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Definitions

• the present invention describes a nonintegrative and non-replicative recombinant lentivirus as well as its uses, in particular for the preparation of a composition intended for gene transfer in vitro, ex vivo or in vivo.
• the invention is useful for the transfer of genes in any mammalian organism, for example in the tissues or cells of the liver, muscle, pancreas and of the central nervous system (including the ocular sphere), and in particular for the treatment of disorders and pathologies such as disorders of the central nervous system, including the ocular sphere.
• Gene transfer into the nervous system has multiple applications, particularly in the experimental (e.g., research) and therapeutic domains.
• this transfer can enable the realization of studies of labeling, toxicity, quality, the construction of pathological models, the restoration of deficits, the expression of therapeutic products (e.g., proteins, RNAs, etc.), etc.
• retroviral vectors derived from oncoretroviruses 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 ex vivo gene transfer or to organs whose cells are mitotically active.
• lentiviral vectors Compared to other commonly developed viral vectors, lentiviral vectors have several practical advantages, including high-grade production facility and increased knowledge of their biology. These vectors are widely used for gene transfer as part of experimental gene therapy protocols. They can effectively transduce many cell types, including quiescent cells of the central nervous system. Lentiviruses are indeed complex retroviruses capable of integrating into the genome of non-mitotically active cells. Examples of such lentiviruses are the HIV-1, HIV-2, SIV, IVF, BIV, VISNA, CAEV and EIAV viruses.
• retroviruses and in particular lentiviruses, resides in particular in the fact that they present a potential risk of insertional mutagenesis since they integrate into the chromatin of the transduced cells and apparently preferential, in the coding sequences. This disadvantage has so far limited the exploitation of this type of vector for gene transfer in vivo.
• Non-integrative and non-replicative lentiviral vectors In order to reduce the risk of insertional mutagenesis, which at present constitutes the major obstacle to the use of these vectors in clinical practice, the inventors have developed a non-integrative and non-replicative lentiviral vector.
• the non-integrative character of this new generation of lentiviral vectors therefore represents a considerable advance in terms of biosecurity, in particular for gene therapy.
• This vector can be used, for example, for the stable expression of a transgene or other nucleic acids in non-dividing cells or for the transient expression of a gene in dividing cells refractory to other cells. methods of transfection or even transduction by other vectors.
• Non-integrative vectors are known in the prior art, the most common of which are adenoviral vectors and herpes vectors.
• the AAV vectors although they integrate only with a relatively low frequency (about 10%), are limited by the size of the transgene that can be cloned there as well as by the mutations they also cause at their insertion site.
• the present invention thus provides a solution to the problems of the prior art and provides new tools and vectors for the transfer of genes into the nervous system.
• the invention lies more particularly in the development of a non-replicative and non-integrative recombinant lentivirus.
• the lentiviruses of the invention generally comprise a mutated integrase and a particular recombinant genome. More preferably, the lentiviruses according to the invention comprise (i) a recombinant genome comprising, between the 5 'and 3' lentiviral LTR sequences, a psi sequence of lentiviral encapsidation, a nuclear export element of RNA, a transgene and, optionally, a promoter and / or a sequence promoting the nuclear import of RNA, as well as (ii) a mutated integrase preventing the integration of said genome into the genome of a host cell.
• the recombinant genome comprises, for example, the 5'LTR-psi-RRE-cPPT CTS-transgene-LTR3 'sequence.
• compositions comprising a lentivirus according to the invention and a pharmaceutically acceptable excipient.
• the invention also relates to methods and compositions for the in vitro, ex vivo and in vivo transfer of target genes into particular cell populations, and also to the treatment of disorders, for example, disorders of the central nervous system, including the system. ocular.
• Another subject of the invention relates to the use of a non-integrative and non-replicative lentivirus as defined above for the preparation of a composition intended for gene transfer in a mammalian cell (preferably human) preferably in a central nervous system (including ocular sphere) cell of a subject in vitro, ex vivo or in vivo.
• a mammalian cell preferably human
• a central nervous system including ocular sphere
• the invention also relates to any method for producing a non-replicative and non-integrative lentivirus as defined above, comprising in particular the introduction of a vector plasmid comprising, in cells, a recombinant genome as defined above , in the presence of the appropriate transcomplementation functions, and in particular of a pol lentiviral region encoding a modified integrase as defined above.
• the method can be carried out by transient transfection of different transcomplementation and envelope plasmids, or in the presence of helper virus, and / or in cell lines expressing one or more of the complementation proteins.
• the invention also relates to a cell line stably expressing a lentiviral integrase, preferably comprising a mutation inducing a loss of integration function of said integrase.
• the mutation within the meaning of the invention, may correspond to point mutations and / or to microdeletions of some bases of the integrase. It preferably corresponds to one or more point mutations affecting a basic region, the C-terminal region (for example a basic region of the C-terminal region) and / or the catalytic Ntegrase site, said mutated integrase being devoid of integrative function. .
