WO2023156530A1 - Gene therapy for neurodegenerative diseases - Google Patents

Abstract

The present invention relates to compositions and methods for gene. The invention also provides novel vectors and methods for delivering a cytoprotective protein in the central nervous system or in neurosensory organs. The invention also relates to the use of these vectors in therapy, especially in the treatment of a neurodegenerative diseases, lysosomal storage diseases, or diseases related to a loss of vision or hearing. Furthermore, the invention relates to a kits and reagents for use in these methods.

Description

GENE THERAPY FOR NEURODEGENERATIVE DISEASES

FIELD OF THE INVENTION

The present invention relates to compositions and methods for gene therapy. The invention also provides novel vectors and methods for delivering a cytoprotective protein in the central nervous system or in neurosensory organs. The invention also relates to the use of these vectors in therapy, especially in the treatment of neurodegenerative diseases, lysosomal storage diseases, or diseases related to a loss of vision or hearing. Furthermore, the invention relates to a kits and reagents for use in these methods.

BACKGROUND OF THE INVENTION

The brain is highly sensitive to changes in redox status. Maintaining redox homeostasis in the brain is critical for the prevention of oxidative damage due to oxidative stress (OS) and nitrosative stress (NS). Downstream markers of OS/NS, in particular lipid peroxidation, have been identified in neurodegenerative diseases (NDD) such as Parkinson’s disease (PD), Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). Protein carbonyls, representing protein oxidation, are also present in the substantia nigra of subject affected with PD, AD or ALS. In addition, and associated with neuroinflammation, superoxide anions generated by the NADPH oxidase system combine with nitric oxide to produce the highly reactive peroxynitrite. Nitration of proteins are thought to contribute to protein misfolding and subsequent formation of protein aggregates, such as alpha-synuclein in PD, betaamyloid plaques in AD, huntingtin in Huntington’s disease (HD) and superoxide dismutase 1 (SOD1) and TAR DNA-binding protein 43 (TDP-43) in ALS. Sustained OS and OD are known to be highly deleterious to the neuronal cells and are responsible, at least in part, for the degeneration or death of neurons that contributes to NDD pathogenesis.

A critical pathway in this regard is the nuclear factor erythroid 2-related factor 2 (NRF2)-antioxidant response element (ARE) pathway. NRF2 is a ubiquitously expressed transcription factor that activates the transcription of several cytoprotective genes implicated in protection from cancer and NDD. NRF2 acts by binding to the ARE enhancer element located in the regulatory regions of genes involved in cellular protection from oxidants, including for example glutamate-cysteine ligase modifier subunit (GCLM), NAD(P)H: quinone oxidoreductase 1 (NQOl), heme oxygenase 1 (HM0X1), and glutathione peroxidase (GPX). Under basal conditions, NRF2 expression is very low, due to its interaction in the cytoplasm with the repressor protein Kelch-like ECH-associated protein 1 (KEAP1), which targets it for ubiquitin/proteasome degradation. In response to oxidative stress (such as for instance in NDD), KEAP1 dissociates from NRF2, which then moves into the nucleus to bind to the ARE regulatory elements and activate the cellular antioxidant response by driving expression of detoxifying and antioxidant genes.

Even though NRF2 has been proposed as a therapeutic target for neurodegenerative diseases (Jimenez- Villegas et al., 2021, Free Radical Biology and Medicine, 173 : 125-141; Johnson & Johnson, 2015, Free Radic Biol Med. 88(Pt B): 253-267), there are currently no approved disease-modifying drugs for any of these diseases that were designed based on their ability to activate NRF2. Even though the multiple sclerosis drug dimethylfumarate (DMF) is a Nrf2 activator, alternative mechanisms of action have been proposed for its activity in multiple sclerosis. In addition, electrophiles such as DMF produce severe systemic side effects, in part due to non-specific S-alkylation of cysteine thiols and resulting depletion of glutathione. The search for non-covalent, brain- and cell -penetrant small molecule activators of NRF2 has so far not yielded any clinically active drug. One of the major reasons for this lack of success is that non-covalent NRF2 activators should disrupt the interaction between NRF2 and KEAP1, which is very difficult to achieve because of the extended surface of this protein-protein interface.

Genetic disruption of Nrf2 aggravates neuronal death in animal models of Huntington’s (HD) and Parkinson’s (PD) diseases. In preclinical in vitro and animal studies, it has been shown that small molecule NRF2 activators (tool compounds) have beneficial effects in NDD animal models including HD, PD and AD. The KEAP1-NRF2 pathway is altered in animal models of ALS and postmortem tissues from ALS patients. NRF2 is also thought to play a role in hearing loss (Li et al., 2021, Frontiers in Pharmacology, 12, Article 620921).

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, known as Lou Gehrig’s disease, with the progressive loss of both upper and lower motor neurons that causes weakness of skeletal muscles, leading to death due to respiratory failure within 2-5 years from onset. The disease mainly occurs in adults, typically between 50 and 60 years and remains incurable. The symptoms typically start in the muscles of arms or legs (spinal onset) or bulbary (bulbar onset). To date, ALS modifying therapy is limited to riluzole (CAS number 1744-22-5), a glutamate release inhibitor, and edavarone, a ROS scavenger. Both drugs produce only very modest effects on survival or disease progression.

Despite decades of research, the details of pathogenesis of ALS remain unclear. Up to 50% of patients with ALS develop cognitive and behavioral impairment, and about 13% of patients have concomitant frontotemporal dementia (FTLD). According to whether there is a family history of ALS, the disease can be roughly divided into two types: familial (5-10%) and sporadic (90-95%). Familial ALS cases show a high heritability and this has enabled the identification of several genetic triggers, of which mutations in superoxide dismutase 1 (SOD1), FUS RNA binding protein (FUS), TAR DNA binding protein (TARDBP) and C9orf72-SMCR8 complex subunit (C9orf72) are the most frequent. The discovery of hexanucleotide GGGGCC repeat expansions in C9orf72 as the major genetic cause of ALS and FTLD proves that these disorders can be extremes on the phenotypic spectrum of a single disease. While such advances have contributed to current understanding of the causes of most cases of familial ALS and their underlying pathophysiological consequences, they only explain a small fraction of sporadic ALS. The etiology of most sporadic ALS cases remains unexplained. In postmortem brains of patients with ALS, the markers of OS and OD are increased. Increased 3- nitrotyrosine levels, a marker of oxidative damage mediated by peroxynitrite, were detected in patients with sporadic as well as familial ALS (Beal et al., Ann. Neurol. 1997, 42, 644-654). The levels of lipid peroxidation, including 4-hydroxynonenal (4-HNE) and 8-hydroxy-20-deoxyguanosine (8-OHdG), also increased in the serum and cerebrospinal fluid (CSF) of patients with ALS (Simpson et al., Neurology, 2004, 62: 1758-1765). Therefore, the regulation of cellular ROS levels is likely important for the prevention and treatment of ALS.

Gene therapy, by correcting a genetic defect or by therapeutic protein delivery has appeared as a new and attractive option to treat ALS. Rodent models in which mutant versions of superoxide dismutase- 1 (SOD1) are overexpressed recapitulate hallmark signs of ALS in patients. Thomsen et al. showed that the knockdown of mutant SOD1 in only the motor cortex of pre symptomatic SOD1^G93A rats through targeted delivery of short hairpin RNA (shRNA) resulted in a significant delay of disease onset, expansion of lifespan, enhanced survival of spinal motor neurons, and maintenance of neuromuscular junctions (Thomsen et al., J. Neurosci., 2014, 34(47): 15587-15600). However, these encouraging results obtained in rats are restricted only to SOD1 ALS associated with SOD1 mutations (SOD1-ALS).

In humans, the safety profile of antisense medicine targeting superoxide dismutase 1 (SOD1) mRNA was first established in 2012 by administering antisense oligonucleotide ISIS 333611 intrathecally to patients diagnosed with SOD 1 -ALS (NTC01041222). Since then, no antisense medicine has been approved by the European Medicines Agency or the Food and Drug Administration. A phase I clinical trial is currently under process for patients diagnosed with C9orf72-ALS. The safety and tolerability of BIIB078 in adults with C9orf72-ALS is under evaluation in NTC03626012 clinical trial. The secondary objectives of this study are to evaluate the pharmacokinetic profile of BIIB078 and to evaluate its effects on clinical function. However, this study is limited to C9orf72-ALS, and will not be transposable to all types of ALS, in particular sporadic ALS.

In another therapeutic approach, growth factor overexpression was used to restore, protect and generate neurons and their functionality. Studies conducted with modified neural progenitor cells (NPC) and neural stem cells (NSC) showed growth factor overexpression and their transplantation improved motor neuron survival. Research conducted with NSC producing glial cell line-derived neurotrophic factor (GDNF) and insulin like growth factor 1 (IGF-1) resulted in improved motor neuron survival in the SOD1-ALS animal model, whereas NSC that express vascular endothelial growth factor (VEGF), neurotrophin-3 (NT-3), and brain derived neurotrophic factor (BDNF) did not elicit therapeutic effets (Gowing et al., Prog Brain Res, 2017, 230:99-132 and Park et al., Exp Mol Med, 2009, 41:487-500).

Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by mutation of the huntingtin gene. Expansion of a triplet CAG repeat leads to expansion of a polyglutamine tract in the huntingtin protein (HTT). This leads to early loss of striatal projection neurons. Mutant huntingtin (mHTT) is neurotoxic to neurons and astrocytes, through gain- and loss-of-fimction mechanisms affecting many cellular functions. The prevalence of HD is 3 to 12 cases per 100,000 people, with the most frequent age of onset in the thirties or forties. Patients develop involuntary movements, chorea, dystonia, cognitive impairment, behavioral abnormalities and personality changes which progress over time. Life expectancy is generally 15 to 20 years after symptom onset. Commonly used therapeutics are monoamine depletors, antipsychotics, antidepressants, and tranquilizers. However, these drugs cannot prevent the psychotic, cognitive, and behavioral dysfunctions associated with HD. In addition to this, their chronic use is limited by their long-term side effects.

Genetically-modified mouse models developed for the study of HD can be grouped into three broad categories based on their CAG repeat numbers, the size and species of origin (mouse or human) of the huntingtin protein, the promoters that drive expression of the HTT proteins, and their background strain. The first two categories aim to study evident/overt phenotypic endpoints; N-terminal transgenic animals carry the 5’ portion of the human HTT gene, which contains the CAG repeats, whereas full-length transgenic models carry the full-length HTT sequence and express full-length HTT protein containing expanded polyglutamine repeats. The third category are knock-in models in which the HD mutation is replicated by directly engineering CAG repeats of varying length into the mouse huntingtin (Htt) genomic locus.

