Lentiviral vector for dopamine synthesis

Abstract

The invention relates to a lentiviral vector comprising a tricistronic expression cassette encoding: - tyrosine hydroxylase of SEQ ID NO 1; - GTP cyclohydrolase I of SEQ ID NO 2; and - aromatic L-amino acid decarboxylase of SEQ ID NO 3, or a functional variant of said enzymes having a nucleic sequence that is at least 80% identical to SEQ ID NOs 1, 2 and 3. The vector is characterised in that the three enzymes are expressed under the control of the same human PGK-1 promoter, or a functional variant of said human PGK-1 promoter having a nucleic sequence that is at least 80% identical to SEQ ID NO 4.

Description

[0001] LENTIVIRAL VECTOR FOR DOPAMINE SYNTHESIS

[0002] TECHNICAL DOMAIN

[0003] The subject of the invention is a gene therapy aimed at restoring dopamine, more particularly two lentiviral vectors constructed from the human immunodeficiency virus type 1 (HIV-1) for their use in the treatment of diseases whose neurological symptoms are due to a dopamine deficiency, in particular pediatric genetic forms or Parkinson's disease.

[0004] PREVIOUS TECHNICS

[0005] Dopamine (DA) is a neurotransmitter, a biochemical molecule that enables communication within the nervous system, and one of those that directly influences behavior. Dopamine reinforces normally beneficial actions such as eating a healthy food by causing the sensation of pleasure, which activates the reward / reinforcement system. It is therefore essential for the survival of the individual. More generally, it plays a role in motivation and risk-taking in mammals, and therefore in humans as well.

[0006] Pediatric dopamine deficiency disorders result from rare genetic diseases affecting the development of dopaminergic neurons, such as Lesch-Nyhan disease.

[0007] Indeed, these pediatric disorders constitute a group of neurological diseases due to DA deficiency in early childhood. Symptoms are dominated by neurological (abnormal movement disorders) and neuropsychiatric disorders. DA deficiency may be the direct consequence of genetic mutations affecting the stages of DA synthesis. This deficiency also affects the development of dopaminergic neurons.

[0008] A lack or deficiency of dopamine can also be caused by the breakdown of dopaminergic neurons, which is the cause of Parkinson's disease.

[0009] Oral administration of pharmacological treatment (dopaminergic) for Parkinson's disease causes, after a few years, sometimes very disabling side effects such as dyskinesia, motor fluctuations and sometimes neuropsychological disorders. These adverse effects are believed to be linked to the intermittent intake of L-DOPA, which causes concentration peaks in the brain and can thus alter its functioning.

[0010] This notion of discontinuous stimulation, characteristic of oral treatments, has directed research efforts towards the development of alternative strategies which would promote continuous and local dopaminergic stimulation.

[0011] Today, many adverse effects are associated with dopaminergic drugs such as: nausea and vomiting; a drop in blood pressure, characterized by a drop in blood pressure which manifests itself by dizziness or malaise; drowsiness during the day, or even sudden falling asleep; hallucinations or abnormal perceptions of reality, most often visual but sometimes auditory; behavioral disorders: gambling addiction, compulsive shopping, hypersexuality; compulsive use of the drug: treatment consumption disorder.

[0012] Gene therapy represents one of the emerging and promising strategies that have evolved over the last decade.

[0013] Various groups have previously attempted to restore dopamine expression through gene therapy approaches, particularly in Parkinson's disease. Most published studies use recombinant vectors derived from adeno-associated virus (rAAV). AAV vectors have provided behavioral benefits in animal models of Parkinson's disease and in early phases of clinical trials. However, using AAV vectors to express multiple transgenes in a single vector is challenging due to the limited packaging capacity of the viral genome.

[0014] Patent application CN 1 13388642 a nucleic acid construct which comprises two or more polynucleotides selected from polynucleotides encoding tyrosine hydroxylase, GTP-cyclohydrolase I and proposes a new strategy for the prevention and treatment of neurodegenerative diseases.

[0015] US patent application 2013 / 01 1631 1 relates to a construct comprising (i) a nucleotide sequence encoding tyrosine hydroxylase (TH), (ii) a nucleotide sequence encoding GTP-cyclohydrolase I (CHI) and (iii) a nucleotide sequence encoding aromatic amino acid dopa decarboxylase (AADC) and its use in the treatment and / or prevention of Parkinson's disease.