• a particular object of the invention thus relates to a method for preparing a non-replicative and non-integrative recombinant lentivirus comprising transfection of a cell using a nonintegrative and non-replicative lentiviral vector system comprising: a) a transcomplementation plasmid, devoid of psi encapsidation signal and comprising a lentiviral gag sequence and a mutated pol lentiviral sequence encoding a non-functional integrase for integration, said plasmid being optionally deleted accessory genes such as vif, nef, vpu and / or vpr, b) an envelope plasmid having a promoter-env-sequence
• a lentiviral vector plasmid comprising a recombinant genome comprising, between the 5 'and 3' lentiviral LTR sequences, a psi sequence of lentiviral encapsidation, a nuclear export element of RNA, a transgene and optionally a a promoter and / or a sequence promoting the nuclear import of RNA, as well as a mutated integrase preventing the integration of said genome into the genome of a host cell, said vector being devoid of any coding sequence of the lentivirus, and the recovery of lentivirus products.
• the present invention describes non-integrative and non-replicative recombinant lentiviruses for the transfer of genes into any mammalian cell, particularly into human cells. It can be dividing cells or quiescent cells, cells belonging to central organs or to peripheral organs, such as the liver, pancreas, muscle, heart, etc.
• a particular object of the The invention relates to the transfer of genes into the nervous system (ocular system included) and in particular into neurons, astrocytic-type glial cells and retinal cells, as well as into cancerous tumors.
• These lentiviral vectors are useful for transfer and expression in vivo of nucleic acid sequences in particular within the nervous system.
• lentiviruses 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 that are initially expressed as a single precursor polypeptide.
• the gag gene codes for internal structural proteins (capsids and nucleocapsid).
• the pol gene codes for reverse transcriptase, integrase and protease.
• the env gene encodes the viral envelope glycoprotein.
• the genome of the lentiviruses also contains an element RRE (Rev Responsive Element) acting in cis responsible for the export out of the nucleus of the viral genomic RNA which will be encapsidated.
• RRE Rev Responsive Element
• the 5 'and 3' LTR sequences serve to promote the transcription and polyadenylation of viral RNAs.
• the LTR contains all the other cis-acting sequences needed for viral replication. Sequences necessary for reverse transcription of the genome (tRNA primer binding site) and encapsidation of viral RNA in particles (site ⁇ ) are adjacent to the 5 'LTR. If the sequences necessary for encapsidation (or packaging of retroviral RNA in infectious virions) are absent from the viral genome, the genomic RNA will not be actively encapsidated.
• the lentiviral genome further includes accessory genes such as vif, vpr, vpu, nave, TAT, REV, etc.
• lentiviral vectors for gene transfer applications has been described, for example, in US Pat. No. 5,665,577, EP 386,882, US 5,981,276, US Pat. No. 6,013,516, and in patent applications WO99 / 58701. and WO02 / 097104.
• These vectors comprise a defective lentiviral genome, that is to say in which at least one of the gag, pol and env genes has been inactivated or deleted.
• the lentiviral vector according to the invention is a non-replicative and non-integrative recombinant lentivirus, that is to say that it is incapable of autonomous replication and specific integration in the transduced cells.
• the invention relates to a non-replicative and non-integrative recombinant lentivirus comprising a recombinant genome comprising, between the 5 'and 3' lentiviral LTR sequences, a psi lentiviral encapsidation sequence, and a nuclear export element of the lentiviral RNA, a transgene and optionally a promoter and / or a sequence promoting the nuclear import of RNA, as well as a mutated integrase preventing the integration of said genome into the genome of a host cell.
• the lentivirus according to the invention may for example comprise the 5'LTR-psi-RRE-cPPT CTS-transgene-LTR3 'sequence.
• a particular object of the invention relates to a lentivirus whose genome is advantageously devoid of any lentiviral coding sequence.
• lentiviruses of the invention comprise a modified integrase.
• the present invention demonstrates, for the first time, that it is possible to produce recombinant non-replicating lentiviruses under conditions that are effective for the expression of a transgene, and whose integration properties are impaired.
• the presence of a modified integrase results from the use, to produce the viruses of the invention, of a modified pol sequence so as to produce a non-functional integrase for integration, but without substantial effect on the previous steps of the invention. vector cycle during cell transduction (so-called class 1 mutation).
• a class 1 mutation preferably consists of one or more point mutations, preferably affecting the nucleic acid portion encoding a basic region, the C-terminal region (preferably a basic region of the C region). -terminal) and / or the catalytic site of integrase.
• the point mutation is preferably translated by the addition of one amino acid to another at the level of the coded sequence of the integrase.
• the mutation is preferably non-conservative in that it renders the integrase non-functional for integration.
• Such a mutation is preferentially chosen from mutants producing a non-functional integrase for integration, while retaining the other functions of the integrase, eg, those participating in the progression of the vector towards the nucleus.
• Examples of mutations affecting HIV-1 and making it possible to obtain a non-functional integrase for integration are the following: H12N, H12C, H16C, H16V, S81R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D116I, D116A, N120G, N120I, N120E, E152G, E152A, D-35-E, K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T (C base region L), K188T, E198A, R199C, R199T, R199A, D202A, K211A, Q214L (214 and 216 belong to the Q region of the C base basic region), Q216L, Q221L, W235F, W
• the mutations affecting the catalytic site preferably concern, with respect to HIV-1, the amino acids 64, 116 and / or 152 of the integrase.
