[go: up one dir, main page]

WO2010037143A1 - Vecteurs et méthodes de traitement des convulsions cérébrales - Google Patents

Vecteurs et méthodes de traitement des convulsions cérébrales Download PDF

Info

Publication number
WO2010037143A1
WO2010037143A1 PCT/US2009/058899 US2009058899W WO2010037143A1 WO 2010037143 A1 WO2010037143 A1 WO 2010037143A1 US 2009058899 W US2009058899 W US 2009058899W WO 2010037143 A1 WO2010037143 A1 WO 2010037143A1
Authority
WO
WIPO (PCT)
Prior art keywords
raav
eaat3
expression
seizures
brain
Prior art date
Application number
PCT/US2009/058899
Other languages
English (en)
Inventor
David J. Poulsen
Original Assignee
The University Of Montana
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The University Of Montana filed Critical The University Of Montana
Publication of WO2010037143A1 publication Critical patent/WO2010037143A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/025—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a parvovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE

Definitions

  • the present invention is directed to methods and therapeutic tools for treating a subject having brain seizures by increasing expression of excitatory amino acid transporter 3 (EAAT3) in GABAergic neurons.
  • EAAT3 excitatory amino acid transporter 3
  • Seizures are symptoms of problems in the brain, which result from abrupt, abnormal electrical activity therein. Focal seizures occur in only one part of the brain. Recurring seizures often result from a brain disorder called epilepsy.
  • Epilepsy is a chronic neurological condition caused by abnormalities in the central nervous system. It is characterized by recurrent seizures originating in the brain and is associated with excessive or abnormal synchronization of neural activity. An estimated 1 % of the population suffers from some form of epilepsy, and more than 2 million people in the US have been diagnosed with epilepsy or experienced a seizure.
  • An epileptic seizure is a brief, excessive surge of electrical activity in the brain that causes a change in consciousness, sensation and behavior.
  • the regulatory systems that maintain the normal balance between excitation and inhibition of the brain's electrical activity break down. There may be a loss of inhibitory nerve cells or an overproduction of an excitatory neurotransmitter. Groups of abnormal cells are activated synchronously, creating a storm of electrical activity.
  • TLE temporal lobe epilepsy
  • TLE causes both simple partial seizures, wherein the afflicted individual does not lose consciousness, and complex partial seizures, which result in a loss of consciousness and memory impairment.
  • the latter form of seizure may secondarily generalize. This occurs in about 60% of people with TLE.
  • seizures spread from the temporal lobe to a wider portion of the brain. The result is a convulsive (grand mal) seizure.
  • anticonvulsive drugs may control seizures in some patients, seizures will remain uncontrolled in 40% of patients despite drug therapy.
  • One treatment approach for these patients is to undergo a phased evaluation for consideration of resective surgery.
  • Resective surgery involves the surgical removal of the portion of the brain responsible for causing seizures.
  • the brain is comprised of four lobes: the frontal, temporal, parietal and occipital. Seizures most often arise from one or both temporal lobes. In the deep front part of the temporal lobes are located the most seizure prone structures in the brain: the hippocampus, and the amygdala. Because of this, temporal lobectomy is one of the most common and one of the most successful types of resective surgery. Typically, one temporal lobe is defined as the site of seizure origin (the epileptogenic region) and the medial temporal lobe including the anterior hippocampus is resected.
  • AED antiepileptic drugs
  • patients undergoing the surgery may require up to a 14 day hospitalization.
  • Post-operation headaches and nausea commonly occur, and most doctors will keep a patient on seizure medication for at least several years. In some cases, the medication is necessary for life, albeit at a reduced dosage. Moreover, the part of the brain removed does not grow back.
  • antiepileptic drugs currently exist for treating seizures, patients taking these drugs display a broad spectrum of side-effects, such as liver damage, nausea, vomiting, double vision, loss of coordination, drowsiness, and headache.
  • the widely used drug carbamazepine for example, exhibits side effects including dizziness, ataxia, drowsiness and reduction of alertness (A. Delcker, Eur.
  • Valproic acid may precipitate metabolic disorders, liver disease, gastrointestinal symptomatology, excessive bodyweight gain and alopecia. See, S. J. Wallace, Drug Saf., 15: 378-93 (1996). Barbiturates precipitate metabolic bone disease and rash. (S. J. Wallace, Drug Saf 15: 378-93 (1996)).
  • improved therapeutic tools and methods of treating a human subject suffering from brain seizures would reduce the severity, frequency, and duration of the brain seizures without the side effects or invasive surgical interventions that accompany current methods of treatment.
  • the present invention is based on the surprising discovery that brain seizures can be treated with minimal or no side effects by increasing the expression of excitatory amino acid transporter 3 (EAAT3) in GABAergic neurons.
  • EAAT3 excitatory amino acid transporter 3
  • the invention is directed to a method of treating a human subject having focal brain seizures.