According to several in vitro studies, NRF2 activation can play a protective role in the reduction of mHtt-induced toxicity, while in HD patients the initiation of the NRF2-ARE system in striatal cells in response to OS failed because of the concurrent activation of the autophagy pathway (Jin et al., 2013, PloS One, 8(3):e57932). Moreover, additional data have confirmed that Htt aggregation directly enhanced ROS generation promoting cell toxicity (Hands et al., 2011, J Biol Chem. 2011 286(52):44512-20). Furthermore, co-transfection ofNRF2 with mHtt in primary striatal neurons led to reduction of the mean lifetime of mHtt N-terminal fragments, and, subsequently, improvement of cell viability, suggesting that NRF2 is likely to decrease mHtt -toxicity by negatively affecting its aggregation (Tsvetkov et al., 2013, Nat Chem Biol., 9(9):586-92).

Another important aspect to be considered when contemplating direct delivery of Nrf2 to cells is its cytoprotective property. Because NRF2 is a cytoprotective protein, it could also protect cancerous cells from dying. Overexpression of Nrf2 could therefore lead to pro-carcinogenic effects, or to protection of cancer cells from chemotherapy (Jenkins and Gouge, 2021, Antioxidants, 10, 1030). It may therefore be important to restrict expression ofNRF2 to certain cell types, using cell-specific promoters. It is not known which cells are more susceptible than others to potential tumor-promoting effects of NRF2. The choice of specific AAV capsid, cellular promoter and biological activity of the Nrf2 transgene product may also be important in determining the pro-carcinogenic potential of AAV vectors expressing NRF2 or NRF2 mutants. Thus, there is a strong need for gene therapy, vectors and methods for treating both familial and sporadic ALS and other neurodegenerative diseases affecting the central nervous system, having good outcomes, efficacy and safety.

SUMMARY OF THE INVENTION

A gene therapy approach is provided herewith that mediates expression of a protein providing protection against oxidative stress. The inventors have developed novel vectors encoding a transcription factor that activates genes involved in cellular protection from oxidants. Long-term expression by recombinant adeno-associated virus (AAV) constructed with a cDNA that encodes a nuclear factor erythroid 2-related factor (NRF2) protein provides strong protection to inappropriate levels of oxidants. Furthermore, by disrupting the interaction of NRF2 with KEAP1, the inventors have developed vectors boosting the antioxidant response in central nervous system tissue.

More particularly, the inventors provide evidence of an improved effect of the NRF2 protein having a substitution T80G or T80L over wild-type NRF2 protein to activate ARE-mediated transcription, with a higher efficiency for the variant carrying the substitution T80G. This NRF2 variant would present the advantage to be less immunogenic than a NRF2 variant having a substitution T80A.

The present invention relates to a recombinant adeno-associated virus (AAV) comprising, in its genome, a polynucleotide encoding a NRF2 protein operably linked to a promoter, wherein the modified NRF2 protein lacks a functional Neh2 domain and wherein the modified NRF2 protein comprises an amino acid substitution T80G or T80L, by reference to SEQ ID NO: 2.

In some embodiments, the recombinant AAV has a tropism for neuronal and/or glial cells, or for a cell of neurosensory organs, such as retinal or inner hair cells.

In some embodiments, the recombinant AAV has a serotype selected from the group comprising or consisting of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh8, AAVrhlO, AAVrh20, AAVrh39, AAVRh74, AAVRHM4-1, AAVhu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 or AAVhu68, or mixtures thereof. In particular embodiments, the recombinant AAV has a AAVrhlO serotype.

In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the promoter is a tissue- or a cell-specific promoter. Optionally, the promoter is a brain-, neural-, eye-, auditory system tissue-, neuronal cell-, astrocyte-, retinal cell-, or inner hair cell-specific promoter. Optionally, the modified NRF2 protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9 or 16, or is a protein having at least 90%, preferably at least 95, 96, 97, 98 or 99% identity to SEQ ID NO: 9 or 16 with the proviso that amino acid at position 80 is G or L.

In a particular aspect, the modified NRF2 protein comprises an amino acid substitution T80G by reference to SEQ ID NO: 2.

The present invention also relates to a method for producing a recombinant AAV comprising: a) culturing cells that have been transfected with plasmids described herein, especially with: i) a plasmid comprising a recombinant AAV according to the present invention, and a polyA; ii) a plasmid comprising polynucleotide (s) encoding Rep and Cap proteins; and iii) a plasmid comprising adenovirus; and b) recovering recombinant AAV from the transfected cells.

Further aspects of the invention include a host cell transduced with a recombinant AAV described herein. Further aspects of the invention include any nucleic acid molecule comprising, or consisting essentially of the genome of a recombinant AAV described herein.

In a further aspect, the present invention relates to a composition comprising a recombinant AAV described herein and a pharmaceutical acceptable carrier. Optionally, the composition is formulated for intravenous, intraparenchymal, or cerebrospinal fluid delivery, more preferably for cerebrospinal fluid delivery. In some embodiments, the composition or pharmaceutical acceptable carrier is formulated for intravenous delivery. In some embodiments, the composition or pharmaceutical acceptable carrier is formulated for delivery to the central nervous system or to the sensory organs. In one particular embodiment, the composition or pharmaceutical acceptable carrier is formulated for intraparenchymal or cerebrospinal fluid delivery. In another particular embodiment, the composition or pharmaceutical acceptable carrier is formulated for delivery to the ear. In still another particular embodiment, the composition or pharmaceutical acceptable carrier is formulated for intraocular delivery.

The invention also relates to a recombinant AAV described herein or a composition comprising it for use in the treatment of a neurodegenerative disease or of a lysosomal storage disease. In some embodiments, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS). In some embodiments, the neurodegenerative disease is Huntington disease (HD). In some embodiments, the lysosomal storage disease is a sphingolipidosis, an oligosaccharidosis, a gangliosidosis, a mucopolysaccharidosis, a neuronal ceroid lipofuscinosis, a sialic acid disorder, or a mucolipidosis. In some embodiments, the lysosomal storage disease is Niemann-Pick disease. In some embodiments, the lysosomal storage disease is Pompe disease. In some embodiments, the recombinant AAV or composition described herein is administered intrathecally. The invention also relates to a recombinant AAV described herein or a composition comprising it for use in the treatment of a disease or disorder that causes a total or partial loss of vision or hearing.

The invention also relates to a method for treating a neurodegenerative disease, in particular amyotrophic lateral sclerosis (ALS) or Huntington disease; or a lysosomal storage disease, in particular Niemann-Pick disease or Pompe disease, in a subject in need thereof, comprising administering to the subject an effective amount of a composition or recombinant AAV described herein. The invention also relates to a method for preventing or treating total or partial loss of vision or hearing, in a subject in need thereof, comprising administering an effective amount of a composition comprising a recombinant AAV described herein.

The invention further relates to the use of a recombinant AAV described herein or a composition comprising it for the manufacture of a medicament for treating a neurodegenerative disease, in particular amyotrophic lateral sclerosis (ALS) or Huntington disease; or a lysosomal storage disease, in particular Niemann-Pick disease or Pompe disease; or a disease or disorder that causes a total or partial loss of vision or hearing.

In some embodiments of the above aspects and embodiments, the subject is a mammal, preferably a human.

In a further aspect, the present invention relates to an in vitro method for delivering a polynucleotide encoding a NRF2 protein as disclosed herein comprising: contacting in vitro a cell with a recombinant AAV described herein.

The present invention also relates to a kit comprising a recombinant AAV, a composition, or a nucleic acid as described herein. In some embodiments, the kit optionally comprises at least one device adapted for cerebrospinal fluid administration or for intraparenchymal administration, or suitable for intraocular or ear administration, and/or instructions for use. In some embodiments, the kit described herein is for use in the treatment of a neurodegenerative disease, in particular amyotrophic lateral sclerosis (ALS) or Huntington disease; or in the treatment a lysosomal storage disease, in particular Niemann-Pick disease or Pompe disease. The kits herein described may also be used in prevention or treatment of a disease or disorder that causes a total or partial loss of vision or hearing.

In addition, any of the methods and uses described herein may be used to increase expression of a NRF2 protein in a subject in need thereof. In some embodiments, the methods and uses described herein increase and/or restore cellular protection from oxidants in the cells of a subject in need thereof.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: schematic representation of a recombinant adeno-associated virus (rAAV) vector construct. Recombinant AAV vector construction is generated by replacing the wild type AAV genome, flanked by two inverted terminal repeats (ITRs), with a transgene expression cassette, consisting of a promoter, the transgene and a polyadenylation (poly A) sequence. The transgene expression cassette capacity is up to approximately 5kb.

Figure 2: schematic representation of the plasmids used for production of a recombinant adeno- associated virus (rAAV) vector construct. Recombinant AAV vector is produced by triple -plasmid- cotransfection into cells. This system comprises a transgene expressing plasmid containing an ITRs- flanked transgene cassette, a packaging plasmid that encodes the rep and cap genes of a specific AAV serotype (generally AAV2), and a helper plasmid that supplies the essential adenovirus helper genes (ex. Ela, Elb, E2a, E4, and VA RNA) to mediate AAV replication.

Figure 3: schematic representation of the expression cassette and flanking region inserted in a recombinant adeno-associated virus (AAV) genome. The designed expression cassette of the recombinant AAV comprises a promoter (ubiquitous, or cell type specific), the human nrf2 cDNA (wildtype or modified) and a polyA sequence for nuclear polyadenylation, flanked by two ITRs of AAV2.

Figure 4: schematic representation of a NRF2 protein and the mutant(s) thereof. Human NRF2 protein contains 605 amino acids (aa). It is composed of six highly conserved domains called NRF2- ECH homology (Neh) domains. Neh2 serves as the binding domain for Keapl with its low -affinity DLG and high affinity ETGE motifs, for its ubiquitination and proteasome-dependent degradation. Nehl is the binding site for small Maf proteins and antioxidant response element (ARE), responsible for the activation of ARE-containing gene expression. Neh3, Neh4 and Neh5 are transactivation domains for NRF2. Neh6 negatively controls Nrf2 stability. Neh7 interacts with Retinoid X Receptor Alpha (RXRa), a nuclear receptor responsible for suppression of Nrf2/ARE signaling pathway. The human Delta-Neh2 NRF2 lacks the Neh2 domain of the human NRF2 protein, comprising from (16-86 aa). Human T80G NRF2 variant contains a single amino acid substitution at position 80 of human NRF2 protein, from Threonine (T) to Glycine (G). Human T80L NRF2 mutant contains a single amino acid substitution at position 80 of human NRF2 protein, from Threonine (T) to Leucine (L).

Figure 5: NRF2 protein expression level. A) NRF2 protein level was detected with the Nano-Gio® HiBiT Dual-Luciferase® Reporter System, at 48 hours post-transfection. Error bar correspond to the SD of signal detection in quadruplicates. B) NRF2 protein expression was also detected with Nano- Glo® HiBiT Blotting System, at 48 hours post-transfection. UNT: Non-transfected negative control; Sulphoraphane: a chemical that stimulates NRF2-mediated signaling pathway, used at 5pM, as a control.