• the mutations affecting the C-terminal portion of this lentivirus are advantageously chosen from the substitution of the RRH motif 262 by AAH, a substitution in the Q region (Q214L and / or Q216L), in the L (K186) region and / or in the L-region.
• a preferred mutation is the substitution of the motif RRK 262 by AAH.
• the lentiviruses of the invention typically comprise a recombinant genome of the 5'LTR-psi-RRE-cPPT CTS- (promoter-) transgene-LTR3 'sequence.
• the transgene is typically placed under the control of a promoter.
• One and / or the other can also be placed upstream of the cPPT CTS element.
• the recombinant genome thus includes cis-acting viral sequences useful for encapsidation and transduction.
• it retains only certain lentiviral sequences, in particular those necessary for encapsidation of the genome (psi sequence of lentiviral origin); a nuclear export element of RNA.
• REV REV sequence for "REV Responsive Element
• REV REV sequence for "REV Responsive Element”
• a genome of lentivirus in particular an HIV, and for example HIV-1 (the RRE sequence, present on viral RNA 1 , interacts with a regulatory element REV), the CTE ("Constitutive Transport Element") of the Mason Pfizer Monkey virus, a SIV, HIV-2 or IVF nuclear export system, or an element equivalent of any other retrovirus (eg the SIV, HIV2 or IVF nuclear export system); a sequence promoting the nuclear import of RNA, for example the flap sequence [cPPT-CTS region (central polypurine tract-central sequence termination); cf.
• cPPT-CTS region central polypurine tract-central sequence termination
• the recombinant genome is preferably deleted from all the lentiviral coding sequences, in particular viral genes coding for the gag, pol and env sequences and accessory genes vif, vpr, vpu and nef.
• the transcomplementing plasmid preferably retains the tat and rev genes.
• the vector plasmid may advantageously comprise a LTR3 'deleted from the U3 enhancer sequence (WO99 / 31251) to improve the expression of the transgene and the safety of the vector.
• Another target may possibly be mutated in addition to the integrase mutations: the att sequences which are located at the ends of the linear genome DNA. If these sequences are mutated, the integration of the genome with integrase is no longer done correctly and there can no longer be integration.
• the genome which is in the form of a linear DNA can be circularized.
• the vectors and plasmids of the invention may be prepared from lentiviruses belonging to different species, in particular HIV-1, HIV-2, SIV, IVF, BIV, VISNA, CAEV and EIAV.
• Particularly preferred serotypes are HIV, in particular HIV-1, FIV, EIAV and SIV.
• the transgene sequence may be placed under the control of a selected promoter and / or enhancer, as well as all the transcriptional, post-transcriptional and post-translational regulatory elements necessary for the proper expression of said transgene.
• the term "transgene” generally refers to any nucleic acid encoding or not. It may be a non-coding sequence such as, for example, an enzyme recognition sequence (specific integration site, site having a particular affinity for a protein, etc.). It is preferably a sequence encoding a given polypeptide or active RNA as such. It may be in particular a cDNA, a gDNA, a synthetic DNA, an RNA, for example an interfering RNA, a ribozyme, etc., or a combination thereof. Typically, the transgene is a DNA comprising a sequence encoding the desired expression product. The transgene may further include one or more transcription termination regions, typically a polyadenylation signal.
• the transgene may be chosen from a catalytic nucleic acid (interfering, antisense, ribozyme), a suicide nucleic acid (eg, encoding a toxin) or a nucleic acid encoding a biologically active peptide, for example a growth factor, a trophic factor, an anti-angiogenic factor, a hormone, a cytokine, an antibody, a receptor, a differentiation factor, a colony stimulating factor, an anti-cancer agent, an enzyme, a neurotransmitter or its precursor, etc.
• a catalytic nucleic acid interfering, antisense, ribozyme
• a suicide nucleic acid eg, encoding a toxin
• a nucleic acid encoding a biologically active peptide for example a growth factor, a trophic factor, an anti-angiogenic factor, a hormone, a cytokine, an antibody, a receptor, a differentiation factor,
• the transgene codes, for example, for the following trophic factors: CNTF, NGF, NT3, NT4, FGF, PDGF, GDNF, etc., or for anti-angiogenic factors or for enzymes restores metabolic activity that is deficient or has a particular metabolic function, for example: TH, AADC, GTPC, ⁇ -glucuronidase, etc.
• the transgene codes, for example, for interfering RNAs (RNAi) for specifically inhibiting the expression of mutated proteins involved in a dominant genetic disease or in an induced disease. by a gain in function, for example a neurodegenerative disease such as mutated SOD (amyotrophic lateral sclerosis), APP proteins, tau, presenilin, or BACE (Alzheimer's disease), ⁇ -synuclein (Parkinson's disease) or Huntingtin (Huntington's disease).