[0016] Patent application EP3937985 proposes a method for improving motor function and reducing dyskinesia in a subject suffering from a neurodegenerative disease or a disease where endogenous dopamine levels are reduced in the subject, using a lentiviral vector derived from the equine infectious anemia virus (EIAV) (ProSavin) that expresses the three enzymes tyrosine hydroxylase (TH), GTP-cyclohydrolase I (CH I ) and aromatic amino acid dopa decarboxylase (AADC), in a single vector. The clinical study related to this application showed a favorable safety profile in patients with Parkinson's disease with moderate improvements in motor behavior. However, despite these motor improvements, clinical data indicate that higher levels of dopamine are required to maximize clinical benefit.There is therefore now a need to improve existing solutions, and in particular to improve the vector described above as indicated by the study investigators.

[0017] TECHNICAL PROBLEM

[0018] Considering the above, a problem that the present invention proposes to solve is to develop a new vector allowing the expression of the three enzymes in a good stoichiometry for an optimal synthesis of dopamine in the target tissue. Thus, the Applicant has discovered, unexpectedly, that the insertion into a lentiviral vector constructed from the human immunodeficiency virus 1 (HIV-1), of a tricistronic expression cassette comprising the coding sequences for TH, AADC and GCH 1 under the control of the human promoter of the gene coding for phosphoglycerate kinase 1 (PGK-1), makes it possible to obtain the expression of these three enzymes in a good stoichiometry for an optimal synthesis of dopamine in the target tissue. It has also discovered that the use of different fusion protein systems in the construction makes it possible to achieve very different dopamine expression profiles in the target tissue.

[0019] TECHNICAL SOLUTION

[0020] The solution of the invention to this technical problem posed has as its first object a lentiviral vector comprising a tricistronic expression cassette coding for: the tyrosine hydroxylase of SEQ ID NO 1, the GTP-cyclohydrolase 1 of SEQ ID NO 2, and the aromatic L-amino acid decarboxylase of SEQ ID NO 3, or a functional variant of said enzymes having a nucleic sequence at least 80% identical to said SEQ ID NO 1, 2 and 3, characterized in that the three enzymes are expressed under the control of the same human PGK-1 promoter, or a functional variant of said human PGK-1 promoter having a nucleic sequence at least 80% identical to said SEQ ID NO 4.

[0021] It also relates to a viral vector according to the invention, for its use as a medicament.

[0022] BENEFITS PROVIDED

[0023] The Applicant has notably been able to develop self-inactivating third-generation lentiviral vectors constructed from the human immunodeficiency virus 1 (HIV-1) comprising the coding sequences of the three enzymes enabling the expression of dopamine. Said sequences are advantageously linked by linker, peptide and IRES sequences and are expressed under the control of the human PGK-1 promoter having a nucleic sequence at least 80% identical to said SEQ ID NO 4. Analysis of the different fusion protein systems has shown that they can give very different dopamine expression profiles in the target tissue.

[0024] The efficacy of one of the vectors in transducing human neurons and inducing neosynthesis and secretion of dopamine was demonstrated on a model of neurons derived from pluripotent stem cells of healthy individuals or those affected by Lesch-Nyhan disease.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The invention and the advantages resulting therefrom will be better understood upon reading the description and the non-limiting embodiments which follow, illustrated with reference to the appended drawings in which: [fig. 1] illustrates the transgene constructs evaluated, as detailed in example 1.

[0027] [Fig. 2] represents a diagram illustrating the synthesis and release of dopamine by human neurons after transduction with a lentiviral vector, measured by ELISA, as detailed in Example 2.

[0028] [Fig. 3] represents immunocytochemistry following transduction of vector number 2 illustrated in Figure 1 into neuronal cells.

[0029] [Fig. 4] represents immunocytochemistry following transduction of vector number 1 illustrated in Figure 1 into neuronal cells.

[0030] DESCRIPTION OF EMBODIMENTS

[0031] In this description, unless otherwise specified, it is understood that, when an interval is given, it includes the upper and lower bounds of said interval.

[0032] The invention relates to a lentiviral vector. Lentiviral vectors are retroviruses created in vitro by transfecting multiple plasmids into mammalian cells. Retroviral particles are harvested from the culture medium and used to stably insert a transgene into the genome of a target cell. Lentiviral vectors can incorporate large transgene cargoes, allowing multiple transgenes to be delivered into the same target cell.

[0033] According to a preferred embodiment, the invention relates to a lentiviral vector included in a composition capable of being administered by human or animal subjects.

[0034] Lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). The non-primate lentivirus group includes the prototype "slow virus," visna / maedi virus (VMV), as well as the related caprine arthritis and encephalitis viruses (CAEV), equine infectious anemia (EIAV), feline immunodeficiency virus (FIV), and bovine immunodeficiency virus (BIV).

[0035] Advantageously, the lentiviral vector according to the invention is a self-inactivating third-generation vector constructed from the human immunodeficiency virus (HIV), preferably HIV-1.

