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  Pharmaceutical Patents  

 

Title:  Methods of gene transfer to astrocytes with mokola virus pseudotyped lentivirus vectors
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 12, 2004
PCT Pub. No.:
 WO02/097104
PCT Pub. Date:
 December 05, 2002


 

George Washington University's Healthcare MBA


Abstract

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 Invention

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 ocular system.

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 active.

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 nervous system.

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.

Pseudotyping

As indicated above, the invention lies in the demonstration of the targeting properties of lentiviruses pseudotyped with particular envelopes.

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 species.

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 REV.

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 retrovirus.

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 plasmids.

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 particularly preferred.

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, etc.


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.
 

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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 patent.
 

 

     
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