  • the method comprises the step of increasing expression of excitatory amino acid transporter 3 (EAAT3) in GABAergic neurons located in the brain of the subject where the focal brain seizures occur.
  • EAAT3 expression is advantageously increased in the GABAergic neurons sufficiently to reduce the severity, frequency, and/or duration of the focal brain seizures to treat the subject having brain seizures.
  • the method comprises directly administering to the hippocampus of the subject a recombinant adeno-associated viral vector (rAAV).
  • rAAV a recombinant adeno-associated viral vector
  • the rAAV used typically comprises an excitatory amino acid transporter 3 (EAAT3) gene operatively linked to a promoter suitable for expression of EAAT3 in GABAergic neurons in the hippocampus, wherein EAAT3 expression is increased sufficiently in the GABAergic neurons to reduce the severity, frequency, and/or duration of the brain seizures in the subject.
  • EAAT3 excitatory amino acid transporter 3
  • Therapeutic tools are also provided herein.
  • the tools are useful in treating humans having chronic brain seizures, e.g. epilepsy.
  • One such tool provided herein is a recombinant adeno-associated viral vector (rAAV) designed specifically to treat brain seizures in human subjects.
  • the rAAV preferably comprises AAV 5' and 3' ITRs flanking a human excitatory amino acid transporter 3 (EAAT3) gene which is operatively linked to a promoter suitable for expression of the EAAT3 in GABAergic neurons of the hippocampus.
  • EAAT3 human excitatory amino acid transporter 3
  • compositions are also provided.
  • the pharmaceutical compositions comprise the recombinant adeno-associated viral vectors (rAAV) designed to treat brain seizures, as described herein, and a pharmaceutically acceptable carrier.
  • rAAV recombinant adeno-associated viral vectors
  • rAAV can also be used in the manufacture of a medicament for the treatment of brain seizures.
  • FIG. 1 Increased functional expression of EAAT3.
  • HC2S2 cultures were transduced with AAV1 -CAG-hEAAT3 and levels of EAAT3 expression compared with non transduced control cultures. All three EAAT3 protein bands are shown: non-transduced control HC2S2 cultures (C); cultures transduced with rAAV1 -CAG-EAAT3 (CE3) relative to ⁇ -actin normalization.
  • FIG. 3 EAAT3 expression regulates intracellular GABA, glutamate and glutamine levels.
  • HPLC analysis of rAAV transduced and control HC2S2 cultures was performed to determine the intracellular concentrations of GABA, glutamine and glutamate.
  • A) GABA levels in HC2S2 cells transduced with rAAV1 -CAG-hEAAT3 were 136% higher than non-transduced controls.
  • C) Glutamine levels in HC2S2 cells transduced with rAAV1 -CAG-hEAAT3 were 146% higher than non-transduced controls.
  • FIG. 4 rAAV mediated targeted expression to GABAergic neurons.
  • rAAV-GAD65-hrGFP was bilaterally injected into the hippocampaus of adult male Sprague-Dawley rats. After 4 weeks, brains were perfused and fixed with 4% paraformalgehyde and 50 ⁇ M sections prepared on a vibratome. Sections were stained with anti-GAD65/67 antibody (red). Colocalization of GFP expression (green) with GAD65 staining indicates targeted expression to GABAergic neurons.
  • FIG. 5 Targeted expression of flag tagged rEAAT3 to GABAergic neurons.
  • rAAV-GAD65-flagEAAT3 was bilaterally injected into the hippocampus of adult male Sprague-Dawley rats. After 4 weeks, 50 ⁇ M vibratome sections were stained with antibody specific for the flag epitope (green) to label recombinant rEAAT3 protein. Sections were dual stained with anti-GAD65/67 antibody (red) to label GABAergic neurons. Colocalization of flag-EAAT3 expression with GAD65 staining indicates targeted expression of the recombinant rEAAT3 to GABAergic neurons.
  • EAAT3 expression altered seizure susceptibility and rate of progression to SE.
  • the pilocarpine induced seizure model was used to evaluate altered state of seizure susceptibility as a function of EAAT3 expression on inhibitory neuron terminals.
  • Control (v), flag-rEAAT3 ( ⁇ ), and EAAT3/AS ( ⁇ ) animals were monitored for progression through distinct behavioral stages based on a modified Racine scale. Time to reach each of the stages was noted, averaged for each treatment group for animals that reached SE.
  • One way ANOVA, n 3-7.
  • FIG. 7 Altered EAAT3 expression on inhibitory presynaptic terminals influences neuroprotection.
  • FIG. 1 Similar neuropathology was observed in cortex following piiocarpine-induced seizures.
  • Rgure Stereotaxic coordinates of electrode placement in the hippocampus of the rat.
  • a reference screw near the bregma was used as a ground, and electrodes were placed at 3.6mm from the bregma, 3.5mm to the right of the midline, and at a depth of 3.2mm.
  • KA and virus injections occurred through a port 2mm medial the electrodes and at a depth of 4mm.
  • Figure 10 Shows a rat with pedestal shown during an EEG a recording
  • FIG. 11 Representative EEG recordings from two rats including their baselines, KA delivery, the 2 weeks after KA delivery, and weeks 1 , 2, and 4 after rAAV delivery.
  • Control rat received AAV8-GAD65-Nu!l and the experimental rat received the AAV8-GAD65-f!ag-rEAAT3, which caused increased expression of rEAAT3 in GABAergic neurons.
  • At two weeks after the injection of virus there is a marked difference in seizure severities between the two groups.
  • the AAV8-GAD65-f!ag-rEAAT3 injected animal showed visibly reduced seizure activity.