[0036] In a preferred embodiment, in particular in a variant where the gene expression construct is a lentiviral vector, the gene expression construct has a size of between 2100 and 5000 nucleotides, in particular between 2700 and 4900 nucleotides, more particularly between 3200 and 4700 nucleotides. In a particular embodiment, the size of the gene expression construct is about 3200 nucleotides, about 3300 nucleotides, about 3400 nucleotides, about 3500 nucleotides, about 3600 nucleotides, about 3700 nucleotides, about 3800 nucleotides, about 3900 nucleotides, about 4000 nucleotides, about 4100 nucleotides or about 4200 nucleotides. The lentiviral vector according to the invention is used for the synthesis of dopamine. Dopamine (DA) is a neurotransmitter, a biochemical molecule that allows communication within the nervous system, and one of those that directly influences behavior.

[0037] Dopamine is synthesized in neurons from circulating tyrosine. The reaction is carried out by two enzymes: tyrosine hydroxylase (TH), a rate-limiting enzyme controlling the production of L-DOPA, and then by DOPA decarboxylase (AADC), which decarboxylates the latter to produce dopamine.

[0038] The lentiviral vector according to the invention comprises a tricistronic expression cassette coding firstly for the tyrosine hydroxylase (TH) of SEQ ID NO 1 or a functional variant of said enzyme having a nucleic sequence at least 80% identical to said SEQ ID NO 1.

[0039] According to a particular embodiment, the nucleic sequence coding for TH is at least 80% identical, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96% 97%, 98% or 99% identical to said SEQ ID NO 1, more preferably it consists of said SEQ ID NO 1.

[0040] An expression cassette comprises a transgene of interest encoding a protein to be expressed and polynucleotide sequences controlling its expression, such as a promoter and a polyadenylation signal. This cassette may also include an intron, defined as a non-coding nucleic acid sequence that can modify the expression level of a gene.

[0041] A cistron is a functional genetic unit, defined by the cis-trans test, which corresponds to a segment of DNA, specifying a polypeptide chain.

[0042] A tricistronic expression cassette is a cassette that will allow the expression of three genes coding for three proteins.

[0043] Tyrosine hydroxylase (TH) is an enzyme, more precisely an oxidoreductase, which catalyzes the following reaction:

[0044] L-tyrosine + tetrahydrobiopterin + O2 L-DOPA + 4a-hydroxytetrahydrobiopterin

[0045] Since L-DOPA is a precursor of dopamine, TH is an essential enzyme for the synthesis of dopamine (DA).

[0046] Nucleotide means any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Polynucleotides include, but are not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single- and triple-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single- or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions. As used herein, the term "polynucleotide(s)" also includes the DNAs or RNAs described above that contain one or more modified bases. Thus, DNA or RNA whose backbones are modified for stability or other reasons are "polynucleotides" as that term is used here.In addition, DNA or RNA comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as that term is used herein. It will be appreciated that a wide variety of modifications have been made to DNA and RNA, which serve many useful purposes known to those skilled in the art. The term "polynucleotide(s)" as used herein encompasses such chemically, enzymatically, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. The term "polynucleotide(s)" also encompasses short polynucleotides often referred to as oligonucleotides.

[0047] A protein sequence means any peptide or protein comprising two or more amino acids linked together by peptide bonds or modified peptide bonds. The term "polypeptide(s)" includes both short chains, commonly referred to as peptides, oligopeptides, and oligomers, and longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 amino acids encoded by the gene. "Polypeptides" include those that are modified by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in the scientific literature and are well known to those skilled in the art. It will be appreciated that the same type of modification may be present to the same or different degrees at several sites in a given polypeptide. Similarly,A given polypeptide can contain many types of modification. Modification can occur anywhere in a polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl ends. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslink formation, cysteine ​​formation, pyroglutamate formation, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, sulfation, addition of amino acids to proteins via transfer RNA, such as arginylation, and ubiquitination. Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched, and unbranched polypeptides may result from natural post-translational processes and may also be made by entirely synthetic methods. Identity means a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by sequence comparison. In the art, "identity" also means the degree of sequence relatedness between the polypeptide or polynucleotide sequences, as the case may be,as determined by the correspondence between the strings of these sequences. The "identity" and "similarity" can be readily calculated by known methods, including, but not limited to, those described in the following references (Computational Molecular Biology, Lesk AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and genome Projects, Smith DW, ed., Academic Press, New York, 1993; Computer Analysis of sequence Data, Part I, Griffin AM, and Griffin HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov M. and Devereux J., eds., M. Stockton Press, New York, 1991; and Carillo H., and Lipman D., SIAM J. Applied Math., 48:1073 (1998)) . Identity determination methods are designed to give the greatest match between the sequences tested. In addition,Identity determination methods are codified in publicly available computer programs. Computer program methods for determining the identity between two sequences include, but are not limited to, the GCG package (Devereux J. et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Altschul SF et al., J. Molec. Biol. 215:403-410 (1990)). The BLAST X program is 12 publicly available from NCBI and other sources (BLAST Manual, Altschul S. et al., NCBI NLM NUH Bethesda, MD 20894; Altschul S. et al., J. Mol Biol. 215:403-410 (1990)).