Figure 6: Transcriptional activation induced by NRF2 protein. The firefly luciferase was detected with Nano-Gio® HiBiT Dual-Luciferase® Reporter System kits, at 48 hours post-transfection. The firefly luciferase signal is expressed as fold change over UNT signal. UNT: Non-transfected negative control, showing the level firefly luciferase induced by endogenous NRF2; Sulphoraphane: a chemical that stimulates NRF2-mediated signaling pathway, used at 5pM, as a control. Error bars correspond to the SD of signal detection in quadruplicates.

Figure 7: The ARE-Luciferase activation efficiency of NRF2. The ratio is expressed as ARE-Firefly luciferase versus HiBiT signal. All the data were collected from the optimal experimental condition: transfection with Ipg NRF2 plasmids and analysed at 48hours post-transfection. Error bars correspond to SEM of 3 independent transfections, n=3. T test of tested plasmid versus WT NRF2: * P<0.5; ** P<0.01. Variations of signal detection from each transfection were also checked in triplicate.

Figure 8: Transcriptional activation of redox regulating genes by NRF2 expression. Error bars correspond to SEM of 3 independent transfections, n=3. Statistical significance between tested condition and UNT condition: *: p<0.05; **: p<0.01; ***: p<0.001 with T test.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to viral vectors expressing NRF2 and their uses for gene therapy. The invention is particularly suitable for treating conditions associated with oxidative stress, such as neurodegenerative diseases. Neurodegenerative diseases share many fundamental processes associated with progressive neuronal dysfunction and death. The underlying pathophysiology common to all forms of neurodegenerative disease seems to involve neuroinflammation, oxidative stress (OS) and nitrosative stress (NS). An imbalance between the production and elimination of reactive oxygen species (ROS) and superoxide radicals, which attacks lipids, protein and DNA, plays an important role in the progression of neuronal loss.

One mechanism by which cells respond to oxidative insults is through the antioxidant response element (ARE), a cis-acting enhancer sequence that regulates the transcription of many cytoprotective genes.

By reprogramming cellular redox homeostasis in neuronal cells, neurodegenerative diseases can be prevented, treated and/or cured regardless of the event(s) causing the disease.

The invention is based at least in part on the discovery that NRF2 expression can lead to successful reprogramming of cellular redox homeostasis in suitable target cells (such as for example neuronal and/or glial cells, retinal cells, or inner hair cells), thereby reducing the deleterious effects of oxidants. Such an effect can be used in the treatment of various conditions such as neurodegenerative diseases, lysosomal storage diseases; or vision or hearing disorders. The invention is also based on the discovery of modified versions of NRF2 protein with enhanced biological activity.

The invention thus relates to recombinant AAV encoding NRF2, their manufacture, compositions comprising the same, and their uses for gene therapy. The invention also relates to modified NRF2 proteins having improved properties. Definitions

The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms.

The indefinite articles “a” and “an”, as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one”.

The term “about” as used herein when referring to a measurable value such as for example an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length, temporal duration, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

A “recombinant adeno-associated virus (AAV)” is a vector derived from AAV, a small Dependoparvovirus with a single -stranded linear DNA genome, essentially lacking pathogenicity, which has been produced using recombinant methods. Recombinant AAVs (rAAVs) preferably contain one or more transgene of interest in their genome that are flanked by at least one, typically two, AAV inverted terminal repeat sequences (ITRs). A rAAV can be in any of a number of forms, including, but not limited to, free DNA, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, or encapsidated in a viral particle, particularly an AAV particle. rAAVs typically contain a recombinant AAV genome packaged in a capsid, preferably an AAV capsid.

An “AAV inverted terminal repeat (ITR)” sequence, a term well-understood in the art, is an approximately 145 -nucleotide sequence that is present at both termini of a native single -stranded AAV genome. The outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, leading to heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter regions of selfcomplementarity (designated A, A', B, B', C, C' and D regions), allowing intrastrand base-pairing to occur within this portion of the ITR.

The term “polynucleotide” or “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate-phosphodiester oligomer. In addition, a doublestranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.

The terms “polypeptide”, “polypeptide fragment”, “peptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full- length proteins and fragments thereof are encompassed by the definition. The terms also include postexpression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, a “polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

As used herein, the term “operably linked” refers to positioning of a regulatory region (e.g. a promoter) and a sequence to be transcribed in a nucleic acid so as to influence transcription or translation of such a sequence. For example, to bring a coding sequence under the control of a promoter, the translation initiation site of the translational reading frame of the polypeptide is typically positioned between one and about fifty nucleotides downstream of the promoter. A promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site or about 2,000 nucleotides upstream of the transcription start site. A promoter typically comprises at least a core (basal) promoter. A promoter also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). The choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression.

The term “promoter” and “promoter sequence” are used interchangeably and refer to a nucleic acid sequence or element capable of controlling the expression (e.g., transcription) of a coding sequence. In general, a coding sequence is located 3' to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be modified or composed of different elements derived from different promoters found in nature, or derive from native promoters or fragments thereof, or comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “ubiquitous promoters.” Promoters that cause a gene to be expressed mainly in a given cell type are commonly referred to as “cell-specific promoters” or “tissue-specific promoters”. Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as “inducible promoters” or “regulatable promoters”. “Constitutive promoters” are defined as promoters active in vivo in all circumstances.

A “cassette” or “gene cassette” or “expression cassette” refers to a polynucleotide sequence that encodes for one or more expression products, and contains the necessary cis-acting elements for expression of these products, which can be inserted into a vector at defined restriction sites.

The term “transgene” refers to a polynucleotide that contains a coding sequence encoding a gene product. The gene product may be a RNA, peptide or protein. In addition to the coding sequence for the gene product, the transgene may include or be associated with one or more elements to facilitate or enhance expression, such as a promoter, enhancer(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s) and/or other functional elements.

The term “transfection” refers to the uptake of polynucleotide by a cell, and a cell has been “transfected” when polynucleotide has been introduced inside the cell. A number of transfection techniques are well known in the art. See, e.g., Graham et al, Virology 52:456 (1973), Sambrook et al, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratories, New York (1989), Davis et al. , Basic Methods in Molecular Biology, Elsevier (1986), and Chu et al, Gene 13: 197 (1981).

The term “transduction” refers to the process by which a virus or a viral vector transfers a nucleic acid molecule into a recipient host cell. For example, transduction of a target cell by a rAAV particle leads to transfer of the rAAV genome contained in that particle into the transduced cell.

A “host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest. A host cell can be a prokaryotic or a eukaryotic cell that may be used as a recipient of an AAV helper construct, an AAV minigene plasmid, an accessory function vector, or other transfer DNA associated with the production of recombinant AAVs. The term includes the progeny of the original cell which has been transduced. Thus, a “host cell” as used herein may refer to a cell which has been transduced with an polynucleotide.

The terms “sequence identity”, “sequence having at least X% identity” and “sequence X% identical to” are used interchangeably to refer to the extent that sequences are identical on a nucleotide -by- nucleotide basis or an amino acid-by-amino acid basis over a window of comparison, preferably over the entire length of said sequences. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison (preferably the window being the full sequence), wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Preferably, sequence identity is determined over the entire length of a reference sequence.

Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement)).

Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1990), J. Mol. Biol. 215: 403-410 and Altschul et al., (1977) Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.

The degree of percent amino acid sequence identity can also be obtained by ClustalW analysis (version W 1.8) by counting the number of identical matches in the alignment and dividing such number of identical matches by the length of the reference sequence.

By “consists essentially of’ is intended that the protein consists of that sequence, but it may also include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, additions, deletions or a mixture thereof, preferably 1, 2, 3, 4, or 5 substitutions, additions, deletions or a mixture thereof. In particular, by “essentially consist in”, it may be intended that the peptide may include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids at the N and/or C-terminal end, preferably 1, 2, 3, 4, or 5 additional amino acids, and/or 1, 2 or 3 substitutions, deletions , additions, or a mixture thereof. Preferably, the number of substitutions, additions, deletions or a mixture thereof depends on the length of the sequence. For instance, the percentage of substitutions, deletions , additions, or a mixture thereof may be no more than 30%, preferably no more than 25, 20,

15, 10 or 5%.

The terms “genome particles (gp)”, “genome equivalents”, or “genome copies (gc)” are used interchangeably in reference to a viral titer, and refer to the number of virions containing the recombinant AAV DNA genome. The number of genome particles can be measured by known procedures, such as described for example, in Clark et al., Hum. Gene Then, 1999, 10: 1031-1039 or in Veldwijk et al., Mol. Then, 2002, 6:272-278.

The terms “viral genome (vg)” refer to the nucleic acid sequence(s) encapsulated in an AAV particle. An AAV genome typically comprises a nucleic acid molecule comprising an expression cassette and at least one AAV ITR.

The term “wild-type”, as used herein, refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally-occurring source. A wild-type gene or gene product (e.g., a polypeptide) is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene.

As used herein, a “modified form” or a “variant” polynucleotide or polypeptide contains at least one sequence alteration as compared to the sequence of the corresponding wild-type or parent polynucleotide or polypeptide. Such alterations, including substitutions, deletions, and/or insertions, are typically non naturally-occurring .

A “neurodegenerative disease” is a condition in which cells of the central or peripheral nervous system progressively degenerate and/or die. Neurodegenerative diseases primarily affect neurons in the central nervous system, typically causing problems with movement (i.e. ataxias), or mental functioning (i.e. dementias). Examples of neurodegenerative diseases are, without limitation, Alzheimer’s disease (AD) and other dementias, Parkinson’s disease (PD) and PD-related disorders, prion disease, motor neuron diseases (MND), Huntington disease (HD), spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA) and Amyotrophic lateral sclerosis (ALS).

A “neurosensory organ” is an organ or a structure that is specialized for receiving external or internal stimuli and transmitting them in the form of nervous impulses to the brain. In human being, eyes, ears, nose, mouth and skin are the neurosensory organs associated with the five senses touch, vision, hearing, smell and taste. Diseases or disorders that affect eyes or ears may result in a total or partial loss of vision or hearing.

As used herein, the terms “treat,” “treating” or “treatment” refer to an action to obtain a beneficial or desired clinical result including, but not limited to, alleviation or amelioration of one or more signs or symptoms of a disease or condition (e.g., regression, partial or complete), diminishing the extent of disease, stabilizing (i.e., not worsening, achieving stable disease) the state of disease, amelioration or palliation of the disease state, diminishing rate of or time of progression, and remission (whether partial or total). “Treatment” of a disease can also mean prolonging survival as compared to expected survival in the absence of treatment. Treatment may be curative or preventive. In certain embodiments, treatment includes one or more of a decrease of physical and mental ability impairment or an increase in the quality of life (QOL) as judged by a qualified individual, e.g., a treating physician, e.g., using accepted assessment tools of pain and QOL. In certain embodiments, “treatment” includes making the neuronal and/or glial cells able to produce enzymes involved in antioxidant response.

The expression “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic effect. A therapeutically effective amount of a rAAV or pharmaceutical composition herein described may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the rAAV or pharmaceutical composition to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response.