• RNAi interfering RNAs
• the transgene is typically placed under control of a transcriptional promoter, which may be homologous to the transgene or heterologous, for example a cellular, viral, synthetic, chimeric, etc. promoter.
• the promoter used may be constitutive or regulated, weak or strong, tissue specific or ubiquitous, dependent on RNA polymerase 2 or 3, etc.
• a viral promoter such as CMV, RSV LTR, TK, etc. is used. or preferably a cell promoter such as PGK, Rho, EF1 ⁇ , etc.
• Tissue specific promoters may be employed. It may be for example promoters ENO, GFAP, NSE, a promoter of I 1 RNA polymerase III such as the promoter U6 or H1, possibly modified, etc.
• the promoter used to direct the expression of the transgene may be, for example, a viral promoter chosen from the promoter of the CMV gene, TK or RSV LTR.
• the promoter present in the envelope plasmid and / or the promoter present in the vector plasmid are identical or different and cellular or viral.
• the lentiviral vectors according to the invention can be prepared in various ways, by transient transfection (s), in stable lines and / or by means of helper viruses.
• the method according to the invention provides, according to a particularly preferred mode, the combination of a minimum of three plasmids (see FIG. 1) to produce a recombinant virion or a recombinant lentivirus: a) a transcomplementation plasmid, devoid of a signal of encapsidation psi and comprising a lentiviral gag sequence and a mutated pol lentiviral sequence encoding a non-functional integrase for integration, said plasmid being optionally deleted from the accessory genes vif, nef, vpu and / or vpr, b) an envelope plasmid comprising a Promoter-env-PoIyA sequence, and c) a lentiviral vector plasmid comprising a recombinant genome, optionally deleted from the LTR3 'or LTR3' enhancer U3 enhancer region, comprising, between the 5 'and 3 LTR sequences
• the three plasmids used do not comprise a homologous sequence sufficient to allow recombination.
• the nucleic acids encoding gag, pol and env may be advantageously 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.
• the trans-complementation plasmid provides a nucleic acid encoding the lentiviral proteins gag and pol. These proteins are derived from a lentivirus and, preferably, are derived from HIV-1.
• the plasmid lacks an encapsidation sequence, an envelope coding sequence, accessory genes, and advantageously also lacks lentiviral LTRs.
• the sequences coding for gag and pol proteins are advantageously placed under the control of a heterologous promoter, for example cellular, viral, etc., which may be constitutive or regulated, weak or strong. It is preferably a transcomplementing plasmid comprising a CMV- ⁇ psi-gag-pol-PolyA sequence.
• the transcomplementation plasmid allows the expression of all the proteins necessary for the formation of empty virions, except the envelope glycoproteins.
• the transcomplementation plasmid may advantageously comprise the TAT and REV genes.
• the transcomplementation plasmid may also further comprise a transcriptional regulatory element selected from WPRE, APP 5'UTR, TAU 3'UTR, and a chromatin insulator sequence such as MAR (Matrix Attachment Region). , SAR (Scaffold Attachment Region), its and its' (Special Chromatin Structure), etc. It is advantageously devoid of accessory genes vif, vpr, vpu and / or nave.
• the gag and pol genes can also be carried by different, possibly separate plasmids.
• several transcomplementation plasmids are used, each encoding one or more of said proteins.
• the mutation in the pol sequence of the transcomplementation plasmid consists of one or more microdeletions of a few bases, preferably one or more point mutations affecting a basic region, the C-terminal region (for example a basic region of the C-terminal region). ), and / or the catalytic region of the sequence of the encoded integrase, as defined above.
• the envelope plasmid provides a nucleic acid that allows the production of the selected envelope glycoprotein (env). It lacks psi encapsidation signal, gag or pol coding sequences and is also devoid of lentiviral LTRs. It has a Promoter-env-PolyA sequence.
• Pseudotyped HIV-1 vectors comprising a different envelope of the wild-type envelope, originating for example from another virus, or, of cellular origin, and thus having a modified tropism
• VSV glycoprotein d Vesicular Stomatitis Virus
• This envelope has advantageous characteristics such as resistance to ultracentrifugation and a very wide tropism.
• the VSV glycoprotein is not labile after ultracentrifugation. This makes it possible to concentrate the viral supernatants and to obtain high infectious titres.
• This envelope also confers virions a very broad tropism including in vitro, allowing the infection of many cell types.
• the receptor of this envelope would be a phosphatidylserine motif, present on the surface of many cells of different species.
• the envelope glycoprotein (env) of vesicular stomatitis virus (VSV-G) is advantageously used in the context of the invention, but any other pseudotype can be used in order to target certain cell populations as well as possible.
• the envelope protein can thus be selected from any envelope glycoprotein of any enveloped virus, for example from rhabdovirus envelope protein, more preferably lyssavirus, even more preferably a rabies virus serogroup virus: Rabies (RAB), Duvenhague (DUV), European bat type 1 (EB-1), European bat type 2 (EB-2), Kotonkan (KOT), Lagos beats (LB), Obodhiang (OBD), Rochambeau (RBU), a coat protein of a Mokola virus serogroup virus (MOK) and any chimeric composition of these envelopes.