[0048] A variant means a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Changes in nucleotides may result in substitutions, additions, deletions, fusions, and / or truncations of amino acids in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide.

[0049] In general, the differences are limited such that the sequences of the reference polypeptide and the variant are broadly very similar and, in many regions, identical. A variant polypeptide and a reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and / or deletions in any combination. A substituted or inserted amino acid residue may or may not be a residue encoded by the genetic code.

[0050] Functional variant means in particular a variant whose initial function is maintained. The present invention also includes variants of each of the polypeptides of the invention, i.e. polypeptides which vary from the references by conservative amino acid substitutions, by which one residue is replaced by another having similar characteristics. Typical conservative amino acid substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues, Asp and Glu; among Asn and Gln; and among the basic residues Lys and Arg; or the aromatic residues Phe and Tyr. These conservative mutations include mutations which exchange one amino acid for another in one of the following groups:

[0051] 1. Small aliphatic residues, non-polar or slightly polar: Ala, Ser, Thr, Pro and Gly; 2. Polar residues, negatively charged and their amides: Asp, Asn, Glu and Gln;

[0052] 3. Polar, positively charged residues: His, Arg and Lys;

[0053] 4. Large, non-polar aliphatic residues: Met, Leu, Ile, Val and Cys; and

[0054] 5. Aromatic residues: Phe, Tyr and Trp.

[0055] These conservative variations may further include the following: Preferably, variants in which several, for example 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted or added in any combination. A variant of a polynucleotide or polypeptide may be of natural origin, such as an allelic variant, or it may be a variant that is not known to be of natural origin. Non-natural variants of polynucleotides and polypeptides may be obtained by mutagenesis techniques, by direct synthesis and by other recombinant methods known to those skilled in the art.

[0056] As mentioned above, in addition to GC content and / or ARF number, sequence optimization may also include decreasing the number of CpG islands in the sequence and / or decreasing the number of splice donor and acceptor sites.

[0057] Of course, as is well known to those skilled in the art, sequence optimization is a balance between all these parameters, meaning that a sequence can be considered optimized if at least one of the above parameters is improved while one or more of the other parameters are not, as long as the optimized sequence leads to an improvement of the transgene, such as an improvement in expression and / or a decrease in the immune response to the transgene in vivo. The expression cassette comprises secondly a coding sequence for GTP-cyclohydrolase 1 (GCH 1 or CH 1 ) of SEQ ID NO 2 or a functional variant of said enzyme at least 80% identical to said SEQ ID NO 2. CH 1 is an enzyme essential for the production of dopamine in nigrostriatal cells. Loss-of-function mutations of GCH 1 result in a significant reduction in dopamine synthesis in nigrostriatal cells.

[0058] According to a particular embodiment, the nucleic sequence coding for GCH1 is at least 80% identical, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96% 97%, 98% or 99% identical to said SEQ ID NO 2, more preferably it consists of said SEQ ID NO 2.

[0059] The tricistronic expression cassette finally comprises a coding sequence for the aromatic L-amino acid decarboxylase of SEQ ID NO 3 or a functional variant of said enzyme at least 80% identical to said SEQ ID NO 3. Aromatic L-amino acid decarboxylase is a lyase, also called DOPA decarboxylase, tryptophan decarboxylase, 5-hydroxytryptophan decarboxylase - or AADC or AAAD (from the English Aromatic L-amino acid decarboxylase), which catalyzes several decarboxylation reactions, including the reaction of the conversion of L-DOPA into dopamine:

[0060] L-DOPA dopamine + CO2

[0061] According to a particular embodiment, the nucleic sequence coding for AADC is at least 80% identical, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96% 97%, 98% or 99% identical to said SEQ ID NO 3, more preferably it consists of said SEQ ID NO 3. The lentiviral vector is characterized in that the three enzymes are expressed under the control of the same human PGK-1 promoter, or a functional variant of said human PGK-1 promoter having a nucleic sequence at least 80% identical to said SEQ ID NO 4.

[0062] The phosphoglycerate kinase 1 (PGK-1) promoter, the promoter of the gene encoding phosphoglycerate kinase 1, used in the invention is derived from a human gene. The advantages of using such a promoter over viral promoters such as those of the cytomegalovirus (CMV) virus are stability and safety during transgene expression.

[0063] According to a particular embodiment, the nucleic sequence coding for the human PGK-1 promoter is at least 80% identical, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96% 97%, 98% or 99% identical to said SEQ ID NO 4, more preferably it consists of said SEQ ID NO 4.

