Methods of gene transfer to astrocytes with mokola virus pseudotyped
United States Patent: 7,749,973
Issued: July 6, 2010
Inventors: Sarkis; Chamsy
(Paris, FR), He; Yi (Paris, FR), Serguera; Che (Paris, FR), Dufour; Noelle
(Mennecy, FR), Mallet; Jacques (Paris, FR)
Assignee: Centre National
de la Recherche Scientifique (Paris, FR)
Appl. No.: 10/477,375
Filed: May 31, 2002
PCT Filed: May 31, 2002
PCT No.: PCT/FR02/01833
371(c)(1),(2),(4) Date: February
PCT Pub. No.: WO02/097104
PCT Pub. Date: December 05,
George Washington University's Healthcare MBA
The invention relates to a defective
lentivirus which is pseudotyped with a lyssavirus envelope of the PV
(rabies virus) or MOK type (Mokola virus), for example, and to the use
thereof, especially in the preparation of a composition for in vivo
transfer of genes in astrocytes and also for the treatment of disorders of
the central nervous system.
Description of the
This application is the US national phase
of international application PCT/FR02/01833 filed 31 May 2002 which
designated the U.S. and claims benefit of FR 01/07239, dated 1 Jun. 2001,
the entire contents of each of which is hereby incorporated by reference.
The present invention relates to gene transfer into the nervous system by
means of pseudotyped viruses. It relates in particular to the use of an
envelope (glycoprotein) of an enveloped virus, for example lyssavirus, for
pseudotyping a defective lentiviral vector and to the targeting of a cell
population or subpopulation of the nervous system. The present invention
also relates to methods and compositions intended for the selective
transfer of target genes into particular nerve cell populations, in
particular into astrocytes, in vitro, ex vivo and in vivo, and also for
the treatment of disorders of the central nervous system and/or of the
Gene transfer into the nervous system has many applications, in
experimental, therapeutic, research fields, etc. Thus, this transfer may
make it possible to carry out labeling, toxicity and quality studies, to
construct pathological models, to restore deficiencies, to express
therapeutic products (for example proteins, RNAs, etc.), etc.
Various approaches have been envisioned in the prior art for this
transfer, such as the use of viral vectors (retroviruses, AAVs,
adenoviruses, etc.), the injection of plasmids, the transplanting of
cells, the implantation of encapsulated cells, etc. Each of these
approaches has advantages and disadvantages, in terms of efficiency,
security, industrial use, selectivity, stability, etc.
Thus, the use of viral vectors is advantageous in terms of transfer
efficiency, associated with the natural properties of infection of
viruses. Among the viruses used, the applicant has been interested more
particularly in retroviruses.
In the first steps of infection, retroviruses deliver their nucleoprotein
core into the cytoplasm of the target cell. Reverse transcription of the
viral genome then takes place, while the core transforms into a
preintegration complex. The complex must reach the nucleus in order to
enable integration of the viral DNA into the chromosomes of the host cell.
Many oncoretrovirus-derived retroviral vectors allow the integration of a
transgene into the genome of target cells, but these vectors are only
capable of transducing dividing cells. This restriction limits their use
to gene transfer ex vivo or to organs in which the cells are mitotically
A means of bypassing this obstacle consists in using lentivirus-derived
vectors. Lentiviruses are complex retroviruses capable of integrating into
the genome of cells which are not mitotically active. These viruses
include in particular HIV-1, HIV-2, SIV, FIV, BIV, VISNA, CAEV and EIAV.
However, drawbacks associated with the use of lentiviruses lie in
particular in the lack of specificity, the potential risks and the
difficulties in production. The drawbacks have up until now limited the
use of this type of vector for gene transfer in vivo.
The present invention brings a solution to the problems of the prior art
and provides novel tools and vectors for gene transfer into the nervous
system. The invention lies in particular in the use of viral envelopes for
the selective transfer of genes into the nervous system, in particular
into certain cell subpopulations of the central nervous system.