  • rEAAT3 Enhanced expression of rEAAT3 in GABAergic neurons significantly delayed the onset and/or severity of pilocarpine-induced seizures and resulted in reduced neuropathology in animals that experienced status epilepticus. In contrast, knock down of rEAAT3 in GABAergic neurons resulted in greater sensitivity to pilocarpine-induced seizures and enhanced neurodegeneration following seizures. Based on these studies and examples, we provide herein methods and therapeutic tools to treat subject's suffering from brain seizures.
  • the present methods successfully treat human subjects suffering from brain seizures by increasing the expression of excitatory amino acid transporter 3 (EAAT3), e.g., GenBank NM_004170; Accession NM_004170; SLC1 A1 NM 004170.4 Homo sapiens solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system Xag), in GABAergic neurons.
  • GABAergic neurons are neurons that convert glutamate to ⁇ -Aminobutyric acid (GABA), typically via the GAD65 and GAD67 enzymes.
  • GABAergic neurons typically also store GABA in synaptic vesicles and release GABA from these vesicles upon stimulation.
  • the increased EAAT3 expression is targeted to GABAergic neurons in the target region wherein the seizures occur.
  • Targeted expression means that preferably at least 50%, more preferably at least 85%, and still more preferably at least 95% of the transduced cells with increased expressing of EAAT3 are GABAergic neurons in the target region. In certain preferred embodiments, 98% to 100% of cells with increased expression of EAAT3 are GABAergic neurons.
  • a focal seizure is localized (partial or focal onset seizures). Focal seizures can be further divided on the extent to which consciousness is affected (simple focal seizures and complex focal seizures). If consciousness is unaffected, then it is a simple focal seizure; otherwise it is a complex focal seizure. A focal seizure may spread within the brain — a process known as secondary generalization.
  • An epileptic seizure is a transient symptom of excessive or synchronous neuronal activity in the brain.
  • the medical syndrome of recurrent, unprovoked seizures is typically termed epilepsy.
  • the invention is directed to a method of treating a human subject having focal brain seizures.
  • the method preferably comprises the step of increasing expression of excitatory amino acid transporter 3 (EAAT3) in GABAergic neurons located in the brain of the subject where the focal brain seizures occur.
  • EAAT3 expression is advantageously increased in the GABAergic neurons sufficiently to reduce the severity, frequency, and/or duration of the focal brain seizures to treat the subject having brain seizures.
  • Increased expression of EAAT3 is compared to the GABAergic neuronal cells prior to transduction.
  • One specific and preferred method of increasing expression of EAAT3 in GABAergic neurons is by directly administering, to a region of the brain where the focal brain seizures occur, a recombinant adeno-associated virus (rAAV) vector having a human EAAT3 gene, preferably by stereotaxic injection, described more fully below.
  • rAAV recombinant adeno-associated virus
  • Another preferred and specific method comprises directly administering to the hippocampus of the subject a recombinant adeno-associated viral vector (rAAV).
  • the rAAV used typically comprises an excitatory amino acid transporter 3 (EAAT3) gene operatively linked to a promoter suitable for expression of EAAT3 in GABAergic neurons in the hippocampus, wherein EAAT3 expression is increased sufficiently in the GABAergic neurons to reduce the severity, frequency, and/or duration of the brain seizures in the subject.
  • EAAT3 excitatory amino acid transporter 3
  • the invention also provides therapeutic tools useful in treating a subject having brain seizures, such as epilepsy.
  • One such tool is a recombinant adeno-associated viral vector (rAAV) designed specifically to treat human brain seizures.
  • the rAAV preferably comprises AAV 5' and 3' ITRs flanking a human excitatory amino acid transporter 3 (EAAT3) gene which is operatively linked to a promoter suitable for expression of the EAAT3 in GABAergic neurons of the hippocampus.
  • the promoter is glutamic acid decarboxylase 65 (GAD65).
  • GAD65 glutamic acid decarboxylase 65
  • the vector further comprises the 3' Woodchuck hepatitis virus post-transcriptional regulation element (WPRE).
  • WPRE Woodchuck hepatitis virus post-transcriptional regulation element
  • AAV At least 13 different AAV serotypes have been identified based on amino acid sequence differences in their respective capsid proteins.
  • the AAV used in the present invention can be any AAV serotype. In a preferred embodiment, however, the serotype comprises serotype 1 , 2, 8 or a mixture of two or more serotypes.
  • the AAV used in the present invention is a derivative of the adeno- associated virus, into which exogenous DNA has been introduced.
  • the construction of infectious recombinant AAV and methods of purification are well known in the art. See, e.g., U.S. Patent Nos.
  • the AAV genome is composed of a linear, single-stranded DNA molecule that contains 4681 bases.
  • the genome includes inverted terminal repeats (ITRs) at each end that function in cis as origins of DNA replication and as packaging signals for the virus.
  • ITRs are approximately 145 bp in length.
  • the internal nonrepeated portion of the genome includes two large open reading frames, known as the AAV rep and cap regions, respectively. These regions code for the viral proteins that provide AAV helper functions, i.e., the proteins involved in replication and packaging of the virion.
  • a family of at least four viral proteins is synthesized from the AAV rep region, Rep 78, Rep 68, Rep 52 and Rep 40, named according to their apparent molecular weight.
  • the rAAV cap region encodes at least three proteins, VP1 , VP2 and VP3.