[0064] In the context of the present invention, a functional variant of the human PGK-1 promoter is a sequence derived therefrom by one or more nucleotide modifications, such as a nucleotide substitution, addition or deletion, which results in an identical or substantially identical expression level (e.g. ± 20%, such as ± 10%, ± 5% or ± 1%) of the tricistronic transgene operably linked thereto. The lentiviral vector according to the invention is preferentially characterized in that the genes encoding tyrosine hydroxylase and aromatic L-amino acid decarboxylase are linked by a linker or a peptide, more preferentially characterized in that the genes encoding tyrosine hydroxylase and GTP-cyclohydrolase 1 are linked directly by a linker or a peptide, advantageously a peptide.

[0065] A linker sequence, or linker, is a nucleic sequence coding for a linker that allows at least two coding sequences to be linked, so that the genome can code for a fusion protein.

[0066] In one embodiment, a suitable linker is a flexible linker. It may comprise amino acid repeats such as glycine-serine repeats. The purpose of the linker is to enable the formation and / or proper functioning of enzymes. It must be sufficiently flexible and long enough to achieve this purpose. Since the coding genes may encode different enzymes, the linker enables the functioning of both enzymes. The coding sequence of the flexible linker may be chosen to promote translational pausing and thus independent folding of protein products.

[0067] The linker according to the invention is advantageously chosen from the category of flexible linkers, preferably GS15 of SEQ ID NO 5.

[0068] The peptide according to the invention is preferentially chosen from the self-cleaving peptides P2A, E2A, F2A and T2A, preferentially P2A of SEQ ID NO 6.

[0069] Self-cleaving 2A peptides, or 2A peptides, are a class of 18 to 22 aa-long peptides that can induce ribosomal skipping during protein translation in a cell. 2A peptides are used to help generate polyproteins by preventing the ribosome from creating a peptide bond. Four members of the 2A peptide family are frequently used in life science research. They are P2A, E2A, F2A, and T2A. F2A is derived from foot-and-mouth disease virus 18; E2A is derived from equine rhinitis A virus; P2A is derived from porcine teschovirus-1 2A; and T2A is derived from asigna virus 2A.

[0070] Preferably, the sequence coding for the aromatic L-amino acid decarboxylase is preceded by an IRES sequence of SEQ ID NO 7 or by a functional variant of said IRES sequence having a nucleic sequence at least 80% identical to said IRES sequence of SEQ ID NO 7.

[0071] IRES (Internal Ribosome Entry Site) are sequences that, in eukaryotic cells, allow the translation of a messenger RNA to start internally. The conventional process of translation of eukaryotic messenger RNAs relies on a ribosome scanning mechanism from the cap located at the 5' end, which scans the mRNA to the first start codon. IRES allow direct recruitment of the ribosome at this start codon, independent of the presence of the cap and the scanning mechanism. IRES are structured regions of the mRNA that interact directly with the ribosome or with translation initiation factors.

[0072] IRES sequences can originate from different viruses selected from poliovirus, rhinovirus, encephalomyocarditis virus, foot-and-mouth disease virus, Kaposi's sarcoma-associated hepes virus, hepatitis A virus, hepatitis C virus, classical swine fever virus, bovine viral diarrhea virus, friendly murine leukemia, Moloney murine leukemia, Rous sarcoma virus, human immunodeficiency virus, Plautia stall intestinal virus, Cricket paralysis virus, triatomine virus, Rhopalosiphum padi virus, Marek's disease virus, preferentially poliovirus.

[0073] According to a particular embodiment, the nucleic sequence coding for said IRES sequence is at least 80% identical, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96% 97%, 98% or 99% identical to said SEQ ID NO 7, more preferably it consists of said SEQ ID NO 7.

[0074] Advantageously, the sequence coding for the aromatic L-amino acid decarboxylase is followed by a WPRE4 sequence of SEQ ID NO 8 or a functional variant of said WPRE4 sequence having a nucleic sequence at least 80% identical to said WPRE4 sequence of SEQ ID NO 8.

[0075] The Woodchuck hepatitis virus (WHV) posttranscriptional regulatory element (WPRE) is a DNA sequence that, when transcribed, creates a tertiary structure that enhances expression. WPRE is a tripartite regulatory element with gamma, alpha, and beta components.

[0076] According to a preferred embodiment of the invention, the WPRE sequence used in the present invention is a truncated sequence.

[0077] According to a particular embodiment, the nucleic sequence coding for WPRE4 is at least 80% identical, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96% 97%, 98% or 99% identical to said SEQ ID NO 8, more preferably it consists of said SEQ ID NO 8.