• Rabies and Mokola viruses are particularly preferred. They have indeed a tropism in the very specific animal of the nervous system (cf WO 02/097104). This type of envelope also allows cell targeting
• the invention uses lentiviral vectors, for example of HIV-1 type, pseudotyped with an envelope of rabies virus or Mokola.
• a vector plasmid of the invention comprises a recombinant nucleic acid comprising between the 5 'and 3' LTRs, the elements psi, RRE (or an equivalent element of another retrovirus), the transgene and optionally a promoter and / or the sequence flap, cPPT CTS. It may comprise, for example, the 5'LTR-psi-RRE-cPPT CTS- (promoter-) transgene-LTR3 'sequence.
• the sequence of the transgene is optionally placed, within the vector plasmid, under the control of the promoter and / or an enhancer, as well as all the transcriptional, post-transcriptional and post-translational regulatory elements necessary for the good expression of this gene.
• This plasmid comprises the viral sequences acting in cis and necessary for the smooth running of the transduction. It retains from the original virus only certain sequences necessary for the encapsidation of the genome (psi sequence of lentiviral origin), possibly the flap sequence (cPPT-CTS region) which allows an efficient nuclear import of the reverse-transcribed vector genome and a 5 'LTR includes allowing the transcription of I-1 RNA vector to be packaged.
• This vector may also be optionally deleted from the enhancer sequence U3 of the 3 'LTR (WO 99/31251). It is also deleted from all viral genes of origin, in particular viral genes coding for the gag, pol and env sequences and accessory genes (vivid, nef, vpr and / or vpu), to improve the safety of the vector.
• the att sequences which are located at the ends of the linear genome are further advantageously mutated, possibly deleted to hinder the handling of the genome by the integrase.
• the promoters used in the transcomplementation plasmid, the envelope plasmid and in the vector plasmid for respectively promoting the expression of gag and pol, the envelope protein, the genome vector mRNA and the transgene are promoters. identical or different chosen advantageously from ubiquitous or specific promoters, for example from the CMV, TK, RSV LTR viral promoters and an RNA polymerase III promoter, such as the U6 or H1 promoter.
• the lentiviruses according to the invention are genetically modified so that certain constituent genes of the native infectious virus are deleted and replaced by a nucleic acid sequence of interest to be introduced into the target cells. After fusion of the virus with the cell membrane, it injects its nucleic acid into the cell. The genetic material thus transferred is then transcribed and possibly translated into proteins within the host cell.
• a preferred vector system comprises: a) a transcomplementation plasmid, devoid of a psi encapsidation signal and comprising a lentiviral gag sequence and a mutated polynucleotide sequence encoding an integrase having a substitution of the RR2 motif 262 by AAH (in a basic region of the C-terminal region of the coded sequence of integrase), which is not functional for integration, said plasmid being devoid of the accessory genes such as vif, nef, vpu and / or vpr, b) an envelope plasmid as defined above, preferably comprising a viral promoter and encoding a VSV-G envelope, more preferably a CMV-VSV-G-PoIyA sequence, and c) a lentiviral vector plasmid, optionally deleted from the LTR3 'or LTR3' enhancer U3 enhancer region, comprising, between the
• the plasmids described above can be introduced into competent cells and the viruses manufactured are harvested.
• the cells used may be any competent cell, in particular eukaryotic cells, in particular mammalian, for example animal or human cells. They can be somatic or embryonic, stem or differentiated. For example, 293 cells, fibroblast cells, hepatocytes, muscle cells (skeletal, cardiac, smooth, blood vessel, etc.), neurons (neurons, glia, astrocytes), epithelial, renal, and ocular cells may be mentioned. etc. It can also be plant cells, yeasts or prokaryotic cells. They may also be cells transformed with SV40 T antigen.
• the invention therefore lies in a method for preparing a recombinant nonintegrative and non-replicative lentivirus, comprising transfection of a population of competent cells with a combination of plasmids as described above, and recovery of the vectors produced.
• the invention thus relates to a particularly advantageous method for producing non-integrative and non-replicative lentiviruses enabling expression in vivo of a transgene, comprising transfection of competent cells using a nonintegrative and non-replicative lentiviral vector system, as described above, comprising: a) at least one transcomplementation plasmid, devoid of signal encapsidation method psi and comprising a gag sequence and / or a pol sequence having a class 1 mutation, allowing, for example, the substitution of the 2 6 2RRK motif by AAH in a basic region and / or in the C-terminal region of the sequence coded integrase, said plasmid being devoid of accessory genes such as vif, nef, vpu and / or vpr, b) an envelope plasmid comprising a promoter sequence (for example CMV) -wrapped (for example VSV-G) -PoIyA,
• the lentiviruses of the invention may also be prepared from encapsidation cell lines producing one or more integrase-modified gag, env and / or pol proteins as indicated above.
• the method of the invention comprises the transfection of only two plasmids (the vector plasmid and the transcomplementation plasmid) in a cell line expressing the chosen env protein.
• the cells used for the preparation of such a line are, for example, the competent cells mentioned above.