The present invention describes, surprisingly, a pseudotyped defective
lentiviral vector and its use for the selective transfer of the genes into
certain nerve cell populations, and in particular into astrocytes, in
vitro, ex vivo and in vivo. Such a targeted gene transfer makes it
possible to locally treat a disease affecting the nervous system.
The invention also lies in the use of lyssavirus envelopes for targeting
lentiviral vectors to particular nerve cell populations. Lyssaviruses
belong to the rhabdovirus family.
The present invention also comprises the preparation of a composition
intended for the transfer of genes into astrocytes in vivo, and also the
use of this composition in the treatment of disorders of the central
DETAILED DESCRIPTION OF THE INVENTION
The present invention describes the pseudotyping of recombinant lentiviral
vectors allowing cellular targeting in the nervous system, and more
particularly in glial cells of the astrocyte type. These pseudotyped viral
vectors are useful for the transfer and the expression in vivo of nucleic
acid sequences within astrocytes.
General Structure of the Vectors
Like other retroviruses, lentiviruses have gag, pol and env genes flanked
by two LTR (Long Terminal Repeat) sequences. Each of these genes encodes
many proteins which are initially expressed in the form of a single
precursor polypeptide. The gag gene encodes the internal structural
proteins (capsids and nucleocapsids). The pol gene encodes the reverse
transcriptase, the integrase and the protease. The env gene encodes the
viral envelope glycoprotein and also contains a cis-acting RRE (Rev
Responsive Element) responsible for exporting the viral RNA out of the
nucleus. The 5' and 3' LTR sequences serve to promote the transcription
and the polyadenylation of the viral RNAs. The LTR contains all the other
cis-acting sequences necessary for viral replication. Sequences necessary
for the reverse transcription of the genome (tRNA primer binding site) and
for encapsidation of the viral RNA into particles (site .PSI.) are
adjacent to the 5' LTR. If the sequences necessary for encapsidation (or
for packaging of the retroviral RNA into infectious virions) are absent
from the viral genome, the genomic RNA will not be actively encapsidated.
The construction of lentiviral vectors for gene transfer applications has
been described, for example, in patents U.S. Pat. No. 5,665,577, EP 386
882, U.S. Pat. Nos. 5,981,276 and 6,013,516 or else in patent application
WO 99/58701. These vectors comprise a defective lentiviral genome, i.e. a
genome in which at least one of the gag, pol and env genes has been
inactivated or deleted. These vectors are encapsidated in a protein
particle composed of the structural lentiviral proteins and in particular
of the envelope glycoprotein.
The recombinant lentiviruses according to the invention are thus
genetically modified in such a way that certain genes constituting the
native infectious virus are eliminated and replaced with a nucleic acid
sequence of interest to be introduced into the target cells. After fusion
of the virus with the cell membrane, said virus injects its nucleic acid
into the cell and, after reverse transcription, said nucleic acid can
integrate into the genome of the host cell. The genetic material thus
transferred is then transcribed and possibly translated into proteins
inside the host cell.
The lentiviruses of the invention can be prepared from various serotypes,
in particular HIV-1, HIV-2, SIV, FIV, BIV, VISNA, CAEV and EIAV.
Particularly preferred serotypes are HIV, FIV and EIAV.
As indicated above, the invention lies in the demonstration of the
targeting properties of lentiviruses pseudotyped with particular
The term "pseudotyping" denotes a recombinant virus comprising an envelope
different from the wild-type envelope, and thus possessing a modified
tropism. In the case of the pseudotyped lentiviruses, they are
lentiviruses which have a heterologous envelope of non-lentiviral origin,
for example originating from another virus, or of cellular origin.
Pseudotyped HIV-1 vectors described in the prior art comprise the envelope
glycoprotein of the vesicular stomatitis virus (VSV). This envelope
exhibits advantageous characteristics, such as resistance to
ultracentrifugation and a very broad tropism. Unlike other envelopes, such
as those of the conventional retroviruses (amphotropic and ecotropic MLV
retroviruses or HIV gp120, but also many others), the VSV glycoprotein is
not labile after ultracentrifugation. This makes it possible to
concentrate the viral supernatents and to obtain high infectious titers.