[0078] According to one embodiment of the invention, said lentiviral vector comprises the construction of the transgene and therefore of the expression cassette in this precise order: PGK-1 - TH

[0079] - linker - CH1 - 1RES - AADC - WPRE.

[0080] According to another embodiment of the invention, said lentiviral vector comprises the construction of the transgene and therefore of the expression cassette in this precise order: PGK-1 - TH

[0081] - peptide - CH1 - 1RES - AADC - WPRE.

[0082] The lentiviral vector according to the invention is preferentially expressed in human neurons.

[0083] The neuron is the brain's basic working unit. It is a specialized cell designed to transmit information to other nerve cells, muscle cells, and glandular cells. The brain's characteristics are largely based on the structural and functional properties of the interconnections between neurons. When neurons receive or send messages, they transmit electrical impulses along the axon. Many axons are covered with a myelin sheath to speed the conduction of nerve impulses. This sheath is produced by specialized cells called glial cells. In the central nervous system (CNS), the glial cells that form the myelin sheath are called oligodendrocytes, and in the peripheral nervous system (PNS), they are called Schwann cells.

[0084] In the context of the invention, the human neurons used are derived from non-embryonic stem cells, more preferably pluripotent stem cells (PSCs). A pluripotent stem cell is capable of differentiating into many different cell types. According to one embodiment, said PSCs may be induced pluripotent stem cells (iPSCs). iPSCs are pluripotent stem cells generated in the laboratory from somatic cells. These induced stem cells have the potential to differentiate into any cell of the human body and therefore have very varied applications in therapy and biomedical research.

[0085] The lentiviral vector according to the invention is preferentially transduced into human neurons.

[0086] The invention also relates to a lentiviral vector according to the invention for its use as a medicament.

[0087] According to an alternative embodiment, the invention relates to the use of a lentiviral vector according to the invention as a medicament.

[0088] According to a preferred embodiment, the invention relates to a pharmaceutical composition comprising a lentiviral vector comprising the tricistronic expression cassette for use as a medicament according to the aspects of the invention described above, and a pharmaceutically acceptable medium. A pharmaceutically acceptable medium includes all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. which are suitable for administration to a mammalian host. The use of such media and agents for pharmaceutical active substances is well known in the art. Additional active ingredients may also be incorporated into the medicament of the present invention.

[0089] According to a first mode of use, the lentiviral vector according to the invention is used in the treatment and / or prevention of diseases whose neurological symptoms are due to a dopamine deficiency, preferably for its use in the treatment and / or prevention of pediatric genetic diseases due to a dopamine deficiency.

[0090] According to the invention, the term "treatment" refers to an amelioration, prophylaxis or reversal of a disease or disorder, or at least one discernible symptom thereof. It also refers to an amelioration, prophylaxis or reversal of at least one measurable physical parameter related to the disease or disorder being treated, which is not necessarily perceptible to the subject. In another embodiment, the term "treatment" refers to the inhibition or slowing of the progression of a disease or disorder, either physically, for example, stabilization of a discernible symptom, physiologically, for example, stabilization of a physical parameter, or both. The term "treatment" also refers to the delaying of the onset of a disease or disorder. In certain particular embodiments of the invention, the compositions of interest are administered as a preventative measure.In this context, the term "prevention" means a reduction in the risk of acquiring a specified disease or disorder.

[0091] A pediatric genetic disease is a disease that affects children from birth to the age of 15.

[0092] Pediatric genetic diseases due to dopamine deficiency include Lewis body diseases, vesicular dopamine transport deficiency, dystonias, Lesch-Nyhan disease, etc.

[0093] Preferably, the lentiviral vector according to the invention is used in the treatment and / or prevention of Lesch-Nyhan disease.

[0094] Lesch-Nyhan disease (LND) is a rare disease with an estimated prevalence of one in 380,000 births. It is a genetic metabolic disorder linked to the X chromosome, involving the HPRT-1 gene, which codes for the HGPRT enzyme (hypoxanthine-guanine-phosphoribosyl-transferase). Mutations in this gene result in a deficiency in the activity of the enzyme, which plays a central role in purine metabolism. This decrease in activity causes a malfunction of the purine recycling pathway, leading to the accumulation of uric acid in the joints (gout) and kidneys (lithiasis), which can be controlled by taking allopurinol. However, these symptoms are generally accompanied by neurological disorders that are still completely unexplained and untreated. These result in significant motor disability with abnormal movements (dystonia) and tone disorders (axial hypotonia).In the most severe cases, behavioral disorders can also occur, manifesting themselves in significant self-mutilation events (lip and finger biting). Studies conducted using brain imaging have identified a decrease in brain dopamine concentration in patients.

[0095] According to another embodiment of the invention, the lentiviral vector according to the invention is used in the treatment and / or prevention of resistant dystonia in children.