• the line used also expresses the env protein, the gag protein and / or pol lentiviral protein, the latter comprising a class 1 mutation.
• the method simply comprises transfection of the vector plasmid.
• the lentivirus products are preferably derived from HIV-1, HIV-2, SIV, IVF 1 BIV, VISNA, CAEV or EIAV.
• the DT40 line established from a hen lymphoma known to be highly recombinogenic and the Cos 7 line (monkey kidney cells immortalized with an SV40 antigen). It may also be HCT116, DLD1 (human lines established from cells derived from colorectal carcinoma), LF1 (human embryonic lung fibroblasts), LL1 (human embryonic skin fibroblasts), TK6 (human lymphoblastic line) ), HaCaT (human keratinocytes), U937 (human monocytes), HCT15, SW480, Colo320, Co115, EB, HbMOO, Rat-1, PC12 (rat photochromocytoma), etc.
• HCT116 human lines established from cells derived from colorectal carcinoma
• LF1 human embryonic lung fibroblasts
• LL1 human embryonic skin fibroblasts
• TK6 human lymphoblastic line
• HaCaT human keratinocytes
• U937 human monocytes
• the plasmids can be introduced into the cells by any technique known to those skilled in the art, adapted to the cell type in question.
• the cells and the vector system are contacted in a suitable device (plate, box, tube, bag, etc.) for a period of time sufficient to allow the transfer of the vector system or plasmid into the cells.
• the vector system or plasmid is introduced into the cells by calcium phosphate precipitation, by electroporation or by using one or more transfection facilitating compounds, such as lipids, polymers, liposomes and peptides, etc. Calcium phosphate precipitation is preferred.
• the cells are cultured in any suitable medium, such as RPMI, DMEM, a specific medium allowing culture in the absence of fetal calf serum, etc.
• a particular subject of the invention also relates to a cell line stably expressing a lentiviral integrase comprising one or more point mutations affecting a basic region, its C-terminal region (for example, a basic region of the C-terminal region). and / or its catalytic site, said integrase being devoid of integrative function, as well as the use of such a cell line for the in vitro preparation of nonintegrative and non-replicative recombinant lentiviruses.
• An object of the invention thus relates to the cells obtained by the implementation of the method and the use of a cell, line or cell population according to the invention for the preparation of a cellular composition intended for the implementation of a method of therapeutic, vaccinal or surgical treatment in humans or animals.
• kits for implementing methods for modifying the genome of cells in vitro or ex vivo comprising a vector as described above.
• viruses and lines according to the invention can be used for example for the expression of a transgene or other nucleic acids preferentially in cells which do not divide or for the transient expression of a gene in cells refractory division to other transfection method or even transduction by other vectors.
• the present application shows that the lentiviral vectors thus obtained are capable of transducing different cell types such as, for example, retinal cells, astrocytes, other glial cells or neurons.
• Other nerve cell subpopulations that may be targeted by vectors of the invention are for example microglial cells, endothelial cells or oligodendrocytes.
• the vectors of the invention may for example be pseudotyped with the Mokola envelope to allow selective transfer to the cells of the pigment epithelium.
• This nonintegrative and non-replicative lentiviral vector is intended to improve the safety and efficiency of gene transfer: by the mutation of integrase, the vector no longer integrates into the genome of the target cell, eliminating the risk of insertional mutagenesis. Moreover, the possible insertion of the flap sequence (cPPT-CTS) into the vector can substantially improve the nuclear import of the DNA genome, allowing a strong expression of the transgene, stable in post-mitotic cells and transient in the cells multiplying.
• cPPT-CTS flap sequence
• non-integrative lentiviral vectors of the invention are of several types and include:
• gene therapy Le., gene transfer in any mammalian cell, particularly in human cells. It can be dividing cells or quiescent cells, cells belonging to central organs or peripheral organs, such as liver, pancreas, muscle, heart, etc. It is preferably a transfer of genes into quiescent cells (which do not divide), in particular in cells of the central nervous system, in particular the brain, the marrow and the ocular sphere, for example in the framework of the treatment of neurodegenerative pathologies or the attacks of the retina, and of a gene transfer in dividing cells for a transient expression (ex: anti-tumor suicide strategy, axonal regrowth strategy to treat the traumas of the spinal cord).
• Gene therapy can allow the expression of proteins, for example neurotrophic factors, enzymes, transcription factors, receptors, etc. It also makes it possible to implement an "oligonucleotide” strategy (antisense or interfering RNA, ribozymes, etc.).
• cell therapy ie, the expression of differentiation factors in progenitor cells to direct the cell to a chosen fate before transplantation or the ex vivo transduction of cells to express a factor of interest, followed by the transplant said cells.
• a particular object of the invention relates to the use of a non-integrative and non-replicative lentivirus according to the invention for the preparation of a composition intended for the transfer of genes for example in the central nervous system (including the ocular sphere) of a subject in vitro, ex vivo or in vivo.
• Another particular object of the invention relates to the use of such a lentivirus for the preparation of a composition intended for the treatment of a disease affecting a central or peripheral organ, for example a disease of the nervous system (including of the ocular sphere).