A “subject” includes a mammal, e.g., a human, including a mammal in need of treatment for a disease or disorder, such as a mammal having been diagnosed with having a disease or disorder or having been determined to be at risk of developing a disease or disorder. In particular examples, a subject is a mammal diagnosed with a neurodegenerative disease, such as ALS, including familial ALS and sporadic ALS, or such as Huntington disease. In other particular examples, a subject is a mammal diagnosed with a lysosomal storage disease, such as Niemann -Pick disease or Pompe disease. In still other particular examples, a subject is a mammal diagnosed with a vision or hearing disorder that causes total or partial loss of vision or hearing.

Gene therapy vectors

The invention provides novel gene delivery constructs. These constructs are based on AAVs. The AAV vectors contain a polynucleotide sequence encoding aNRF2 protein.

In a first aspect, the present invention relates to a recombinant adeno-associated virus (AAV) comprising in its genome, a polynucleotide encoding a NRF2 protein operably linked to a promoter.

AAV

Adeno-associated viruses (AAV) are small, non-enve loped, icosahedral viruses. The genomic organization of all known AAVs is very similar. The genome of AAV is a linear, single stranded DNA molecule of 4.7 kilobases (kb) to 6 kb in length. The AAV genome contains two open reading frames (ORFs) flanked by short inverted terminal repeats (ITRs) that contains, inter alia, c/.s-acting sequences required for virus replication, rescue, packaging and integration. The two ORFs encode for the non- structural replication (Rep) and the capsid (Cap) proteins. Four related proteins are expressed from the rep gene; Rep78 and Rep68 are transcribed from the p5 promoter while a downstream promoter, pl9, directs the expression of Rep52 and Rep40. Rep78 and Rep68 are directly involved in AAV replication as well as regulation of viral gene expression. The cap gene is transcribed from a third viral promoter, p40. The capsid is composed of three proteins of overlapping sequence (VP1, -2 and -3); the smallest (VP-3) is the most abundant. The AAV VP proteins are known to determine the cellular tropism of the AAV virion.

Because the inverted terminal repeats (ITRs) are the only AAV sequences required in cis for viral replication and packaging, most rAAV vectors dispense with the viral genes encoding the Rep and Cap proteins and contain only the transgene(s), e.g., therapeutic gene(s), inserted between the terminal repeats.

In a typical embodiment, the genome of the recombinant AAV comprises one or more AAV inverted terminal repeat (ITR) sequences and a transgene, such as illustrated in Figure 1. More particularly, the genome of a recombinant AAV of the invention preferably comprises one or two AAV ITR sequences and a polynucleotide encoding aNRF2 protein. Preferably, the NRF2-coding polynucleotide is operably linked to a promoter and, optionally, to a polyA sequence (forming an expression cassette). Typically, one of the 2 ITRs is located 5’ to the NRF2-coding polynucleotide or expression cassette, and the other ITR is located 3 ’to the polynucleotide or cassette. The genome of the rAAV is preferably encapsidated in an AAV capsid or pseudocapsid.

To date, at dozens of different major serotypes of AAVs with variations in their surface properties have been isolated from human or non-human primates (NHP) and characterized. The serotype influences (or determines) viral tropism and may thus be adjusted depending on the contemplated uses.

The rAAV herein described, may be of any one of the known serotypes of AAV, for example, an AAV 1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh8, AAVrhlO, AAVrh20, AAVrh39, AAVRh74, AAVRHM4-1, AAVhu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSCI, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSCIO, AAV.HSCI 1, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, or AAVhu68, of mixtures thereof.

In a particular embodiment, the rAAV of the invention is an AAVrhlO. AAVrhlO have been demonstrated to efficiently transduce neurons and/or astrocytes in the central nervous system. AAVrh.10 vectors have also been shown to cross the blood-brain barrier and transduce neurons and astrocytes in the neonatal mouse central nervous system (Zhang et al., Molecular Therapy, 2011, 19: 1440-1448). In addition, AAVrh. 10 vectors have activity upon injection into the brain of rodents (Sondhi et al., Mol Then, 2007, 15(3):481 -491), and there is no natural disease with AAV serotype 10 in the human population. Examples of AAVrh.10 are described e.g., in PCT Patent Application Publication No. W02003/042397. An exemplary AAVrh.10 cap polynucleotide sequence is provided as SEQ ID NO:59 in PCT Patent Application Publication No. W02003/042397, with the sequence encoding VP1 at nucleotides 845-3061, VP2 at nucleotides 1256-3061, and VP3 at 1454-3061. An exemplary AAVrh.10 cap polypeptide sequence is provided as amino acid s 1-738 of SEQ ID NO: 81 of PCT Patent Application Publication No. W02003/042397, with the VP1 sequence at amino acids 1-738, VP2 at amino acids 138-738, and VP3 at amino acids 203-738.

In a particular embodiment, the invention relates to a recombinant AAV comprising one or two AAVrh. 10 ITR sequences, a polynucleotide encoding a NRF2 protein, and an AAVrh. 10 CAP protein.

In another particular embodiment, the rAAV of the invention is an AAV9. AAV9 have been demonstrated to efficiently transduce neurons and/or astrocytes in the central nervous system.

In a particular embodiment, the invention relates to a recombinant AAV comprising one or two AAV9 ITR sequences, a polynucleotide encoding a NRF2 protein, and an AAV9 CAP protein.

In further particular embodiments, the rAAV of the invention is an AAV2 or AAV8. AAV2 and AAV8 have been demonstrated to efficiently transduce retinal cells, in particular the photoreceptors (rods and cones), the interneuron cells (amacrine cells, bipolar cells and horizontal cells), the retinal ganglion cells, or the retinal pigment epithelium cells. Such serotypes are thus useful for treating eye diseases or disorders.

In a particular embodiment, the invention relates to a recombinant AAV comprising one or two AAV2 ITR sequences, a polynucleotide encoding a NRF2 protein, and an AAV2 CAP protein.

In a particular embodiment, the invention relates to a recombinant AAV comprising one or two AAV8 ITR sequences, a polynucleotide encoding a NRF2 protein, and an AAV8 CAP protein.

In further particular embodiments, the rAAV of the invention is an AAV5. AAV5 has been demonstrated to efficiently transduce inner hair cells. Such serotype is thus useful within the context of the present invention, such as for treating hearing disorders.

In a particular embodiment, the invention relates to a recombinant AAV comprising one or two AAV5 ITR sequences, a polynucleotide encoding a NRF2 protein, and an AAV5 CAP protein.

AAV6 and AAV5 also represent particular embodiments of the invention. These serotypes are particularly suitable for use for treating lysosomal storage diseases (Hudry and Vandenberghe, Neuron 101, 2019, 839-855).

In some embodiments, the rAAV of the invention are pseudotyped, i.e., have a capsid and a genome originating from different AAV serotypes. The ability of rep and ITR sequences to cross-complement corresponding sequences of other serotypes allows for the production of pseudotyped rAAV particles comprising the capsid protein of one serotype and the rep and/or ITR sequences of another serotype. For example, a 2/rhlO rAAV particle has ITRs from AAV2 and a capsid from AAVrhlO. Other examples of pseudotyped rAAV of the invention include, but are not limited to rAAV2/9, rAAV2/8, rAAV2/5 and rAAV2/6. NRF2

As indicated, the invention relates to rAAVs encoding a NRF2 protein.

Nuclear factor erythroid 2(NFE2)-related factor 2 (NRF2), also known as HEBP1, Nrf-2 or IMDDHH, is a transcription factor which is a member of a small family of basic leucine zipper (bZIP) proteins. NRF2 is considered the responsible regulator of 100-200 target genes involved in cellular responses to oxidative and/or electrophilic stress. Targets of NRF2 include genes that influence and produce glutathione (GSH) mediators, antioxidants, and genes controlling efflux pumps. Nrf2 (OMIM 600492) is located on chromosome 2q31.2, comprises 5 exons and encodes a nuclear transcription factor comprised of seven highly conserved domains referred to as NRF2-ECH homology (Neh). In the N- terminal region ofNRF2 is the Neh2 domain encoded by exon 2 of the Nrf2 gene. This KEAPl-binding domain negatively regulates NRF2 levels in cells through binding of KEAP1, which is mediated by two motifs, DLG and ETGE, leading to ubiquitination and degradation. The Neh4 and Neh5 domains, encoded by exon 3 and exons 4 and 5 respectively, are required for transactivation of downstream target genes through recruitment of CREB-binding protein. The last four Neh domains are encoded by exon 5 and serve as DNA binding domains or binding for ubiquitination. The Neh7 domain is a repressor domain for RXRa, which inhibits NRF2-ARE signaling through direct interaction. The Neh6 domain is a b-TrCP binding domain which regulates KEAP1 -independent degradation and stability of NRF2. The Nehl domain is a DNA binding domain which dimerizes with small Maf proteins which facilitate binding to ARE elements of target genes for activation. In the C-terminal region of NRF2, the Neh3 domain is involved in transcriptional activation and is a highly conserved region that is essential for its transcriptional activity.

Within the context of the present invention, the term “NRF2 protein” designates preferably a human NRF2 protein. Examples of sequences of human NRF2 protein isoforms are available in the public databases, such as under the accession numbers NP_006155.2 (isoform 1), NP_001300830.1 (isoform 2), NP_001138885.1 (isoform 3), NP_00I30083I.l (isoform 4), NP_001300832.1 (isoform 5) and NP_001300833.1 (isoform 6). The invention may be implemented with any human NRF2 protein or variant thereof.

Preferably, the NRF2 protein is a human NRF2 protein of isoform 1, or a variant thereof.

An illustrative amino acid sequence of a human NRF2 isoform 1 is provided herein as SEQ ID NO: 2.

In a particular aspect, the NRF2 protein comprises, consists essentially of, or consists of SEQ ID NO: 2 or a variant thereof, preferably a variant having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% amino acid sequence identity to SEQ ID NO: 2.

“Variants” of a reference NRF2 protein may differ from said NRF2 protein by one, two, three, four, five, six, seven, eight, nine, ten or more amino acid residues. Variation(s) may be substitution(s), deletion(s), and/or addition(s) of one or more amino acid residues. In some embodiments, the NRF2 protein is a full-length NRF2 protein. By “full-length” NRF2 protein is meant a NRF2 protein comprising from N- to C-ter Neh2, Neh4, Neh5, Neh7, Neh6, Nehl and Neh3 domains. Typically, a full length NRF2 protein comprises or consists of about 605 amino acid residues. In a particular embodiment, the NRF2 protein is a protein comprising, consisting essentially of, or consisting of SEQ ID NO: 2 or a protein having at least 90%, preferably at least 95, 96, 97, 98 or 99% identity to SEQ ID NO: 2. The percentage of identity is calculated over the entire length of the sequence of SEQ ID NO: 2.

In some aspects, the NRF2 protein is a modified NRF2 protein that lacks a functional Neh2 domain. Typically, such a protein lacks, or has a reduced ability to bind KEAP1, preferably while retaining a transactivation activity.