Moreover, this envelope confers on the virions a very broad tropism, in
particular in vitro, allowing the infection of a very large number of cell
types. The receptor for this envelope is thought to be a
phosphatidylserine motif present at the surface of many cells of various
A particular characteristic of the invention consists in using an envelope
(glycoprotein) of a lyssavirus, and in particular of a virus of the rabies
virus serogroup: Rabies (RAB) (Tuffereau C., Benejean J., Blondel D.,
Kieffer B., Flamand A.; Low-affinity nerve-growth factor receptor (P75NTR)
can serve as a receptor for rabies virus; EMBO J., 1998 December 15 ;17
(24): 7250-9); Duvenhague (DUV), European bat type 1 (EB-1), European bat
type 2 (EB-2), Lagos bat (LB), Mokola (MOK) (Bourhy H, Kissi B, Tordo N.
Molecular diversity of the Lyssavirus genus. Virology. 1993
May;194(1):70-81.) (Badrane H, Bahloul C, Perrin P, Tordo N. Evidence of
two Lyssavirus phylogroups with distinct pathogenicity and immunogenicity.
J Virol. 2001 April;75(7):3268-76), Kotonkan (KOT), Obodhiang (OBD) and
Rochambeau (RBU) (Della-Porta AJ, Brown F. The physicochemical
characterization of bovine ephemeral fever virus as a member of the family
Rhabdoviridae. J Gen Virol. 1979 July;44(1):99-112.) or any chimeric
composition of these envelopes.
Unlike VSV, the present application shows that the rabies and Mokola
viruses have a tropism in animals which is very specific for the nervous
system. The system of entry of these viruses also depends on specific
receptors. For rabies, several candidates have been proposed as receptors:
acetylcholine receptors (nicotinic receptors in particular), NCAM
molecules (140 and 180 kDa) and also low-affinity NGF receptors (p75).
In a preferred embodiment, the invention uses lentiviral vectors, for
example of the HIV type, pseudotyped with an envelope of the PV (rabies
virus) or MOK (Mokola virus) type.
The invention in fact shows, surprisingly, that this type of envelope
allows cellular targeting, in particular the targeting of glial cells of
the astrocyte type.
The MOK and PV pseudotypes can also be ultracentrifuged just like VSV, and
therefore make it possible to concentrate supernatents. They also make it
possible, surprisingly, to obtain a tropism which is restricted relative
to VSV, and therefore a cellular targeting in vivo in the CNS. It is also
possible to obtain stable clones without an induction system for the PV
and MOK glycoproteins. These stable clones can be used to develop stable
encapsidation lines for the production of lentiviral vectors pseudotyped
with one of these two envelopes.
The invention therefore relates to lyssavirus (in particular PV and/or MOK)
envelopes which make it possible to pseudotype lentiviral vectors for
specifically transducing astrocytes.
It also relates to the use of these pseudotyped lentiviral vectors, for
preparing a composition intended for gene transfer into astrocytes in
vivo. According to a particularly advantageous characteristic of the
invention, the defective lentivirus used is the human immunodeficiency
virus. According to another particularly advantageous characteristic, the
lentivirus used comprises one or more viral proteins selected from TAT and
Production of Recombinant Pseudotyped Lentiviral Vectors
The lentiviral vectors according to the invention can be prepared in
various ways, by transient transfection(s) into stable lines and/or by
means of helper viruses.
The method according to the invention provides, according to a
particularly preferred embodiment, for the combination of a minimum of
three vectors in order to produce a recombinant virion or a recombinant
A first vector provides the lentiviral vector comprising the cis-acting
viral sequences necessary for the correct functioning of the viral cycle.
Such sequences include one or more lentiviral LTRs, a Psi packaging
sequence of lentiviral origin, reverse transcription signals, a promoter
and/or an enhancer and/or polyadenylation sequences. The first vector also
contains a cloning site for a heterologous nucleic acid sequence to be
transferred into a nonproliferative cell. In this vector, the LTRs can
also be modified so as to improve the expression of the transgene or the
safety of the vector. Thus, it is possible to modify, for example, the
sequence of the 3' LTR by eliminating the U3 region (WO 99/31251).