  • VP1 , VP2 and VP3 proteins
  • rAAV vectors can be engineered to carry an exogenous nucleotide sequence of interest, preferably EAAT3, more preferably human EAAT3, by deleting, in whole or in part, the internal portion of the rAAV genome and inserting the DNA sequence of interest between the ITRs.
  • the ITRs remain functional in such vectors allowing replication and packaging of the rAAV containing the heterologous nucleotide sequence of interest.
  • the heterologous nucleotide sequence is also typically linked to a promoter sequence capable of driving gene expression in the patient's target cells under the certain conditions. Termination signals, such as polyadenylation sites, can also be included in the vector.
  • susceptible cells are co- transfected with the rAAV-derived vector and a suitable rAAV-derived helper virus or plasmid.
  • the vector retains from rAAV essentially only the recognition signals for replication and packaging.
  • the rAAV-derived sequences do not necessarily have to correspond exactly with their wild-type prototypes.
  • the rAAV vectors of the present invention may feature mutated inverted terminal repeats, etc., provided that the vector can still be replicated and packaged and still infect GABAergic neuronal cells.
  • the rAAV is produced using the plasmid-based methods and the virus is purified on iodixanol gradients as described in Zolotukhin, Methods 28: 158-167 (2002).
  • Viruses are titered by the number of genomic particles per ml. Titers of the rAAV can vary, particularly depending upon the target cell, but preferably the rAAV used is a high-titer virus of at least 10 9 gp/ ⁇ l and more preferably, at least 10 10 gp/ ⁇ l or alternatively at least 10 11 gp/ ⁇ l. Methods of producing high- titer viruses are also well known in the art. See, e.g., U.S. Patent Nos. 6,632,670 (teach methods of generating high-titer, contaminant free, recombinant rAAV vectors in large quantities); 7,015,026.
  • exogenous DNA any heterologous DNA, i.e., not normally found in wild-type rAAV that can be inserted into the rAAV for transfer into the target cell, e.g. EAAT3.
  • the coding sequence must be "operably linked” to a promoter sequence functional in the target cell.
  • operatively linked is meant that the promoter can drive expression of the exogenous DNA, as is known in the art, and can include the appropriate orientation of the promoter relative to the exogenous DNA.
  • the exogenous DNA preferably has all appropriate sequences for expression.
  • the DNA can include, for example, expression control sequences, such as an enhancer, and necessary information processing sites.
  • the exogenous DNA will have a length of about up to 5,000 bases.
  • the DNA is 4,500 bases.
  • the promoter can be any desired promoter, selected by known considerations, such as the level of expression of the DNA operatively linked to the promoter and the GABAergic neuronal cells in which the DNA is to be expressed. Promoters can be an exogenous or an endogenous promoter.
  • the promoter is designed for expression in GABAergic neuronal cells, e.g., glutamic acid decarboxylase 65 (GAD65) (e.g., GenBank NT_008705.16, Bases 26444235-26445738; Accession NT_008705.16, bases 26555235-26445738; GenBank AF090195.1 , Bases 1233-2733; Accession AF090195.1 Bases 1233-2733).
  • GABAergic neuronal cells e.g., glutamic acid decarboxylase 65 (GAD65) (e.g., GenBank NT_008705.16, Bases 26444235-26445738; Accession NT_008705.16, bases 26555235-26445738; GenBank AF090195.1 , Bases 1233-2733; Accession AF090195.1 Bases 1233-2733).
  • GABAergic neuronal cells e.g., G
  • the promoter is one that will not induce expression in other cells located near the site of administration.
  • the promoter can be said to be specific to have GABAergic neuronal cell specific expression.
  • the promoter will not induce expression in other cells that might be transduced.
  • tissue-specific promoter one identifies a protein which is expressed only (or primarily) in that tissue, and then isolates the gene encoding that protein.
  • the gene may be a normal cellular gene, or a viral gene of a virus, which infects that cell).
  • the promoter of that gene is likely to retain the desired tissue-specific activity when linked to another gene.
  • tissue specificity of a promoter may be associated with a particular genetic element, which may be modified, or transferred into a second promoter.
  • Delivery can be accomplished by any standard means for administering rAAV. For example, by simply contacting the rAAV, optionally contained in a desired liquid such as tissue culture medium, or a buffered saline solution, with the target cell.
  • the rAAV can be allowed to remain in contact with the target cell for any desired length of time, and typically the rAAV is administered and allowed to remain for a time sufficient to effectively transduce the target cell.
  • the rAAV may be delivered by any suitable method. Preferably delivery is via direct injection of the virus. In one embodiment the rAAV is directly administered to a region of the brain of the subject where the focal brain seizures occur. In another embodiment, the rAAV is directly administered into the hippocampus.
  • a “therapeutically effective amount” will fall in a relatively broad range that can be determined through clinical trials. For example, for in vivo injection. More importantly is the number of genomic particles (gp) administered to the hippocampus or region where the focal seizures occur.
  • the rAAV is administered such that 10 9 to 10 14 gp, more preferably 10 10 to 10 12 gp, and most preferably around 10 11 gp are administered to the region.
