Title: Method for in vitro
propagation of the agent responsible for transmissible spongiform
United States Patent: 7,001,767
Issued: February 21, 2006
Blanquet Grossard; Francoise (St Etienne de St Geoire,
FR); Cesbron; Jean-Yves (Meylan, FR); Lemaire Vieille; Catherine (St
Martin D'uriage, FR); Follet; Jerome (Lille, FR)
Assignee: Universite Joseph Fourier (Grenoble
1) (Grenoble, FR)
Appl. No.: 380800
Filed: September 20, 2001
PCT Filed: September 20, 2001
PCT NO: PCT/FR01/02933
371 Date: May 12, 2003
102(e) Date: May 12, 2003
PCT PUB.NO.: WO02/24871
PCT PUB. Date: March 28, 2002
Web Seminars -- Pharm/Biotech/etc.
The invention concerns a method for in
vitro propagation of the agent responsible for transmissible spongiform
encephalopathies, or prion, comprising steps which consist in: providing a
culture or a glial cell line and in infecting the culture or line with the
agent responsible for transmissible spongiform encephalopathies or prion.
Said method enables to obtain infected cell line capable of being used to
assess the efficacy of a molecule in the reduction or inhibition of the
infectiosity of the prion or for detecting the prion.
Description of the Invention
The invention relates to a method for in
vitro propagation of the agent responsible for transmissible spongiform
encephalopathies (TSEs), or prion, making it possible to conserve the
infecting capacity of the prion.
TSEs include mainly scrapie, bovine spongiform encephalopathy,
Creutzfeldt-Jakob disease and Kuru in humans.
These neurodegenerative diseases are characterized by thy appearance of an
abnormal form of the PrPc protein naturally present at the
surface of certain cells, and in particular of neurones.
The PrPc protein, or prion protein, is a cell surface
sialoglycoprotein which plays a primordial role in transmissible
spongiform encephalopathies in humans and animals. In these diseases, PrPc
is converted to a pathogenic form called PrPsc, which is
thought to be an abnormal isoform of PrPc, which has been shown
to be essential to infectivity and which is today considered to be the
infectious agent of these diseases.
The process of transformation of PrPc to PrPsc
remains undetermined and few tools are currently available for studying
the mechanism of action of this infectious agent, in particular for the
purpose of detecting it and searching for therapeutic treatments to treat
the diseases which it causes.
According to the publication by C. Lemaire-Vieille et al., PNAS, vol. 97,
No. 10 of May 9, 2000, Epithelial and endothelial expression of the
green fluorescent protein reporter gene under the control of bovine prion
protein (PrP) gene regulatory sequences in transgenic mice,
the authors of the studies obtained several transgenic mouse lines which
express the reporter gene of a fluorescent protein (gfp), under the
control of regulatory sequences of the bovine PrP protein gene. Exposure
to UV radiation of histological sections derived from organs taken from
these mice makes it possible to locate the production of fluorescence and
therefore to identify the cells capable of expressing the PrP gene. The
authors thus observed that the sites of production were cerebellar
Purkinje cells, lymphocytes, epithelial cells of the medullar region of
the thymus, keratinocytes and endothelial cells of the blood capillaries
of the intestinal villi.
These studies have therefore made it possible to have a study model from
which it is then possible to determine the sites of expression of the
The authors of the present invention have discovered that the glial cells
of the nervous system, and in particular the glial cells of the peripheral
nervous system and the Schwann cells, constitute a suitable site for the
in vitro propagation of the prion.
To date, only very few cell lines are known which are capable of
perpetuating the infectious capacity of the agent responsible for TSEs;
these are the N2a cell line derived from mouse neuroblastoma cells
[Butler, J. Virol. (1988) 62, 1558-1564], the PC12 cell line derived from
rat pheochromocytoma cells [Rubenstein, J. Gen. Virol. (1984) 65,
2191-2198], the GT1 cell line derived from neuronal cells from the
hypothalamus [Schatz, J. Virol. (1997) 11, 8821-8831] and the 1C11 cell
line derived from neuroectoderm cells [Mouillet-Richard, Microbes Infect.
(1999) 12, 969-976].
In the article V. M. Roikhel et al., Acta Virologica, vol. 31, No. 1
(1987) 36-42, the authors demonstrated that cells derived from the rat
Gasser's node, NGUK-I cells, could be infected in vitro and conserve their
infectious capacity. The NGUK-I cells were characterized and have a growth
profile which is that of astrocytes and a morphological profile which is
that of epithelial cells (A. P. Avtsyn et al., Tsitologiia (January 1989)
None of the cells previously mentioned are glial cells and the discovery
according to the present invention is surprising in that glial cells are
not neuronal cells and that the infectivity which develops therein, as is
shown below, is strong.
Thus, a first object of the invention is a method for in vitro propagation
of the prion, according to which a glial cell culture or a glial cell line
is provided and the culture or the line is infected with the agent
responsible for TSEs, or prion. More particularly, said agent is of murine
origin. Advantageously, the glial cells are glial cells of the peripheral
nervous system or Schwann cells.
Before describing the invention in greater detail, some terms used in the
description and the claims are defined below.
According to the invention, the expression "propagation of the prion in a
cell culture" is understood to mean that, after infection, or infestation,
of at least one cell of the starting cell culture or of the starting cell
line, the infectious capacity of the prion is conserved in the derived
cells, i.e. the cells resulting from subcultures.
The prion used to infect the starting culture or line is chosen from prion
strains such as the Chandler strain or the RML (Rocky Mountain Laboratory)
strain, or from the cells of a primary culture, i.e. cells taken directly
from an infected tissue. By way of example, they may be brain tissues
taken post-mortem from mice.
Preferential variants of the method above and of other subjects of the
invention are now presented.
The method is advantageously carried out using a culture or a line of
Schwann cells of murine origin; a preferred line is the MSC-80 line.
The invention also relates to a line of glial cells infected with the
prion, capable of propagating the prion; the cells of the line are
advantageously derived from Schwann cells, and more particularly derived
from cells of the murine Schwann cell line MSC-80, after infection with
Another subject of the invention is a method of evaluating the
effectiveness of a molecule, of an agent or of a composition in decreasing
or inhibiting the infectiousness of the prion, according to which method a
culture of glial cells or of glial cells of the peripheral nervous system
infected with the prion or a cell line of the invention, or an extract
thereof, is brought into contact with said molecule, said agent or said
composition, at predetermined doses, and the decrease in or the inhibition
of the infectiousness of the prion is detected. The detection step can be
carried out in Tga 20 mice.
The invention also provides a method for detecting and, optionally,
quantifying the prion, in a biological sample, according to which said
sample is brought into contact with a culture or a line of glial cells or
of glial cells of the peripheral nervous system, under conditions which
allow the infection thereof, and the infection or noninfection of said
line with said sample is detected. The glial cells are preferably Schwann
cells, and in particular the glial cell line is the MSC-80 line.
Yet another subject of the invention is the use of the MSC-80 cell line
for propagating the prion in vitro.
Claim 1 of 5 Claims
1. An isolated line of Schwann
cells infected with a prion, capable of propagating the prion, wherein said
Schwann cells are of the peripheral nervous system.
If you want to learn more
about this patent, please go directly to the U.S.
Patent and Trademark Office Web site to access the full