As discussed above, the binding of NRF2 to KEAP1 negatively regulates NRF2 levels in cells, leading to ubiquitination and degradation. KEAP1 binds a single NRF2 protein at a high-affinity ETGE-site and a low-affinity DLG-site contained in the Neh2 domain of NRF2 protein (the Neh2 domain typically corresponds to amino acids 16-86 of a human NRF2 protein). ETGE-site and DLG-site correspond respectively to amino acids 79-82 and 29-31 of human NRF2 protein. The binding of KEAP1 to the ETGE motif (SEQ ID NO: 17) is 100-fold higher than to the DLG motif.

In a particular aspect, a modified NRF2 protein lacking a functional Neh2 domain is a NRF2 protein wherein the Neh2 domain has been modified to disrupt (e.g., suppress or reduce) the interaction of NRF2 with KEAP 1. Preferably, the modified NRF2 protein retains a transactivation activity. The Neh2 domain can be rendered unfiinctional by deleting all or part of the Neh2 domain, or by introducing mutation(s), substitution(s) and/or addition(s) at one or more amino acid positions in the Neh2 domain.

In a particular aspect, the modified (human) NRF2 protein has a complete deletion of the Neh2 domain, i.e., a deletion that corresponds to or spans amino acids 16-86 of a human NRF2 protein. A NRF2 protein lacking the entire Neh2 domain is typically 534 amino acids long. A particular example of such a modified NRF2 protein is a protein comprising, consisting essentially of, or consisting of SEQ ID NO: 4, or a protein having at least 90%, preferably at least 95, 96, 97, 98 or 99% identity to SEQ ID NO: 4. The percentage of identity is calculated over the entire length of the sequence of SEQ ID NO: 4.

In another particular aspect, the modified (human) NRF2 protein has a partial deletion of the Neh2 domain, i.e., a deletion that corresponds to, or spans, a part only of amino acids 16-86, by reference to a human NRF2 protein. By “part” of the Neh2 domain is meant a deletion of at least one amino acid or any portion of the Neh2 domain, such as any portion of at least 2 consecutive amino acid resides. In a particular embodiment, the part of Neh2 is or comprises at least the hydrophilic region of the Neh2 domain that corresponds to amino acids 33-73 of the NRF2 protein, or the ETGE-site, or the DLG-site. The deletion of the hydrophilic region of the Neh2 domain of the NRF2 protein is sufficient to reduce or prevent binding of the KEAP1 repressor. Other examples of NRF2 protein variants are for instance, the Ml and M2 mutants reported by Itoh et al. (Genes Dev., 1999, 13(l):76-86).

In another aspect, the interaction between NRF2 and KEAP1 proteins is disrupted by introducing one or more point mutations within the Neh2 domain, more preferably in the ETGE-site and/or DLG-site and/or hydrophilic region. The mutation is preferably a substitution of an amino acid by another one. As for example, modified NRF2 proteins of the invention comprise at least one mutation at an amino acid position chosen among aa 29, 30, 31, 79, 80, 81 and 82, by reference to a human NRF2 protein of SEQ ID NO: 2. In a particular aspect, amino acid E79 and/or E82 is/are substituted, preferably with non- acidic amino acids.

In another particular embodiment, amino acid T80 of is substituted by any other amino acid, preferably T80G or T80L. Preferably, amino acid T80 of is substituted by a glycine (T80G). Alternatively, amino acid T80 of is substituted by a leucine (T80L).

In some aspects, the modified NRF2 protein lacking a functional Neh2 domain is a modified human NRF2 protein comprising at least one substitution or deletion at an amino acid position chosen among 29, 30, 31, 79, 80, 81 and 82, by reference to SEQ ID NO: 2.

In some embodiments, the modified NRF2 protein lacking a functional Neh2 domain is a modified human NRF2 protein comprising an amino acid substitution T80G or T80L, by reference to SEQ ID NO: 2. Examples of such proteins are provided as SEQ ID NOs: 9 and 16, which represent particular aspects of the invention.

In this regard, a further aspect of the present invention also relates to a protein comprising, consisting, or consisting essentially of the sequence of SEQ ID NO: 9 or 16, or a protein having at least 90%, preferably at least 95, 96, 97, 98 or 99% identity to SEQ ID NO: 9 or 16 with the proviso that amino acid at position 80 is G or L. The percentage of identity is calculated over the entire length of the sequence of SEQ ID NO: 9 or 16. rAAVs of the invention comprise any polynucleotide encoding a NRF2 protein as herein described. The polynucleotide may comprise (i) a sequence of gene #4780 or a sequence complementary thereto; (ii) a natural variant of a sequence of (i) such as a polymorphism; or (iii) a sequence having at least 90% identity, preferably at least 95, 96, 97, 98 or 99% to a sequence of (i) or (ii). Examples of polynucleotide encoding a human NRF2 protein are available in the public databases, such as under the accession numbers NM_006164.5 (isoform 1), NM_001313901.1 (isoform 2), NM_001145413.3 (isoform 3), NM_001313902.2 (isoform 4), NM_001313903.2 (isoform 5) and NM_001313904.1 (isoform6). Preferably, the polynucleotide encodes a human NRF2 protein of isoform 1, or a variant thereof. An illustrative nucleic acid sequence encoding a human NRF2 protein of isoform 1 is provided herein as SEQ ID NO: 1. Examples of polynucleotides encoding a NRF2 protein are or comprise any one of SEQ ID NOs: 1, 3, 5-8, 10-15, or any variants thereof, particularly variants having at least 90% identity, preferably at least 95, 96, 97, 98 or 99% thereto over the entire length thereof.

SEQ ID NO: 1 is an example of a polynucleotide encoding a full-length NRF2 protein.

SEQ ID NOs: 3, 5-8, 10-15 are examples of polynucleotides encoding modified human NRF2 proteins lacking a functional Neh2 domain. SEQ ID NO: 3 encodes a NRF2 protein with a deletion of all Neh2 domain, while SEQ ID NOs: 5-8 and 10-15 encode NRF2 proteins with mutation in Neh2 domain.

Promoter

Preferably, the polynucleotide encoding a NRF2 protein as herein described is under the control of a promoter sequence. Depending on the contemplated use, the promoter is preferably a promoter functional in one or more cells of the CNS or of a neurosensory organ. The promoter may be a constitutive promoter, an inducible promoter, a ubiquitous promoter, or a tissue- or a cell-specific promoter.

More preferably, the polynucleotide encoding a NRF2 protein is operably linked to a constitutive promoter, an inducible promoter, a ubiquitous promoter, or a tissue- or a cell-specific promoter.

In some embodiments, the promoter is a constitutive promoter. Examples of constitutive promoters suitable for use in the invention include, without limitation, the cytomegalovirus (CMV) early enhancer, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) immediate early promoter (optionally with the CMV enhancer) and the simian virus 40 (SV40) promoter.

In some embodiment, the promoter is an inducible promoter. Example of inducible promoters suitable for use in the invention include, without limitation, metal ion inducible promoters such as for example the zinc-inducible metallothionein (MT) promoter; steroid hormone inducible promoters such as for example the dexamethasone (Dex) -inducible mouse mammary tumor virus (MMTV) promoter, or the growth hormone promoter; the ecdysone insect promoter; the tetracycline -inducible system; promoters which would be inducible by a helper virus such as for example adenovirus early gene promoter inducible by adenovirus E1A protein, or the adenovirus major late promoter.

A ubiquitous promoter enables the expression of the polynucleotide in many types of cells, whereas a tissue-specific promoter may direct the expression of the polynucleotide preferentially, predominantly, or specifically in a desired tissue of interest, such as specific organs (e.g., brain or spinal cord) or particular cell types (e.g., oligodendrocytes, astrocytes, neurons, microglial cells, ependymal cells; cells of the retina such as for example photoreceptors (rods and cones), interneuron cells (amacrine cells, bipolar cells and horizontal cells), retinal ganglion cells, retinal pigment epithelium cells; and/or inner hair cells. In some embodiments, the promoter is a ubiquitous promoter. Examples of ubiquitous promoters suitable for use in the invention include, without limitation, the chicken P-actin (CAG) promoter, the phosphoglycerate kinase-1 (PGK) promoter, the early growth response factor-1 (EGR1) promoter, the eIF4Al promoter, the human ferritin heavy chain (FerH) promoter, the human ferritin light chain (FerL) promoter, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, the b-KIN promoter, the phosphoglycerate kinase 1 (PKG-1) promoter, the ROSA26 promoter, the ubiquitin C (UbiC) promoter, and the elongation factor 1 -alpha promoter (EFl -alpha) promoter. In a particular embodiment, the ubiquitous promoter of the rAAV vector herein described is the CAG promoter.

In some embodiments, the promoter is a tissue- or cell-specific promoter. Examples of tissue- or cellspecific promoters suitable for use in the invention include, without limitation, the synapsin-1 promoter, the Vga promoter, the Vglut2 (glutamatatergic neurons), the NSE/RU5’ promoter, the Neurofilament light chain promoter (neurons), the glial fibrillary acidic protein (GFAP) promoter gfa2 or its truncated 681-bp sequence variant gfaABCID promoter, the Nrl promoter, the Crx promoter, the Rax promoter, the cone opsin promoter, the interphotoreceptor retinoid binding protein (IRBP156) promoter, the rhodopsin kinase (RK) promoter, the neural leucine zipper (NRLL) promoter, the cone arrestin promoter, the Cabp5 promoter, the Cralbp promoter, the Ndrg4 promoter, the clusterin promoter, the Hes 1 promoter, the vimentin promoter, the cluster differentiation (CD44) promoter, or any other suitable tissue- or cell-specific promoter as describes for instance in Table 2 of WO2021/202651.

In some embodiments, the polynucleotide encoding a NRF2 protein is operably linked to a promoter suitable for expression of the polynucleotide encoding a NRF2 protein preferentially, or specifically, in one or more cells of the CNS. In some embodiments, the one or more cells of the CNS comprise one or more cells of the brain or spinal cord. In some embodiments, the one or more cells of the CNS is an oligodendrocyte, astrocyte, neuron, microglial cell, ependymal cell, and/or a Purkinje cell. In some embodiments, the cell of the brain or spinal cord is a neuron. In this particular embodiment, the cellspecific promoter of the rAAV vector herein described can be the synapsin-1 promoter, the Vga promoter, the Vglut2 promoter, the NSE/RU5’ promoter or the Neurofilament light chain promoter. In some embodiments, the cell of the brain or spinal cord is an astrocyte. In this particular embodiment, the cell-specific promoter of the rAAV vector herein described can be the glial fibrillary acidic protein (GFAP) promoter gfa2 or its truncated 681-bp sequence variant gfaABCID promoter.