  • the rAAV is preferably administered by stereotaxic microinjection.
  • patients will have the stereotactic frame base fixed in place (screwed into the skull).
  • the brain with stereotactic frame base (MRI-compatible with fiducial markings) will be imaged using high resolution MRI.
  • the MRI images will then be transferred to a computer which runs stereotactic software.
  • a series of coronal, sagittal and axial images will be used to determine the target (site of rAAV vector injection) and trajectory.
  • the software directly translates the trajectory into 3 dimensional coordinates appropriate for the stereotactic frame.
  • Burr holes are drilled above the entry site and the stereotactic apparatus positioned with the needle implanted at the given depth.
  • the rAAV vector will then be injected at the target sites. Since the rAAV vector will integrate into the target cells, rather than producing viral particles, the subsequent spread of the vector will be minor, and mainly a function of passive diffusion from the site of injection, prior to integration.
  • the degree of diffusion may be controlled by adjusting the ratio of vector to fluid carrier.
  • the rAAV vectors are designed to target GABAergic neurons in the subject's brain, more particularly in the region of the brain where the seizures are occurring.
  • the rAAV can be associated with a homing agent that binds specifically to a surface receptor of the cell.
  • the vector may be conjugated to a ligand which is specific to GABAergic neurons cells.
  • the conjugation may be covalent, e.g., a crosslinking agent such as glutaraldehyde, or noncovalent, e.g., the binding of an avidinated ligand to a biotinylated vector.
  • Another form of covalent conjugation is provided by engineering the helper virus used to prepare the vector stock so that one of the encoded coat proteins is a chimera of a native AAV coat protein and a peptide or protein ligand, such that the ligand is exposed on the surface.
  • transduction denotes the delivery of a DNA molecule to a recipient cell either in vivo or in vitro, via rAAV, preferably, as used herein, delivery of EAAT3 to GABAergic neuronal cells.
  • compositions preferably comprise the recombinant adeno-associated viral vector (rAAV) designed to treat brain seizures, as described herein, and a pharmaceutically acceptable carrier.
  • rAAV adeno-associated viral vector
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which an active ingredient is administered. Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the transfer virus is directed.
  • one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline).
  • buffering solutions e.g., phosphate buffered saline.
  • Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water.
  • the selection of the carrier is not a limitation of the present invention.
  • compositions of the invention may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers.
  • suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
  • Suitable chemical stabilizers include gelatin and albumin.
  • Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • Preferred excipients confer a protective effect on the rAAV such that loss of rAAV, as well as the loss of transduceability resulting from formulation procedures, packaging, storage, transport, and the like, is minimized.
  • Plasmid Design and Virus Preparation cDNA was produced from rat cortical brain tissue using the Cells-to cDNA Il kit (Ambion, Austin, TX) and used as template for PCR amplification. Primers were designed for amplification of the rat GAD65 promoter sequence (Skak, Gene 236:231 -241 (1999)).
  • the upstream primer contained a Kpnl restriction site: ⁇ '-GGTACCGGCGCTCCGCAG-S' (SEQ ID NO:1 ) and the downstream primer containing a BcM restriction site: 5'- TGATCAGGGTTCTGCTAGTCTGG-3' (SEQ ID NO:3).
  • Amplified products were cloned into the TOPO Blunt PCR cloning plasmid (Invitrogen, Carlsbad, CA) and fragments confirmed by double stranded sequence analysis.
  • the GAD65 promoter sequence was subcloned into the Kpnl and BamHI sites of the pAM-hrGFP-WPRE vector.
  • Two additional GAD65 plasmids were designed with the hrGFP sequence replaced by either the rEAAT3 sequence tagged with a flag epitope (DYKDDDK) at the 5' end (flag-rEAAT3), or the rEAAT3 in the antisense orientation (rEAAT3/AS).
  • Rat cDNA was used to PCR amplify the flag-rEAAT3 sequence with an upstream primer containing an Xhol restriction site and the flag epitope:5'- CTCGAGATGGATTATAAAGATGACGATGACAAATGTATGGGGAAG CCCACGAG-3' (SEQ ID NO:3) and a downstream primer containing a Hindlll restriction site: ⁇ '-AAGCTTAGGCATCTAAGGCCAGGC-S' (SEQ ID NO:4). All constructs contained the 3' Woodchuck hepatitis virus post-transcriptional regulation element (WPRE).
  • WPRE Woodchuck hepatitis virus post-transcriptional regulation element
  • the WPRE construct has been shown to enhance transgene expression (Loeb, Hum Gene Ther 10:2295-2305 (1999); Paterna, Gene Ther 7:1304-131 1 (2000); Hlavaty, Virology 341 :1 -1 1 (2005)).
  • the GAD65 promoter and expression cassettes are flanked by rAAV serotype 2 (rAAV2) inverted terminal repeats.
  • rAAV2 rAAV2
  • Prior studies have shown chimeric rAAV at select serotype ratios increases overall rAAV mediated transduction (Rabinowitz, J Virol 78:4421 -4432 (2004)). For this study, we used a 3:1 ratio of rAAV serotype 1 to serotype 2.
  • the three pAM-GAD65 plasmids, containing the hrGFP, rEAAT3/AS and flag-rEAAT3 expression cassettes, and one cassette containing the CAG promoter (pAM-CAG-hEAAT3) were used in the production of recombinant rAAV.