[0096] Dystonia is a movement disorder that can affect adults and children and is defined by sustained, involuntary, tonic muscle contraction, resulting in repetitive twisting movements or abnormal postures. It is secondary to a dysfunction of the central nervous system, involving, in particular, the basal ganglia, which are a deep structure of the brain, but certainly affecting an entire intracerebral network. The thalamus, which is another brain structure allowing the planning and implementation of movements, also seems to be involved.

[0097] An imbalance between different neurotransmitters, including dopamine, leads to dopamine deficiency.

[0098] According to a second mode of use, the lentiviral vector according to the invention is used in the treatment and / or prevention of Parkinson's disease.

[0099] Parkinson's disease is a progressive degenerative brain disorder associated with motor symptoms (slow movements, tremors, rigidity, and imbalance) and other complications, including cognitive, mental health, sleep, and pain and sensory disturbances. Motor impairments, such as dyskinesias (involuntary movements) and dystonias (painful involuntary muscle contractions), contribute to speech and mobility problems and lead to restrictions in many areas of life. The progression of these symptoms leads to high rates of disability and care needs. Many people with the disease also develop dementia during the course of their illness. It is characterized by the destruction of certain neurons in the brain and the accumulation of protein clumps that are toxic to nerve cells.

[0100] Parkinson's disease is primarily caused by a progressive degeneration of dopamine neurons in the brain, dopamine being a neurotransmitter involved in the control of many functions such as voluntary movements, cognition, motivation and affects. This neuronal death is associated with three essential phenomena: a. an accumulation of clumps called Lewy bodies, made up largely of the protein a-synuclein, b. abnormal activity of mitochondria, which are the "power plants" of cells, c. inflammation of the brain tissue, which is believed to be linked to several types of immune cells: microglial cells of innate immunity, and T lymphocytes of adaptive immunity.

[0101] Dopaminergic neurons in the brain's nigrostriatal pathway (emerging from the substantia nigra), responsible for coordinating movements, are those that degenerate preferentially and massively: this explains why Parkinson's disease often first manifests itself through motor symptoms. But the disease is not restricted to the cells of the substantia nigra. Indeed, the presence of Lewy bodies - or synucleinopathy - is observed in other brain dopaminergic neurons, notably in the olfactory bulb or the ventral tegmental area (mesocortical and mesolimbic pathways), as well as in neurons of the intestine (enteric nervous system).

[0102] The present invention will now be illustrated by means of the following examples:

[0103] Example 1:

[0104] Two new lentiviral vectors constructed from HIV-1 have been generated. These vectors are third-generation self-inactivating vectors.

[0105] The coding sequences for the three enzymes, TH, CH1 and AADC, enabling the expression of DA are linked by different systems and are expressed under the control of the human PGK-1 promoter.

[0106] The transgene constructs evaluated are illustrated in Figure 1. A lentiviral vector constructed from HIV-1 expressing the eGFP protein under the control of the human PGK-1 promoter was used as a control vector to establish the transduction conditions. Two lentiviral vectors constructed from HIV-1 expressing the three enzymes enabling DA expression were designed and evaluated.

[0107] HIV-1 vector number 1 contains a construct between the TH and CH 1 coding sequences using a flexible GS15 linker with AADC expression downstream of a poliovirus IRES internal ribosome entry site sequence.

[0108] HIV-1 vector number 2 contains a fusion construct between the TH and CH 1 coding sequences using a 2A peptide with AADC expression downstream of a poliovirus IRES internal ribosome entry site sequence.

[0109] All three vectors contain the post-transcriptional regulatory element of woodchuck hepatitis virus.

[0110] Example 2: In vitro validation of vector efficacy:

[0111] Non-dopaminergic neurons were differentiated from human pluripotent stem cells from healthy individuals or patients with Lesch-Nyhan disease, a severe form of pediatric genetic dopamine deficiency. These neurons were transduced with the HIV-1 TH-AADC-CGH lentiviral vector to assess de novo TH and AADC expression, as evidence of vector integration, as well as de novo dopamine synthesis and release (Figure 2). A negative control comprising TSSM buffer from the vector preparation was used. DA concentration (ng / ml) was measured by ELISA.

[0112] We can observe the significant expression of DA in human neurons in vitro after transduction of the lentiviral vector compared to the negative control. Indeed, the dopamine concentration increases from 0 to at least 70 ng / ml and can go up to more than 190 ng / ml.

[0113] We can therefore say that the lentiviral vectors according to the invention have significant efficacy and allow the synthesis of dopamine in human neuronal cells.