According to a preferred embodiment of the invention, it is a vector
plasmid comprising a recombinant lentiviral genome of sequence
LTR-psi-RRE-flap-Promoter-transgene-LTR which allows expression of the
vector RNA which will be encapsidated in the virions. The transgene may
encode, for example, the following trophic factors: CNTF, NGF, NT3, NT4,
FGF, PDGF, etc., or enzymes which restore a deficient metabolic activity,
for example: TH, AADC, GTPC, .beta.-glucuronidase, etc. The transgene is
typically placed under the control of a transcriptional promoter, which
may be homologous with respect to the transgene or heterologous, for
example a viral, cellular, synthetic, etc. promoter. The promoter used may
be constitutive or regulated, weak or strong, tissue-specific or
ubiquitous, etc. Use is typically made of a viral promoter such as CMV,
LTR, TK, etc, or a cellular promoter such as PGK, Rho, etc.
Tissue-specific promoters can be used, even though the vectors of the
invention already confer an expression selectivity characteristic.
A second vector, for trans-complementation, provides a nucleic acid
encoding a gag lentiviral protein and a pol lentiviral protein. These two
proteins are derived from a lentivirus and preferably originate from HIV.
The second vector is devoid of encapsidation sequence, of sequence
encoding an envelope and, advantageously, is also devoid of lentiviral
LTRs. As a result, the sequences encoding gag and pol proteins are
advantageously placed under control of a heterologous promoter, for
example a viral, cellular, etc. promoter, which may be constitutive or
regulated, weak or strong. It is preferably a trans-complementing plasmid
comprising a sequence CMV-.DELTA.psi-gag-pol-.DELTA.env-PolyA. This
plasmid allows the expression of all the proteins necessary for the
formation of empty virions, except the envelope glycoproteins. It is
understood that the gag and pol genes may also be carried by different
A third vector provides a nucleic acid which allows the production of the
chosen envelope (env) glycoprotein.
This envelope may be chosen from the envelopes mentioned above, in
particular an envelope of a rhabdovirus, more particularly of a lyssavirus,
even more preferably MOK or PV. This vector is devoid of encapsidation
sequence and of sequences encoding gag or pol and, advantageously, is also
devoid of lentiviral LTRs.
Advantageously, the three vectors used do not contain any homologous
sequence sufficient to allow a recombination. The nucleic acids encoding
gag, pot and env may advantageously be cDNAs prepared according to
conventional techniques, from sequences of the viral genes available in
the prior art and on databases, as illustrated in the examples.
For the production of the replication-defective recombinant viruses, the
vectors described above are introduced into competent cells and the
viruses produced are harvested. The cells used may be any competent cell,
preferably mammalian cell, for example animal or human cell, which is non
pathogenic. Mention may, for example, be made of 293 cells, embryonic
cells, fibroblasts, muscle cells, etc.
The invention therefore lies in a method for preparing a pseudotyped
defective recombinant lentivirus, comprising transfection of a population
of competent cells with a combination of vectors as described above, and
recovery of the viruses produced.
The invention thus relates to a particularly advantageous method for
producing lentiviruses capable of targeting the in vivo expression of a
transgene, in human astrocytes, comprising transfection of competent cells
with: a) a vector plasmid comprising a sequence
LTR-psi-RRE-flap-Promoter-transgene-LTR(.DELTA.U3), b) a
trans-complementing plasmid comprising a sequence
CMV-.DELTA.psi-gag-pol-.DELTA.env-PolyA, c) an envelope plasmid comprising
a sequence CMV-env-PolyA, the envelope being an envelope of the Mokola
virus or of the rabies virus.
The lentiviruses of the invention may also be prepared from an
encapsidation cell line producing one or more gag, pol and env proteins.
According to a particular embodiment, use is made of a cell line
expressing the env protein in a stable manner (i.e. from a sequence
integrated into its genome). Indeed, unlike the lentiviral envelopes, the
MOK or PV envelopes, for example, do not exhibit any significant toxicity
for the cells and can be expressed stably and constitutively (or in a
regulated manner) in a line.