In some embodiments, the polynucleotide encoding a NRF2 protein is operably linked to a promoter suitable for expression of the polynucleotide encoding a NRF2 protein preferentially, or specifically, in one or more cells of the eye. In some embodiments, the one or more cells of the eye comprise one or more cells of the retina. In some embodiments, the one or more cells of the retina is a photoreceptor (rod and cone), an interneuron cell (amacrine cell, bipolar cell and horizontal cell), a retinal ganglion cell, or a retinal pigment epithelium cell. In this particular embodiment, the cell-specific promoter of the rAAV vector herein described is selected from the group consisting of an Nr 1 promoter, a Crx promoter, a Rax promoter, a cone opsin promoter, an interphotoreceptor retinoid binding protein (IRBP156) promoter, a rhodopsin kinase (RK) promoter, a neural leucine zipper (NRLL) promoter, a cone arrestin promoter, a Cabp5 promoter, a Cralbp promoter, an Ndrg4 promoter, a clusterin promoter, a Hesl promoter, a vimentin promoter and a cluster differentiation (CD44) promoter.

In some embodiments, the polynucleotide encoding a NRF2 protein is operably linked to a promoter suitable for expression of the polynucleotide encoding a NRF2 protein preferentially, or specifically, in one or more cells of the hearing organ (i.e. cochlea). In some embodiments, the one or more cells of the hearing organ comprise one or more cells of the auditory cells, such as for example, inner /outer hair cells, and/or auditory nerve, such as for example, spiral ganglion neurons. In this particular embodiment, the promoter of the rAAV vector herein described can be CMV or CAG promoter.

Other elements

The rAAv may also comprise in its genome other regulatory elements such as for example enhancers, other elements that contribute to accurate or efficient transcription or translation such as for example internal ribosomal entry sites (IRES) and other expression control elements (e.g. transcription termination signals, such as for example polyadenylation signals and poly-U sequences).

The promoter, transgene, and other regulatory elements are typically operably linked.

Preferred rAAVs

A preferred rAAV of the invention is of an AAV9 or AAV/rhlO serotype and comprises, in its genome, a polynucleotide encoding a NRF2 protein, under the control of a ubiquitous or constitutive promoter herein described, preferably under the control of a CAG promoter.

Another preferred rAAV of the invention is of an AAV9 or AAV/rhlO serotype and comprises, in its genome, a polynucleotide encoding a NRF2 protein, under the control of a promoter selected from a synapsin-1 promoter, a Vga promoter, a Vglut2 promoter, a NSE/RU5’ promoter, or a Neurofilament light chain promoter, preferably under the control of a synapsin-1 promoter.

Still another preferred rAAV of the invention is of an AAV9 or AAV/rhlO serotype and comprises, in its genome, a polynucleotide encoding a NRF2 protein, under the control of a glial fibrillary acidic protein (GFAP) promoter gfa2 or its truncated 681-bp sequence variant gfaABCID, preferably under the control of a glial fibrillary acidic protein (GFAP) promoter gfa2.

Another preferred rAAV of the invention is of an AAV2 or AAV8 serotype and comprises, in its genome, a polynucleotide encoding a NRF2 protein, under the control of a ubiquitous or constitutive promoter herein described, preferably under the control of a CAG promoter.

Still another preferred rAAV of the invention is of an AAV2 or AAV8 serotype and comprises, in its genome, a polynucleotide encoding a NRF2 protein, under the control of a promoter selected from an Nrl promoter, a Crx promoter, a Rax promoter, a cone opsin promoter, an interphotoreceptor retinoid binding protein (IRBP 156) promoter, a rhodopsin kinase (RK) promoter, a neural leucine zipper (NRLL) promoter, a cone arrestin promoter, a Cabp5 promoter, a Cralbp promoter, an Ndrg4 promoter, a clusterin promoter, a Hes 1 promoter, a vimentin promoter and a cluster differentiation (CD44) promoter.

Another preferred rAAV of the invention is of an AAV5 or AAV6 serotype and comprises, in its genome, a polynucleotide encoding a NRF2 protein, under the control of a ubiquitous or constitutive promoter herein described, preferably under the control of a CAG promoter.

In each of said preferred constructs, the encoded NRF2 protein is more preferably a human protein, even more preferably a modified human NRF2 protein lacking a functional Neh2 domain.

Another object of the present invention thus relates to a nucleic acid molecule comprising, consisting essentially of, or consisting of the genome of a rAAV herein described.

Method for producing gene therapy vector

Gene therapy vectors of the present invention may be produced by methods known per se in the art. Methods have been described for instance in WO03/042397, U.S. Patent No. 6,632,670, etc. rAAV vectors are produced in competent host cells. Typically, the method requires distinct components including: a cis plasmid carrying an ITR-flanked target gene expression cassette (i.e. the vector plasmid); one or more trans plasmid(s) supplying AAV structural (cap) and/or packaging (rep) proteins (i.e. a packaging plasmid(s)); and a helper plasmid supplying the adenoviral helper genes. Figure 2 illustrates a schematic representation of the plasmids used for the production of rAAVs herein described. After cotransfection of the plasmids into competent host cells, the rAAV vectors are produced and the transfected host cells are harvested. rAAV vectors are then recovered from the transfected cells.

In another aspect, the present invention relates to a method for producing a recombinant AAV comprising (the steps of): a) culturing cells that have been transfected with plasmids herein described; and b) recovering recombinant AAV from the transfected cells.

The cell usable in the method of the invention can be a mammalian cell or an insect cell. In some embodiment, the cell is or derived from a human embryonic kidney cell line, such as for example 293, 293T, 2C4 or 3B1 cell. In some embodiment, the cell is sf9 cell.

In order to generate recombinant AAV vector stocks, standard approaches provide the AAV rep and cap gene products on a packaging plasmid that is used to co-transfect a suitable cell together with the AAV vector plasmid encoding the NRF2 protein. In some embodiments, standard approaches provide the AAV rep and cap gene products on a packaging plasmid that is used to co-transfect a suitable cell together with the AAV vector plasmid encoding the NRF2 protein and together with the helper plasmid providing helper functions.

In one embodiment, gene therapy vector of the present invention is produced by the transfection of two or three plasmids into a 293 or 293T human embryonic kidney cell line. In some embodiments, polynucleotide encoding a NRF2 protein is provided by a first plasmid, and the capsid proteins (e.g. from AAVrh.10), replication genes (e.g. from AAV2) and helper functions (e.g. derived from adenovirus serotype 5) are all provided in trans by a second plasmid. In some embodiments, polynucleotide encoding a NRF2 protein is provided by a first plasmid, the capsid proteins (e.g. from AAVrh.10) and replication genes (e.g. from AAV2) are provided in trans by a second plasmid, and helper functions (e.g. derived from adenovirus serotype 5) are provided by a third plasmid. In particular embodiments, the first plasmid comprises an expression cassette of the present invention, including the flanking ITRs.

In some embodiments, a method for producing a recombinant AAV comprises (the steps of): a) culturing cells that have been transfected with: i) a plasmid carrying an ITR-flanked polynucleotide encoding a NRF2 protein operably linked to a promoter, and a polyA; ii) a plasmid comprising polynucleotide(s) encoding Rep and Cap proteins; and iii) a plasmid comprising adenovirus components; and b) recovering recombinant AAV from the transfected cells.

In particular embodiments, AAV rep and cap genes are provided on a replicating plasmid that contains the AAV ITR sequences. In some embodiments, the rep proteins activate ITR as an origin of replication, leading to replication of the plasmid. The origin of replication may include, but is not limited to, the SV40 origin of replication, the Epstein-Barr (EBV) origin of replication, the ColEl origin of replication, as well as others known to those skilled in the art. Where, for example, an origin of replication requires an activating protein, e.g., SV40 origin requiring T antigen, EBV origin requiring EBNA protein, the activating protein may be provided by stable transfection so as to create a cell line source, (e.g., 293T cells), or by transient transfection with a plasmid containing the appropriate gene.

In other embodiments, AAV rep and cap genes may be provided on a non- replicating plasmid, which does not contain an origin of replication. Such non-replicating plasmid further insures that the replication apparatus of the cell is directed to replicating recombinant AAV genomes, in order to optimize production of virus. The levels of the AAV proteins encoding by such non-replicating plasmids may be modulated by use of particular promoters to drive the expression of these genes. Such promoters include, inter aha, AAV promoters, as well as promoters from exogenous sources, e.g., CMV, RSV, MMTV, E1A, EFla, actin, cytokeratin 14, cytokeratin 18, PGK, as well as others known to those skilled in the art. Levels of rep and cap proteins produced by these helper plasmids may be individually regulated by the choice of a promoter for each gene that is optimally suited to the level of protein desired. Standard recombinant DNA techniques may be employed to construct the helper plasmids used to produce viral vector of the present disclosure (see e.g., Current Protocols in Molecular Biology, Ausubel., F. et al., eds, Wiley and Sons, New York 1995), including the utilization of compatible restriction sites at the borders of the genes and AAV ITR sequences (where used) or DNA linker sequences which contain restriction sites, as well as other methods known to those skilled in the art.

Following cell culture, the gene therapy vector (i.e. rAAV) is released from cells using mechanical means, such as, for instance, by freeze thaw cycles and purified for example by an iodixanol step gradient followed by ion exchange chromatography on Hi -Trap QHP columns. The resulting gene therapy vector may be concentrated by spin column. The purified vector may be stored frozen (at or below -60°C), e.g., in phosphate buffered saline. Other known methods can be used by the skilled person to produce and purify gene therapy vectors.

Characterization of the final formulated vector may be achieved through SDS-PAGE and Western blot for capsid protein, real time PCR for transgene DNA, Western analysis, in vivo and in vitro general and specific adventitious viruses, and enzymatic assay for functional gene transfer.

Compositions

Also provided herein are compositions comprising a rAAV herein described. In particular, the present invention also relates to a composition comprising a rAAV of the invention and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any carrier, excipient or diluent suitable for pharmaceutical use. Pharmaceutically acceptable carriers are well known in the art (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). For example, one acceptable carrier includes saline, which may be formulated with a variety of 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 carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and/or by the use of surfactants. The selection of the carrier is not a limitation of the present disclosure.

The prevention of the action of microorganisms can be brought about by various antibacterial and/or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Prolonged absorption of the injectable compositions can be accomplished by including in the compositions of agents that delay absorption, for example, aluminum monostearate and gelatin. Injectable compositions can be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. T1

The pharmaceutical carrier can be formulated for delivery in the central nervous system, preferably in the brain or the cerebrospinal fluid. In some embodiments, the pharmaceutical carrier is formulated for intravenous, intraparenchymal, or cerebrospinal fluid delivery; more preferably for cerebrospinal fluid delivery.

The pharmaceutical carrier can also be formulated for delivery in a neurosensory organ, preferably the eyes or the ears.

In some embodiments, the pharmaceutical carrier is suitable and formulated for delivery to the ear, e.g., to the cochlea. In some embodiments, the composition is formulated as ear drops.

In some embodiments, the pharmaceutical carrier is suitable and formulated for delivery to the eye, e.g., intraocular delivery. In some embodiments, the pharmaceutical carrier is suitable and formulated for administration to the eye by subretinal injection, such as buffered saline or other buffers, e.g., HEPES, to maintain pH at appropriate physiological levels.