• the non-integrative and non-replicative lentiviruses according to the invention may be used for the manufacture of a pharmaceutical composition intended to treat, for example, a neurodegenerative disease and in particular Alzheimer's disease, Parkinson's disease. , Huntington's disease, ALS or ADS, age-related macular degeneration (AMD), eye degeneration, or central nervous system trauma (stroke, epilepsy, spinal cord injury or trauma, etc.).
• a neurodegenerative disease and in particular Alzheimer's disease, Parkinson's disease.
• Huntington's disease ALS or ADS
• AMD age-related macular degeneration
• eye degeneration eye degeneration
• central nervous system trauma stroke, epilepsy, spinal cord injury or trauma, etc.
• diseases affecting the central nervous system diseases affecting the central nervous system (mucopolysaccharidoses, etc.), glioblastomas or astrocytomas, metabolic diseases affecting the nervous system (mucopolysaccharidoses, Charcot-Marie, etc.) or diseases affecting the ocular sphere (AMD, retinitis pigmentosa , glaucoma, etc.).
• the non-integrative and non-replicative recombinant lentiviruses are used for the manufacture of a pharmaceutical composition for treating retinitis pigmentosa.
• retinitis pigmentosa is a term used to denote a heterogeneous group of ocular disorders characterized by progressive degeneration of rods and cones (nerve cells of the retina) by apoptosis. With an incidence of 1 in 3,000 individuals, it is the leading cause of blindness. The transduction of the cells of the pigment epithelium and the photoreceptors is of crucial interest in this type of pathology.
• a gene replacement strategy requires the transduction of photoreceptors or pigment epithelium, whereas a neuroprotection strategy may benefit from transduction of the pigment epithelium. Indeed, this route has the advantage of not modifying the nerve cells but only the pigment epithelium which will then synthesize a diffusible trophic factor, such as GDNF, and secrete it in the photoreceptor environment to be protected.
• GDNF diffusible trophic factor
• Another object of the invention resides in the combined use of several lentiviruses, for the purpose of transferring and expressing several nucleic acids in the cells of the nervous system.
• the combined use may include sequential administrations of the different viruses, or simultaneous administration.
• the invention can allow the transport and expression of multiple nucleic acids in nerve cells, such as for example catalytic nucleic acids (interfering, antisense, ribozymes, etc.), nucleic acids encoding factors growth factors, trophic factors, cytokines, colony stimulating factors, anti-cancer agents, toxins, enzymes, neurotransmitters or their precursors, etc.
• the pharmaceutical composition containing the lentivirus according to the invention can be administered to a patient intracerebrally or systemically taking into account the particular tropism of pseudotyped lentiviral vectors using a suitable envelope glycoprotein.
• it may be intracerebral administration, for example intra-striatal, in the hypocampus or the black substance, intravenously, intra-arterially, intravenously, in the subretinal space, etc.
• Preferred modes of injection are intracerebral injection and injection into the subretinal space.
• composition is advantageously administered in a proportion of 10 2 to 10 10 , typically from 10 3 to 10 8 particles effective for transduction (as determined by transduction of cells by serial dilutions of the vector stock) or, in genome equivalent, on the order of 10 5 to 10 13 copies [titre determined by reverse transcription-PCR (polymerase chain reaction) quantitative on the vector RNA genome or by quantitative PCR on the DNA strand associated with the vector RNA genome].
• Lentiviruses can be packaged in any suitable solution, such as saline, isotonic, buffered, optionally combined with stabilizing agents such as isogenic albumin or any other stabilizing protein, glycerol, etc., as well as adjuvants such as polybrene or DEAE dextran, etc.
• Figure 1 System for producing lentiviral vectors by transfection of three plasmids: a vector plasmid carrying the GFP transgene under the control of the human cytomegalovirus (hCMV) promoter as well as the central flap sequence [central polypurine tract-central sequence termination (cPPT- CTS)] involved in the nuclear import, the RRE (REV Responsive Element) sequence that interacts with a REV regulatory element and the encapsidation sequence psi ( ⁇ ), the U3 region of the 3 'LTR has been deleted from the promoter sequence ( ⁇ U3); a transcomplementation plasmid expressing the proteins necessary for the early phases of the replicative cycle of HIV-1 (GAG and POL), regulatory elements (TAT, REV) under control of the CMV promoter and deleted sequence ⁇ ; an envelope plasmid expressing the vesicular stomatitis virus envelope glycoprotein (VSV-G) under the control of the CMV promoter.
• GFP obtained after transduction of cell lines (293T and HeLa) by a nonintegrative lentiviral vector.
• the percentage of transduction is determined by FACS 72h after incubation of the cells in the presence of different doses (volume in microliter per well) of a integrative lentiviral vector INWT CMV GFP and non-integrative IN N CMV GFP.
• Figure 4 Expression of GFP over time after transduction of cell lines (293T and MT4) with lentiviral vectors integrative INWT CMV GFP or nonintegrative IN N CMV GFP.