  • HEK293T cells cultures were transfected with the pAM-GAD65 and pAM-CAG constructs using Polyfect Transfection Reagent (Qiagen, Valencia, CA) (Selkirk, Eur J Neurosci 21 : 2291 -2296 (2005)).
  • the plasmids used in the transfection were pF ⁇ 6 (adenoviral helper plasmid), pH21 (cap gene for rAAV serotype 1 and rep gene for serotype 2) and the rAAV plasmid containing the CAG promoter upstream from the hEAAT3 sequence.
  • plasmids were used in the transfection were pF ⁇ 6' pH21 , pRVI (cap and rep genes for rAAV serotype 2), and the rAAV plasmid containing the GAD65 promoter upstream from the flag-rEAAT3, rEAAT3/AS, or hrGFP gene sequence, flanked by rAAV2 inverted terminal repeats.
  • Virus was isolated from HEK293T cells through repeated freeze-thaw cycles, incubation for 30 minutes at 37 0 C with 5OU benzonase (Novagen, Madison, Wl) and 0.5% sodium deoxycholate, briefly sonicated and further purified by iodixonol density gradient centrifugation as previously reported (Zolotukhin, Gene Ther 6:973-985 (1999)).
  • the titer (genomic particles/ml (gp/ml)) of the final virus isolate was determined by quantitative real time- polymerase chain reaction (RT-PCR) using an ABI Prism 7700 with primer and probe sets specific for the cis-acting WPRE enhancer sequence.
  • HC2S2 cells are an immortalized rat hippocampal GABAergic cell line (kindly provided by Dr. Fred H. Gage, SaIk Institute, La JoIIa, CA) and were cultured as previously reported (Hoshimaru, Proc Natl Acad Sci USA 93:1518- 1523 (1996); Asahi, J Neurosci Res 52:699-708 (1998)).
  • D M E IWF- 12 50:50
  • glutamine N2 supplement
  • bFGF 20ng/ml
  • amphotercin B in flasks coated with poly-L-ornithine and mouse laminin
  • D M E IWF- 12 50:50
  • glutamine N2 supplement
  • bFGF 20ng/ml
  • amphotercin B in flasks coated with poly-L-ornithine and mouse laminin
  • cells were cultured on coated 12 well plates at 1 .0 x 10 4 cells/well in differentiation media (D M E IWF- 12 (50:50) containing glutamine, N2 supplement (Invitrogen, Carlsbad, CA), bFGF at 2ng/ml, amphotercin B and 20 ⁇ g/ml tetracyline).
  • Tetracycline was added daily to cell culture media at 20 ⁇ g/ml. After 3 days, cells stopped dividing and began to send out neuronal processes. HC2S2 cells were transduced with rAAV1 -CAG-hEAAT3 on day 3 in culture with 5.0 x 10 5 gp/cell. Cells were maintained in culture media, which was changed every other day for 7 days.
  • [ 3 H]D-Aspartic-acid (PerkinElmer, Boston, MA) uptake was used to assess functional hEAAT3 activity. Seven days post transduction with rAAV1 - CAG-hEAAT3, differentiation media was removed and HC2S2 cells were gently rinsed with pre-warmed sodium-containing HEPES buffer (2OmM HEPES, 120 mM NaCI, 5mM KCI, 1 mM KH 2 PO 4 , 2mM CaCI 2 , 1 OmM D- glucos ⁇ , pH 7.4) or a sodium-free HEPES Buffer (2OmM HEPES, 120 mM choline, 5mM KCI, 1 mM KH 2 PO 4 , 2mM CaCI 2 , 1 OmM D-glucose, pH 7.4).
  • pre-warmed sodium-containing HEPES buffer 2OmM HEPES, 120 mM NaCI, 5mM KCI, 1 mM KH 2 PO 4 , 2mM
  • HPLC analysis was performed to evaluate the total intracellular concentrations of specific amino acids in control or rAAV1 -CAG-hEAAT3 transduced HC2S2 cultures.
  • Cells were lysed in 0.1 N perchloric acid solution, centrifuged at 14,00Og for 10 minutes at 4 0 C and supernatant was retained. Protein concentrations were determined using the Bio-Rad DC microplate protein assay (Hercules, CA). Samples (10 ⁇ l_) were derivatized with o-phtaldialdehyde (OPA) solution (90 ⁇ l_) and 40 ⁇ l of the derivatized sample was loaded for HPLC analysis.
  • OPA o-phtaldialdehyde
  • Detection of amino acid content was performed by HPLC using an Amersham Bioscience AKTA purifier system (GE Healthcare, Piscataway, NJ) equipped with a Spherisord 5 ⁇ M ODS1 reverse phase column (4.6x250mm, Waters Corporation, Tokyo Japan). Amino acid levels were quantified by fluorescence detection at 440 nm (emission) and 330nm (excitation). Mobile phase A was composed of 0.03M sodium acetate and 1% tetrahydrofuran. Mobile phase B was compose of 0.02M sodium acetate in 80% acetonitrile. Amino acids were separated using a linear increase in concentration of mobile phase B from 0% to 40% over 40 minutes at a flow rate of 0.8 ml/min. Retention time for GABA, glutamate and glutamine (Sigma, St. Louis, MO) were 29, 8, and 23 minutes respectively as confirmed by amino acid standards. Specific concentrations were determined through analysis of amino acid standards over an applicable range of concentrations.
  • the three rAAV1/2-GAD65p vectors (hrGFP, rEAAT3/AS, and flag-rEAAT3, 1 x 10 13 gp/ml) were diluted 1 :1 with 25% mannitol (American Regent Inc., Shirley, NY) and 8 ⁇ l was delivered to each hippocampus at a rate of 0.5 ⁇ l/min.
  • pilocarpine 100mg/ml, BioChemika, Buchs, Switzerland
  • atropine 100mg/ml, Sigma, St. Louis, MO
  • IP intraparitoneal
  • Pilocarpine solution was delivered I. P. at a concentration of 300mg/kg body weight.
  • Fluoro-Jade B Histo-Chem, Inc., Jefferson, AR
  • Fluoro-Jade B was used to evaluate the level of seizure mediated damage. Tissues were processed as previously published (Wang, J Spinal Cord Med 23:31 -39 (2000)). Briefly, cryosectioned tissue was incubated in 1 % sodium hydroxide and 80% ethanol solution for 5 minutes, rinsed in 70% ethanol for 2 minutes, and rinsed in distilled water for an additional 2 minutes. Tissue was then incubated with gentle agitation in 0.06% potassium permanganate for 10 minutes, rinsed with distilled water and incubated in 0.0004% FluoroJade B working solution for 20 minutes.