Optionally, the composition may further comprise other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. Exemplary physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline. A variety of such known carriers are provided in U.S. Pat. Publication No. 7,629,322, incorporated herein by reference. In one embodiment, the carrier is an isotonic sodium chloride solution. In another embodiment, the carrier is balanced salt solution. In one embodiment, the carrier includes tween.

In some embodiments, the pharmaceutical composition of the present invention is used as a solo therapy. In other embodiments, the pharmaceutical composition of the present disclosure is used in combination therapy. The combination therapy may be in combination with one or more neuroprotective agents such as small molecule compounds, peptides, proteins and antibodies which have been tested for their effect on the central nervous system.

The composition preferably comprises or is used at an effective dosage of rAAV, e.g., a dosage adapted to produce amount ofNRF2 sufficient to affect oxidative stress.

Dosage value may vary with the nature and severity of the disease to be alleviated. The dosage may also be adjusted based on the serotype of the rAAV and/or administration route. For example AAV9 or AAVrhlO are better at transducing neurons as compared to AAV2. Other parameters such as for example, body weight of the subject or cerebrospinal fluid volume may be used to adjust dosage.

In particular embodiments, the rAAV or composition comprises or is administered at a dose of about 10⁸ to 10¹⁵ viral genome (vg) per administration.

In various embodiments, the composition comprising the rAAV vector of the invention can be administered as a bolus or by continuous infusion over time. In some embodiments, the volume of composition comprising the rAAV vector that is administered is about 50 pL to 20 mL. In some embodiments, the infusion rate for administration of the composition comprising the rAAV vector is about 0.5 pL/min. to about 1 mL/min. The lowest volume and slowest infusion rate are particularly well adapted for intraparenchymal administration. The lowest volume and slowest infusion rate are particularly well adapted for intra-cerebrospinal or intravenous administration.

The rAAV vectors or composition comprising it can be administered in a one time treatment.

Alternatively, the rAAV vectors or composition comprising it can be administered repeatedly, such as daily, weekly or monthly, for instance. The duration of the treatment can be for at least one week, one month, one year or more, depending on the disease and subject.

Methods of treatment

Recombinant AAV herein described, or compositions comprising it, may be used for delivering a transcription factor that activates the expression of genes involved in the cellular antioxidant response. Provided herein are efficient methods for in vitro or in vivo delivering of NRF2 protein in target cells or tissue.

When NRF2 protein is delivered using an AAV vector to a cell suffering from pathology due to oxidative stress, in sufficient amounts to overcome repression by endogenous KEAP1, or in a form that is insensitive to repression by KEAP1, it will move to the nucleus of the transduced cell and bind to ARE elements present in the regulatory regions of many antioxidant genes. Binding of NRF2 or KEAP1- independent NRF2 variants will cause transactivation of these genes and lead to expression of several antioxidative proteins, such as glutamate-cysteine ligase modifier subunit (GCLM), NAD(P)H: quinone oxidoreductase 1 (NQOl), heme oxygenase 1 (HM0X1), and glutathione peroxidase (GPX). Increased levels of these and other NRF2-induced proteins will protect the cell from oxidative stress and lead to improved cellular function and increased cell survival.

In another aspect, the present invention relates to an in vitro method for delivering a polynucleotide encoding a NRF2 protein comprising (the step of): contacting in vitro a cell with a recombinant AAV herein described. The transduced cell is a quiescent or dividing cell, a differentiated or undifferentiated cell having a function in the central nervous system or in neurosensory organs, in particular in the brain or spinal cord (e.g. oligodendrocyte, astrocyte, neuron, microglial cell, ependymal cell), in the eye (e.g. rod or cone photoreceptor, an interneuron cell, a retinal ganglion cell, or a retinal pigment epithelium cell) or in the ear (i.e. an inner hair cell). The cell is in contact with the rAAV in sufficient amount to provide sufficient levels of transgene transfer and expression. As a consequence, the transduced cell produces exogenous NRF2 protein, in an amount that reduces the effects of the oxidative stress by activating the cellular antioxidant response.

Another object of the present invention thus relates to a host cell transduced with a rAAV herein described. The host cell is preferably a cell of the brain or spinal cord, such as for example an oligodendrocyte, astrocyte, neuron, microglial cell, or ependymal cell, or a cell of the retina, such as for example a photoreceptor (rod and cone), an interneuron cell (amacrine cell, bipolar cell and horizontal cell), a retinal ganglion cell, or a retinal pigment epithelium cell, and/or a inner hair cell. In some embodiments, the host cell is a neuron and/or an astrocyte. In some embodiments, the host cell is a retina cell. In some embodiments, the host cell is an inner hair cell. rAAV vectors and packaged viral particles containing the rAAV vectors can be in the form of a medicament or a composition and may be used in the manufacture of a medicament or a composition comprising rAAVs herein described as active ingredient.

In still another aspect, the present invention relates to a recombinant AAV herein described or a composition comprising it for use in the treatment of a neurodegenerative disease, in particular amyotrophic lateral sclerosis (ALS) or Huntington’s disease; of a lysosomal storage disease, in particular Niemann-Pick disease or Pompe disease; or a disease of disorder that effects vision or hearing. In some embodiments, the lysosomal storage disease is a sphingolipidosis, an oligosaccharidosis, a gangliosidosis, a mucopolysaccharidosis, a neuronal ceroid lipofuscinosis, a sialic acid disorder, or a mucolipidosis. The application of the present invention is used, for example, to delay the onset of memory or cognitive dysfunctions, to reduce the incidence of a neurodegenerative disease, to reduce the decline neuromuscular coordination, to extend time to mechanical ventilation, and/or to extend the lifespan for a subject.

In particular embodiments, the rAAV for use in a method for the treatment of a neurodegenerative disease, or of a lysosomal storage disease, is of serotype AAV9 or AAVrhlO, and comprises, in its genome, a polynucleotide encoding aNRF2 protein as herein described, and is administered by injection into the cerebrospinal fluid (CSF) or into the brain parenchyma to a subject in need thereof. When considering Huntington disease, the intra-parenchymal delivery of the rAAV as herein disclosed occurs into specific brain nuclei, e.g. striatum. rAAV of serotype AAV5 or AAV6 can also be used in a method for the treatment of a lysosomal storage disease.

Recombinant AAV herein described or a composition comprising it may also be used in the treatment of diseases or disorders that affect neurosensory organs associated with vision or hearing, in particular eyes and ears.

In another aspect, the present invention relates to a recombinant AAV herein described or a composition comprising it for use in the treatment of diseases or disorders that may cause a total or partial loss of vision. Example of diseases or disorders that affect vision, include without being limited to glaucoma, optic neuritis, optic neuropathy, retinitis pigmentosa, ischemic optic neuropathy, compressive optic neuropathy, infiltrative optic neuropathy, tramautic optic neuropathy, mitochondrial optic neuropathy, nutritional optic neuropathy, toxic optic neuropathy, hereditary optic neuropathy including Leber’s hereditary optic neuropathy, dominant optic atrophy, Behr’s syndrome, and Berk- Tabatznki syndrome. Still other eye diseases or disorders include macular degeneration, age-related macular degeneration (AMD), geographic atrophy, wet AMD, dry AMD, drusen formation, dry eye, diabetic retinopathy, vitreoretinopathy, comeal inflammation, uveitis and ocular hypertension. The application of the present invention is used, for example, to prevent drusen formation; to prevent visual loss or slow the rate of visual loss; to prevent or slow the rate of choroidal neovascularization; to improve visual acuity and/or contrast sensitivity; to prevent or reduce the rate of photoreceptor or RPE (retina pigmented epithelium) cell atrophy or apoptosis; to reduce intraocular hypertension.

In particular embodiments, the rAAV for use in a method for the treatment of an disease or disorder that affects vision is of serotype AAV2, AAV8 or AAVrhlO, and comprises, in its genome, a polynucleotide encoding a NRF2 protein as herein described, and is administered by intravitreal injection, subretinal injection, injection into the anterior chamber of the eye, injection or application locally to the cornea, subconjunctival injection, sub-tenon injection, or eye drops to a subject in need thereof.

In another aspect, the present invention relates to a recombinant AAV herein described or a composition comprising it for use in the treatment of hearing impairment, in particular hearing loss. There are two main types of hearing loss: conductive hearing loss and sensorineural hearing loss. Conductive hearing loss can occur when sound is not conducted efficiently through the outer ear canal to the eardrum and the tiny bones (ossicles) of the middle ear. Sensorineural hearing loss can occur when there is damage to the inner ear, cochlea, or hearing nerve.

Overt reactive oxygen species (ROSs) can be produced due to noise and certain ototoxic drugs that can damage the inner and outer hair cells of the ear, resulting in either temporary or permanent hearing loss. Exposure to blast waves and continuous noise not only damaged the inner ear, but caused cell death in the hippocampus, suppressed neurogenesis and impaired memory function. Aging as well can manifest in mitochondrial dysfunction leading to hearing loss.

In particular embodiments, the rAAV for use in a method for the treatment of hearing impairment, in particular hearing loss, is of serotype AAV1, AAV5, AAV8 or AAVrhlO, and comprises, in its genome, a polynucleotide encoding aNRF2 protein as herein described, and is administered by intratympanic (in the middle ear), intracochlear, or parenteral route (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intrathecal) to a subject in need thereof.

In yet another aspect, the present invention also relates to a method for treating a neurodegenerative disease, in particular amyotrophic lateral sclerosis (ALS) or Huntington’s disease; or a lysosomal storage disease, in particular Niemann-Pick disease or Pompe disease, in a subject comprising administering an effective amount of a composition comprising a recombinant AAV described herein to the central nervous system, in particular to the cerebrospinal fluid or to the brain parenchyma. In some embodiments, the NRF2 protein is a full-length protein. In some embodiments, the NRF2 protein lacks a functional Neh2 domain. In some embodiments, the NRF2 protein has a modified Neh2 domain as herein disclosed. In some embodiments, the subject has been diagnosed with a neurodegenerative disease or a lysosomal storage disease, e.g. through mental or neuropsychological tests, brain imaging, electromyogram, blood and spinal fluid analysis, muscle biopsy, etc. In some embodiments, the subject has been diagnosed with a lysosomal storage disease.

In another aspect, the present invention also relates to a method for treating eye diseases or disorders that may cause a total or partial loss of vision in a subject, comprising administering an effective amount of a composition comprising a recombinant AAV described herein to the eye, in particular to neuronal cell types of the eye such as, for example, the photoreceptors (rods and cones), the interneuron cells (amacrine cells, bipolar cells and horizontal cells) the retinal ganglion cells, or the retinal pigment epithelium cells. In some embodiments, the NRF2 protein is a full-length protein. In some embodiments, the NRF2 protein lacks a functional Neh2 domain. In some embodiments, the NRF2 protein has a modified Neh2 domain as herein disclosed. In some embodiments, the subject has been diagnosed with an eye disease or disorder, e.g. through visual acuity test, pupil dilatation, ophthalmoscopy or fundus photography, fundus angiography, optical coherence tomography, etc.