[0114] Example 3: Evaluation of rHIV vectors synthesizing dopamine in an in vitro model of human iPS neurons derived from healthy individuals and those with Lesch-Nyhan disease:

[0115] To carry out this experiment, the Applicant chose to work with 4 human cell lines: 2 from healthy individuals and 2 from individuals suffering from Lesch-Nyhan disease to verify that the vector has the capacity to function in both healthy and diseased lines.

[0116] The efficacy of the in vitro vectors on human neurons generated from iPS for these 4 cell lines was tested.

[0117] The neurons, which were generated in vitro for this experiment, are cortical neurons that do not produce basal dopamine in order to assess the effectiveness of the vectors in producing and releasing dopamine.

[0118] Several criteria were evaluated:

[0119] Toxicity of vectors on human neurons

[0120] Ability of vectors to express the different enzymes necessary for dopamine synthesis

[0121] Effectiveness of vectors to produce and release dopamine

[0122] The described vectors were tested for their ability to express tyrosine hydroxylase (TH) and aromatic L-amino acid dopa decarboxylase (AADC) in the cell bodies of human neurites and neurons derived from healthy subjects or patients with Lesch-Nyhan disease and for their ability to produce dopamine.

[0123] The applicant showed that the way in which the three reading frames coding for the different enzymes are linked affects their expression level and their colocalization within the cell bodies of human neurites and neurons.

[0124] Indeed, the rHIV_GS vector in which the reading frames coding for the TH and CH1 enzymes are linked by a linker leads to an altered expression of the TH enzyme with a weak or even an absence of colocalization of the AADC enzyme (see figure 4) in the cell bodies of human neurites and neurons. On the other hand, the rHIV-2A vector in which the reading frames coding for the TH and CH1 enzymes are linked by a self-cleaving peptide leads to a very strong expression of the TH enzyme with a perfect colocalization with the AADC enzyme (see figure 3) in the cell bodies of human neurites and neurons. Although these two vectors allow the synthesis of dopamine, the Applicant has shown that only the rHIV2A vector allows a very good expression of the TH and AADC enzymes and a perfect colocalization within the cell bodies of human neurites and neurons from healthy subjects or patients suffering from Lesch-Nyhan disease.

Claims

CLAIMS 1. Lentiviral vector comprising a tricistronic expression cassette coding for: tyrosine hydroxylase (TH) of SEQ ID NO 1, GTP-cyclohydrolase 1 (GCH1) of SEQ ID NO 2, and aromatic L-amino acid decarboxylase (AADC) of SEQ ID NO 3, or a functional variant of said enzymes having a nucleic sequence at least 80% identical to said SEQ ID NO 1, 2 and 3, characterized in that the three enzymes are expressed under the control of the same human PGK-1 promoter, or a functional variant of said human PGK-1 promoter having a nucleic sequence at least 80% identical to said SEQ ID NO 4.

2. Lentiviral vector according to claim 1, characterized in that it is constructed from the human immunodeficiency virus (HIV).

3. Lentiviral vector according to claim 1 or 2, characterized in that the genes coding for tyrosine hydroxylase and GTP-cyclohydrolase 1 are linked by a linker or a peptide.

4. Lentiviral vector according to claim 3, characterized in that the linker is chosen from the category of flexible linkers, preferably GSI 5 of SEQ ID NO 5.

5. Lentiviral vector according to claim 3, characterized in that the peptide is chosen from the self-cleaving peptides P2A, E2A, F2A and T2A, preferentially P2A of SEQ ID NO 6.

6. Lentiviral vector according to one of the preceding claims, characterized in that the sequence coding for the aromatic L-amino acid decarboxylase is preceded by an IRES sequence of SEQ ID NO 7 or by a functional variant of said IRES sequence having a nucleic sequence at least 80% identical to said IRES sequence of SEQ ID NO 7.

7. Lentiviral vector according to one of the preceding claims, characterized in that the sequence coding for the aromatic L-amino acid decarboxylase is followed by a WPRE4 sequence of SEQ ID NO 8 or a functional variant of said WPRE4 sequence having a nucleic sequence at least 80% identical to said WPRE4 sequence of SEQ ID NO 8.

8. Lentiviral vector according to one of the preceding claims, characterized in that said lentiviral vector comprises the expression cassette in this precise order: PGK-1 -TH - peptide - CH 1 - 1RES - AADC - WPRE.

9. Lentiviral vector according to one of the preceding claims, characterized in that it is transduced into human neurons.

10. Lentiviral vector according to one of the preceding claims, for use as a medicament.

11. Lentiviral vector according to claim 10, for its use in the treatment and / or prevention of diseases whose neurological symptoms are due to a dopamine deficiency.

12. Lentiviral vector according to claim 11, for its use in the treatment and / or prevention of pediatric genetic diseases due to dopamine deficiency.

13. Lentiviral vector according to claim 10 or 11, for use in the treatment and / or prevention of Parkinson's disease.

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