As a result, in a particular embodiment, the method of the invention
comprises transfecting only two vectors (the lentiviral vector and the
trans-complementing vector) into a cell line expressing the chosen env
protein. The cells used to prepare such a line are, for example, the
competent cells mentioned above. Such a line also constitutes a subject of
the present application.
According to another embodiment, the line used also expresses the
lentiviral gag and pol proteins. In this case, the method simply comprises
transfection of the lentiviral vector.
In this regard, it is possible to use a lentiviral encapsidation cell such
as that described in patent application WO 99/58701, in which the gag and
pol genes of HIV are present on separate constructs.
Preferably, the lentiviruses produced are derived from the HIV-1, HIV-2,
SIV, FIV,BIV, VISNA, CAEV or EIAV virus.
Surprisingly, the present application shows that the pseudotyped
lentiviral vectors thus obtained are capable of preferentially infecting
certain nerve cell subpopulations, in particular astrocytes, in vivo. The
term "preferentially" should be understood to mean that the lentiviruses
according to the invention target essentially astrocytes but are,
nevertheless, capable of transfecting other cell types, such as other
glial cells or neurons. Other nerve cell subpopulations which may be
targeted by vectors of the invention are, for example, microglial cells,
endothelial cells or oligodendrocytes. In a particular application
concerning gene transfer into the eye, the vectors of the invention
pseudotyped with the Mokola envelope allow selective transfer to the cells
of the pigment epithelium.
The lentiviruses according to the invention may, finally, be used for
producing a pharmaceutical composition intended to treat a disease of the
nervous system or a neurodegenerative disease, and in particular
Alzheimer's disease, Parkinson's disease, Huntington's Chorea, SLA or SMA,
ocular degenerations or else traumas of the central nervous system
(cerebral attack, epilepsy, spinal cord lesions, etc.), diseases affecting
the central nervous system (MPS, etc.), glioblastomas or astrocytomas, or
metabolic diseases affecting the nervous system (mucopolysaccharidosis,
Charcot-Marie-Tooth disease, etc.).
Another subject of the invention lies in the combined use of several
lentiviruses pseudotyped with a different lyssavirus envelope, for the
purpose of transferring and expressing a nucleic acid wherein the cells of
the nervous system. The combined use may comprise sequential
administrations of the various viruses, or a simultaneous administration.
As indicated above, the invention may allow the transport and the
expression of multiple nucleic acids wherein nerve cells, such as, for
example, catalytic nucleic acids (antisense nucleic acids, ribozymes,
etc.), nucleic acids encoding growth factors, trophic factors, cytokines,
colony stimulating factors, anticancer agents, toxins, enzymes,
neurotransmitters or their precursors, etc.
The pharmaceutical composition containing the lentivirus according to the
invention may be administered to a patient intracerebrally or systemically
given the particular tropism of the pseudotyped lentiviral vectors, in
particular for astrocytes. Thus, it may be an administration given
intracerebrally, intra-striatally, intra-venously, intra-arterially, into
the subretinal space, etc. Preferred modes of injection are intracerebral
injection and injection into the subretinal space. For the use for gene
transfer into the eye, viruses pseudotyped with a Mokola envelope are most
The composition is advantageously administered at a rate of 10.sup.2 to
10.sup.9 particles per dose, typically of 10.sup.3 to 10.sup.8. The
lentiviruses may be conditioned in any suitable solution, such as a
saline, isotonic, buffered solution, optionally combined with stabilizing
agents such as isogenic albumin or any other stabilizing protein,
glycerol, etc., and also adjuvants such as polybrene or DEAE dextrans,
Claim 1 of 5 Claims
1. A method for the transfer of a gene
into the astrocytes of an individual comprising injecting into said
individual, by direct injection, a composition comprising a defective
lentivirus comprising said gene, said defective lentivirus being
pseudotyped with a Mokola (MOK) envelope, so that said gene is transferred
into the astrocytes of said individual.
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