can so direct expression. Thus, we include promoters from animal sources, especially mammalian sources, as well as from viral sources.
A promoter which expresses in all layers of the epidermis is suitable but it is preferred if the said promoter is a keratinocyte-selective promoter, more preferably a suprabasal cell-selective promoter.
By “suprabasal cell-selective promoter” we include all nucleic acid elements that direct expression selectively in the suprabasal layer of the epidermis of an animal.
The organisation of the basement membrane, basal layer and suprabasal layer of the epidermis of a mammal is shown diagrammatically in FIG.
1
.
A suprabasal cell is a cell of the epidermis. That is, if the single layer of cells adherent to the basement membrane (ie adjacent to the dermis) are defined as basal, all the other keratinocyte cells are suprabasal. A suprabasal cell is identified by its location and by expression of differentiation markers such as keratin-1, keratin-10, loricrin, filaggrin and involu
Carroll Joseph M.
Watt Fiona M.
Baker Anne-Marie
Imperial Cancer Research Technology Limited
Nixon & Vanderhyde P.C.
Priebe Scott D.
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