• the percentage of GFP + cells was determined by FACS (A: 293T, B: MT4) 3 days, 6 days, 9 days and 12 or 15 days after transduction (MOI 5).
• FACS A: 293T, B: MT4 3 days, 6 days, 9 days and 12 or 15 days after transduction (MOI 5).
• MOI 5 For the points scored "goal”: the cells were treated with sodium butyrate 5mM 24h before the analysis.
• A Immunocytochemical analysis of expression in control (non transduced) neurons transduced by the integrative vector (IN WT ) OR transduced by the non-integrative vector (IN N ) 3, 9 and 16 days after transduction (x20 magnification).
• Figure 7 Expression of GFP in vivo after injection into the striatum of mice.
• the brain was removed 10 days after stereotaxic injection of the INN CMV GFP vector into the mouse striatum, cut with cryostat in 20 ⁇ m thick section and analyzed after immunohistofluorescence. confocal microscope.
• B GFP / NeuN Comarking: a neuron expressing GFP (x16 magnification).
• A Expression of GFP in vivo in the rat after sub-retinal injection of 66ng p24 of nonintegrative lentiviral vector INN CMV GFP. Fluorescence microscopy (x2.5) on retinas mounted flat 2 weeks after injection.
• B Expression of GFP in vivo in the dog after sub-retinal injection of 2.5 ⁇ g p24 non-integrative lentiviral vector INN CMV GFP. Fluorescent light angiography on dog vigil 1 month after injection.
• the integrase (fused to haemagglutinin) mutated or non-mutated sequences in the transcomplementation plasmid used for the production of the lentiviral vectors (plasmid p8.91 INWT and p8.91 INN) were used.
• HIV-1 derived vector stocks expressing green fluorescent protein (GFP) under control of the early human cytomegalovirus viral promoter (hCMV) and carrying normal integrase (INWT CMV GFP vector) or mutated (INN CMV GFP) were then been produced.
• a study on the efficacy of CMG GFP INN vectors for directing the expression of the GFP transgene in nerve cells was finally performed first in vitro and then in vivo.
• Inhibition of RT thus makes it possible to reduce the percentage of GFP + cells, either after transduction by the integrative vector INWT CMV GFP or by the INN CMV GFP vector. Most of the GFP + cells observed in the absence of the reverse transcriptase inhibitor are therefore efficiently transduced by both types of vectors and do not result from a pseudotransduction mechanism.
• the percentage of GFP-expressing cells, 293T or MT4 is relatively stable when they are transduced with the INWT CMV GFP integrative control vector, this percentage decreasing slightly at the last evaluated points in 293T cells ( Figure 4). ).
• the treatment of sodium butyrate cells at the end of the experiment makes it possible to reduce the percentage of 293T GFP + cells to the level initially measured, which suggests a reactivation of the promoter governing the expression of the transgene and shows the stability in the integrated vector time in the cell population analyzed.
• the expression in cells transduced by the mutant integrase-bearing vector shows a profile close to that observed after transduction with an adenoviral vector. Indeed, the percentage of GFP + cells decreases over time ( Figure 4) and can not be reduced to the initial level by treatment of sodium butyrate cells. This result suggests that, as in the case of transduction by an adenoviral vector, the genome of the INN CMV GFP vectors is removed from the initially transduced cells by successive dilution at each cell division.
• Transgene expression persists for up to 25 days after transduction (Figure 5C), so episomal forms are relatively stable in the nucleus of the transduced cells and allow expression of the transgene for at least 25 days in quiescent cells. supports the hypothesis of a decrease in GFP expression over time in dividing cells by diluting genomes episomal vectors at each mitosis, rather than by degradation.
• the INN CMV GFP vector was injected into the mouse striatum. Ten days after this injection, the expression of GFP could be evidence. This expression lasts for a period of at least one month following the injection. This result demonstrates the effectiveness of non-integrative vectors in allowing and maintaining the expression of a transgene in CNS cells ( Figure 6).
• GFP / GFAP glial fibrillary acidic protein, astrocyte marker
• GFP / NeuN neuroon marker
• GDNF Glial-derived neurotrophic factor
• retinitis pigmentosa refers to a heterogeneous group of ocular disorders characterized by progressive degeneration, apoptosis, rods and cones (nerve cells of the retina). With an incidence of one in 3000, this is the leading cause of blindness.
• the transduction of the cells of the pigment epithelium and the photoreceptors is of crucial interest in this type of pathology.
• a gene replacement strategy requires the transduction of photoreceptors or pigment epithelium, whereas a neuroprotection strategy may benefit from transduction of the pigment epithelium.
• this pathway has the advantage of not modifying the nerve cells but only the pigment epithelium which will then synthesize a diffusible neurotrophic factor, such as GDNF, and secrete it in the photoreceptor environment to be protected.
• GDNF diffusible neurotrophic factor
• results indicated above show the efficacy and the therapeutic potential of the non-integrative lentiviral vectors according to the invention capable of allowing the expression of a transgene in vitro, ex vivo and in vivo in the central nervous system (brain and retina ), in rodents as well as in large animals.

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