  • Tissues were rinsed with distilled water and allowed to dry in a 37 0 C incubator over-night. Slides were immersed in xylene for 1 minute and coverslipped with DPX. Tissue was imaged using an Olympus IMT-2 inverted fluorescent microscope attached to an Olympus digital camera (Olympus, Melville, NY) and captured using Magnafire SP imaging software package (Optronics, Goleta, CA). Total fluorescent intensity per region of interest was determined using Image Pro Plus software (MediaCybernetics, Silver Springs, MD).
  • HC2S2 cells are transformed rat hippocampal neurons under the control of the tet operon. Under normal culture conditions the cells grow and exhibit characteristics of a transformed cell line. However, in the presence of tetracycline, HC2S2 cells stop dividing and exhibit characteristics of differentiated adult rat hippocampal GABAergic neurons.
  • GAD65 is localized to GABAergic.
  • a robust patter of GFP expression was observed throughout the CA1 -CA3 and hilus regions, which showed strong colocalization with GAD65 ( Figure 4).
  • rEAAT3 gene sequence was subcloned into this same rAAV vector in both the sense and antisense orientation to facilitate the overexpression and knock down of rEAAT3 in GABAergic neurons.
  • rEAAT3 is normally expressed on inhibitory presynaptic terminals and co-localizes with GAD65 (Conti, Cereb Cortex 8:108-1 16 (1998)).
  • GAD65 Conti, Cereb Cortex 8:108-1 16 (1998).
  • Adult male Sprague-Dawley rats were injected with rAAV vectors carrying either the rEAAT3 gene in the antisense orientation (rEAAT3/AS) or in the sense orientation fused with a flag epitope tag at the N-terminus (flag-rEAAT3).
  • rEAAT3/AS antisense orientation
  • flag-rEAAT3 flag epitope tag at the N-terminus
  • Brain tissue transduced with rAAV1 /2-GAD65- Flag-rEAAT3 was dual stained with antibodies against GAD65 and the flag epitope. Again, robust expression of flag-rEAAT3 was detected throughout the CA1 -CA3 region with strong colocalization with GAD65 staining ( Figure 5). Images taken of tissue transduced with rAAV1/2-GAD65-rEAAT3/AS did not reveal detectable differences in rEAAT3 expression (data not shown). Due to the limited sensitivity of standard immunohistochemical techniques, knock down of this protein could not be efficiently detected. This may be due to the antisense construct knocking down, but not completely knocking out rEAAT3 expression within inhibitory neurons. In addition, rEAAT3 present in postsynaptic terminals of glutamatergic pyramidal neurons would not be altered and would be detected in immunohistochemical staining.
  • Virus injected groups and non-transduced controls were monitored for progression through distinct behavioral stages leading to status epilepticus (SE) after receiving IP injections of pilocarpine (300mg/kg) (Racine, Electroencephalogr Clin Neurophysiol 32:295-299 (1972a); Racine, Electroencephalogr Clin Neurophysiol 32:281 -294 (1972b); Sperk, Prog Neurobiol 42:1 -32 (1994)).
  • SE status epilepticus
  • EAAT3 By altering the expression of EAAT3 we were able to change the threshold of seizure susceptibility, severity, duration, frequency and the rate of progression to SE.
  • Glutamate transport from extracellular to intracellular spaces is one of the primary mechanisms for preventing excitotoxicity and reducing neuronal damage caused by epilepsy. While various types of glutamate transporters are found in both neuronal and glial synaptic terminals, only excitatory amino acid transporter 3 (EAAT3) is uniquely located in the presynaptic terminals of inhibitory GABAergic neurons. Rats with significantly reduced EAAT3 activity have reduced GABA synthesis, develop EEG seizures correlated with behavioral freezing episodes, limbic hyperexcitability and decreased tonic inhibition (Sepkuty, J. Neurosci, 22:6372-6379 (2002)).
  • EAAT3 excitatory amino acid transporter 3
  • a bipolar depth probe composed of two twisted wires and a guide cannula port (Plastics One, Roanoke, VA) were implanted in the hippocampus of male Sprague Dawley rats (300-45Og) (Fig. 9).
  • a surface electrode placed in the skull and in contact with the undisrupted dura served as a ground. All three electrodes were collected into a 363 Plastics One pedestal and cemented to the skull of the animal. 300ng of kanic acid (KA) was delivered directly into the hippocampus to establish spontaneous but clinically unapparent seizures. Rats then underwent weekly 24-hour EEG recordings that allowed unrestricted movement while recording (Fig. 10).
  • an AAV vector was directly delivered into the hippocampus.
  • the vector included the GAD65 promoter that ensured selective expression of either an EAAT3 expression cassette or a null expression cassette in only GABAergic neurons.
  • Weekly 24-hr recordings were continued for four weeks after delivery of the virus (Fig. 1 1 ).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des outils thérapeutiques et des méthodes utiles dans le traitement d’un sujet souffrant de convulsions cérébrales. Les méthodes comprennent l’étape consistant à augmenter suffisamment l’expression du transporteur d’acides aminés excitatoires de type 3 (EAAT3) dans les neurones GABAergiques localisés dans le cerveau du sujet pour réduire la gravité, la fréquence et/ou la durée des convulsions cérébrales. Les outils thérapeutiques comprennent un vecteur viral adéno-associé recombinant (rAAV) composé d’ITR d’AAV en 5' et 3' flanquant le gène du transporteur d’acides aminés excitatoires de type 3 (EAAT3) qui est lié de manière fonctionnelle à un promoteur approprié pour l’expression de l’EAAT3 dans les neurones GABAergiques de l’hippocampe.
PCT/US2009/058899 2008-09-29 2009-09-29 Vecteurs et méthodes de traitement des convulsions cérébrales WO2010037143A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US10109408P 2008-09-29 2008-09-29
US61/101,094 2008-09-29

Publications (1)

Publication Number Publication Date
WO2010037143A1 true WO2010037143A1 (fr) 2010-04-01

Family

ID=42060155

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2009/058899 WO2010037143A1 (fr) 2008-09-29 2009-09-29 Vecteurs et méthodes de traitement des convulsions cérébrales

Country Status (1)

Country Link
WO (1) WO2010037143A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017122789A1 (fr) * 2016-01-15 2017-07-20 学校法人自治医科大学 Virion viral adéno-associé pour son utilisation dans le traitement de l'épilepsie
US10287607B2 (en) 2017-04-03 2019-05-14 Encoded Therapeutics, Inc. Tissue selective transgene expression
CN112640847A (zh) * 2020-12-30 2021-04-13 重庆医科大学附属第一医院 一种内源性癫痫发作动物模型及其构建方法
CN117122688A (zh) * 2023-08-31 2023-11-28 中国科学院脑科学与智能技术卓越创新中心 作用于前脑兴奋性神经元的prrt2及其上调剂的应用
US12083188B2 (en) 2017-12-01 2024-09-10 Encoded Therapeutics, Inc. Engineered DNA binding proteins

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020098473A1 (en) * 2000-07-25 2002-07-25 Edwards Robert H. Novel glutamate transporters
US6458571B1 (en) * 1993-10-20 2002-10-01 Oregon Health & Science University Excitatory amino acid transporter 3, EAAT3, polypeptides
US20050025746A1 (en) * 2000-05-23 2005-02-03 Neurologix, Inc. Glutamic acid decarboxylase (GAD) based delivery systems
US20070116686A1 (en) * 2002-12-18 2007-05-24 Nobuaki Tamamaki Method for separating precursor cells producing gabaergic neuron alone
US20070259825A1 (en) * 2004-08-31 2007-11-08 Johns Hopkins University Transgenic Mammals and Cell Lines for the Identification of Glutamate Transporter Modulators
US20080057539A1 (en) * 1999-10-23 2008-03-06 The Johns Hopkins University Glutamate transporter associated proteins and methods of use thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6458571B1 (en) * 1993-10-20 2002-10-01 Oregon Health & Science University Excitatory amino acid transporter 3, EAAT3, polypeptides
US20080057539A1 (en) * 1999-10-23 2008-03-06 The Johns Hopkins University Glutamate transporter associated proteins and methods of use thereof
US20050025746A1 (en) * 2000-05-23 2005-02-03 Neurologix, Inc. Glutamic acid decarboxylase (GAD) based delivery systems
US20020098473A1 (en) * 2000-07-25 2002-07-25 Edwards Robert H. Novel glutamate transporters
US20070116686A1 (en) * 2002-12-18 2007-05-24 Nobuaki Tamamaki Method for separating precursor cells producing gabaergic neuron alone
US20070259825A1 (en) * 2004-08-31 2007-11-08 Johns Hopkins University Transgenic Mammals and Cell Lines for the Identification of Glutamate Transporter Modulators

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017122789A1 (fr) * 2016-01-15 2017-07-20 学校法人自治医科大学 Virion viral adéno-associé pour son utilisation dans le traitement de l'épilepsie
JPWO2017122789A1 (ja) * 2016-01-15 2018-11-29 学校法人自治医科大学 てんかん治療のためのアデノ随伴ウイルスビリオン
US12263231B2 (en) 2016-01-15 2025-04-01 Jichi Medical University Adeno-associated virus virions for treatment of epilepsy
US10287607B2 (en) 2017-04-03 2019-05-14 Encoded Therapeutics, Inc. Tissue selective transgene expression
US10287608B2 (en) 2017-04-03 2019-05-14 Encoded Therapeutics, Inc. Tissue selective transgene expression
US10519465B2 (en) 2017-04-03 2019-12-31 Encoded Therapeutics, Inc. Tissue selective transgene expression
US12083188B2 (en) 2017-12-01 2024-09-10 Encoded Therapeutics, Inc. Engineered DNA binding proteins
CN112640847A (zh) * 2020-12-30 2021-04-13 重庆医科大学附属第一医院 一种内源性癫痫发作动物模型及其构建方法
CN117122688A (zh) * 2023-08-31 2023-11-28 中国科学院脑科学与智能技术卓越创新中心 作用于前脑兴奋性神经元的prrt2及其上调剂的应用
CN117122688B (zh) * 2023-08-31 2024-06-04 中国科学院脑科学与智能技术卓越创新中心 作用于前脑兴奋性神经元的prrt2及其上调剂的应用

Similar Documents

Publication Publication Date Title
US12281321B2 (en) Frataxin expression constructs having engineered promoters and methods of use thereof
JP2023052805A (ja) Ａａｖを送達するための組成物および方法
JP2022123145A (ja) 認知保護を提供しつつ疾患の発症及び進行を遅らせるための遺伝子治療を用いた神経変性疾患の治療方法
AU2019310459A1 (en) Systems and methods for producing gene therapy formulations
EP4124658A2 (fr) Traitement de la sclérose latérale amyotrophique (sla)
WO2020077165A1 (fr) Compositions et procédés pour l'administration d'aav
AU2018261003A2 (en) Compositions and methods of treating Huntington's Disease
EP3254702B1 (fr) Virus aav/xbp1s-ha, procédé de traitement génétique et utilisation de celui-ci pour l'optimisation et l'amélioration des capacités cognitives, de la mémoire et de l'apprentissage
KR20220004696A (ko) 폼페병의 치료에 유용한 조성물
AU2008316398A1 (en) Splice variants of GDNF and uses thereof
WO2021247995A2 (fr) Compositions et méthodes de traitement de la douleur neuropathique
WO2010037143A1 (fr) Vecteurs et méthodes de traitement des convulsions cérébrales
KR20230123460A (ko) Neurod1 벡터
TW202449167A (zh) 用於治療肌肉萎縮性脊髓側索硬化症之組成物及方法
CN110461367A (zh) 用于缓解或治疗疼痛的组合物
US20230126157A1 (en) Mirna-485 inhibitor for gene upregulation
US20240390521A1 (en) Chimeric modified ion channels and uses thereof for treatment of trigeminal nerve disorders
KR20230123925A (ko) Neurod1 및 dlx2 벡터
CA3192936A1 (fr) Compositions et procedes d'inhibition de l'agregation d'alpha-synucleine
TW202509227A (zh) 用於治療與cdkl5缺乏相關之病症之組合物及方法
KR20220003566A (ko) 신규한 유형의 효소 조성물
WO2023143366A1 (fr) Variant de virus adéno-associé et son application dans le traitement de maladies
WO2024229037A1 (fr) Capsides et procédés de ciblage de neurones
WO2025038955A9 (fr) Systèmes et procédés pour améliorer la sécurité d'une thérapie génique médiée par aav à intéine divisée
WO2024178364A1 (fr) Thérapie génique pour le traitement de la douleur

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09817063

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09817063

Country of ref document: EP

Kind code of ref document: A1