In another aspect, the present invention also relates to a method for treating hearing impairment, in particular hearing loss, in a subject, comprising administering an effective amount of a composition comprising a recombinant AAV described herein to the hearing organ, in particular to auditory cells (inner and outer hair cells), and/or to the auditory nerve (spiral ganglion neurons) . In some embodiments, the NRF2 protein is a full-length protein. In some embodiments, the NRF2 protein lacks a functional Neh2 domain. In some embodiments, the NRF2 protein has a modified Neh2 domain as herein disclosed. In some embodiments, the subject has been diagnosed with hearing impairment, in particular hearing loss, e.g. through clinically audiological testing for sensorineural hearing impairment, e.g., by recording the auditory brainstem response (AB) and otoacoustic emissions (OAEs), etc.

In a particular aspect, the present invention also relates to a modified NRF2 protein comprising, consisting essentially of, or consisting of SEQ ID NO: 9 or 16, or a variant thereof, for use as a medicament.

In another aspect, the present invention relates to a modified NRF2 protein comprising, consisting or consisting essentially of a sequence of SEQ ID NO: 9 or 16, wherein the Neh2 domain comprises an amino acid substitution T80G or T80L, for use in the treatment of in the treatment of a neurodegenerative disease, in particular amyotrophic lateral sclerosis (ALS) or Huntington’s disease; a lysosomal storage disease, in particular Niemann-Pick disease or Pompe disease; or a disease or disorder that causes a total or partial loss of vision or hearing.

In some embodiments, the rAAV or the composition comprising it is administered by systemic route, in particular by intravenous route. Alternatively, the rAAV or the composition comprising it is administered directly to subject’s central nervous system. For example, in some embodiments, the rAAV or the composition comprising it is administered into the cerebrospinal fluid (CSF) of the subject by intrathecal, intracerebroventricular or intracistemal (e.g. intra-cistema magna) route. CSF is a clear fluid that surrounds the brain and spinal cord. It cushions the brain and spinal cord from injury and also serves as a nutrient delivery and waste removal system for the brain. Delivery of rAAV vectors into the CSF results in widespread delivery to the CNS.

Intrathecal route refers to injection of medication into the cerebrospinal fluid at the level of the lumbar spine intrathecal space, via a catheter, needle or other suitable injection device. Intrathecal administration can thus be distinguished from systemic administration, such as for example intravenous route, and from epidural administration. Injection into the ventricles refers to intra-cerebrospinal fluid administration. Intrathecal administration will be referred to herein as spinal intrathecal injection when the administration occurs in the spine, and intracranial intrathecal delivery when the administration occurs in the brain.

The rAAV or the composition comprising it can be administered into the cerebrospinal fluid intrathecally through spinal or lumbar delivery into the subarachnoid space; through intracistemal delivery; or through intracerebroventricular (ICV) delivery (administration into the cerebral ventricles), etc.

In some embodiments, the rAAV or the composition comprising it can also be administered into the brain through intraparenchymal route.

Intracranial administration, including intraparenchymal and intracerebroventricular delivery, is performed through one or more burr holes drilled in the skull, using a delivery device, optionally comprising a catheter and an infusion pump.

Neurosensory organs, such as for example eye and ear, are well suited for local rAAV delivery. The compartimentalized nature of the eye limits the dose and restricts systemic spread of the vector. Various injection routes to administer to the eye are available and depend on the target cell type.

Alternatively, the rAAV or the composition comprising it can be administered to the eye by subretinal, intravitreal, intracameral, suprachoroidal or topical route.

Like the eye, the ear presents an opportunity to deliver a rAAV in the proximity of the neural target tissue in the cochlea. Various injection routes to administer to the ear are available.

Alternatively, the rAAV or the composition comprising it can be administered to the ear via a round window membrane, an oval window, a transcanal or a cochleostomy procedure.

In some embodiments, the rAAV vector is administered at a single site. In some embodiments, the rAAV vector is administered at more than one sites. Kits

The rAAVs described herein may be assembled into pharmaceutical kits to facilitate their use in therapeutic applications. A kit may include one or more containers housing the components of the present invention and instructions for use. Specifically, such kits may include one or more rAAV described herein, along with instructions describing the intended application and/or uses thereof. As used herein, "instructions" can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the kit. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g. videotape, DVD, etc.), Internet, and/or web-based communications, etc.

In another aspect, the present invention relates to a kit comprising a recombinant AAV herein described, and optionally at least one pharmaceutical excipient. In a particular embodiment, the pharmaceutical excipient is suitable for intravenous administration; for cerebrospinal fluid administration; for intraparenchymal administration; for delivery to the ear; or for intraocular delivery. Optionally, the kit may further comprise instructions for use.

In other aspects, the present invention relates to a kit comprising a recombinant AAV or composition of the invention and a delivery device, such as a syringe or canula, for instance. For example, the kit may contain a pre-filled syringe, vial, tube or other container, typically with a pre -determined dose of rAAV or composition.

The kit may also include other components, depending on the specific application, for example, containers, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the reagents prior to administration etc.

The following examples are provided in order to demonstrate and further illustrate certain preferred aspects of the present invention and are not to be construed as limiting the scope thereof.

EXAMPLES

EXAMPLE 1 - rAAV construction

Expression cassettes (NFR2 or NRF2 variants) included in plasmid vectors are cloned using traditional method by restriction enzyme-ligation based molecular cloning. Briefly, two pieces of DNA that have complementary or compatible overhangs after restriction digestion can then be fused together during a ligation reaction with T4 DNA ligase. Recombinant DNA is then transformed into competent cells and a colony screening is performed to identify the transformed colonies containing the gene of interest.

NFR2 variants are obtained by site-directed mutagenesis using In-Fusion cloning technology. The InFusion system utilizes mutagenic oligonucleotide primers to generate desired mutations. Adherent HEK293 cells seeding in CellStack 5 chambers are co-transfected with the vector plasmid (an ITRs-flanked transgene cassette coding for NRF2 or NRF2 variants), a packaging plasmid that encodes the rep and cap genes of a selected AAV serotype and a helper plasmid (containing helper genes from adenovirus). The virus is harvested 3 days post-transfection. rAAV are purified from supernatant by PEG-precipitation and double CsCl-gradient ultracentrifugation. The viral suspension is formulated in DPBS IX buffer and stored at <-70°C in polypropylene low-binding cryovials. rAAV vector genome is tittered by quantitative polymerase chain reaction (qPCR). rAAV are constructed based on AAV2, AAV9 and AAVlOrh serotype. For each serotype, several rAAVs are prepared containing a polynucleotide sequence encoding SEQ ID NO: 2, SEQ D NO: 4, SEQ ID NO: 9, and SEQ ID NO: 16, respectively.

EXAMPLE 2 - Expression of NRF2 protein in vitro

2.1 - ARE-luciferase cell line

ARE-luciferase cell line is a cell line stably expressing luciferase reporter gene under the control of the antioxidant response element (ARE). Cells are seeded at about 4 x 10⁴ cells/cm² and incubated with empty or unrelated AAV vector as a control or with different amounts of AAV vectors expressing NRF2 or NRF2 variants. At different time intervals thereafter (e.g. 24h, 48h and 72h), cells are treated with the Bright-Glo Kit (Promega) or similar commercial kits according to manufacturer’s instructions and the luminescence signal is recorded.

This experiment can be used to demonstrate functional expression of NRF2 variants of the invention.

2.2 - Expression in neurons and astrocytes

To determine activity of the rAAV of the invention in cells of the central nervous system, primary cultures of neurons or astrocytes are transiently transfected with an ARE luciferase reporter plasmid, using a kit from BPSBioscience. This reporter contains a firefly luciferase gene under the control of multimerized ARE responsive elements located upstream of a minimal promoter. The ARE reporter is premixed with a constitutively expressing Renilla (sea pansy) luciferase vector that serves as an internal control for transfection efficiency. The BPSBioscience kit also includes a non-inducible firefly luciferase vector premixed with constitutively expressing Renilla luciferase vector as negative control. The non-inducible luciferase vector contains a firefly luciferase gene under the control of a minimal promoter, without any additional response elements. This negative control is critical to determining pathway-specific effects and background luciferase activity. Transiently transfected cells are then incubated with empty or unrelated AAV vector as a control or with different amounts of AAV vectors expressing NRF2 or NRF2 variants. At different time intervals thereafter (e.g. 24h, 48h and 72h), cells are treated with the Bright-Glo Kit (Promega) or similar commercial kits according to manufacturer’s instructions and the luminescence signal is recorded. This experiment can be used to demonstrate functional expression of NRF2 variants of the invention in primary nervous cells.

EXAMPLE 3 - In vivo efficacy in a mouse model of ALS

Substitution of alanine for glycine at position 93 of SOD1 (SOD1G93A) is a mutation which causes ALS in people and transgenic expression of SOD1G93A in mice induces paralysis and premature mortality. The activity of AAV vectors expressing NRF2 or mutant forms of NRF2 is compared to that of empty or unrelated AAV vectors by performing a standardized litter-matched and gender-balanced efficacy study as described (Scott et al. 2008, Vieira etal. 2017). AAV vectors are delivered at 30 to 60 days of age by injection into the cerebrospinal fluid via the lateral ventricle or the cistema magna. The relative transduction of CNS tissue and transgene expression is quantified in the brain and spinal cord. One month after injection, mice are sacrificed by an overdose of ketamine/xylazine, and cleared by transcardiac perfusion with ice cold PBS. Cerebrum, cerebellum and spinal tissues are collected for analysis of vector genome by quantitative PCR (qPCR) and vector genome transcripts by RT-qPCR.

The C9 B AC-25 500 mouse model is used to evaluate the therapeutic effects AAV vectors expressing NRF2 or mutant forms of NRF2 in a model of C9ALS/FTD. AAV vectors are delivered by injection into the cerebrospinal fluid via the lateral ventricle or the cistema magna and biochemical parameters and behaviors of the mice are recorded at various timepoints thereafter. The relative transduction of CNS tissue and transgene expression is quantified in the brain and spinal cord. One month after injection, mice are sacrificed by an overdose of ketamine/xylazine, and cleared by transcardiac perfusion with ice cold PBS. Cerebrum, cerebellum and spinal tissues are collected for analysis of vector genome by quantitative PCR (qPCR) and vector genome transcripts by RT-qPCR.

EXAMPLE 4 - In vivo efficacy in a mouse model of HD

The R6/2 mouse model is used to evaluate the therapeutic effects AAV vectors expressing NRF2 or mutant forms of NRF2 in a model of Huntington disease. AAV vectors are delivered by injection into the striatum and biochemical parameters and behaviors of the mice are recorded at various timepoints thereafter. The relative transduction of CNS tissue and transgene expression is quantified in the brain and spinal cord One month after injection, mice are sacrificed by an overdose of ketamine/xylazine, and cleared by transcardiac perfusion with ice cold PBS. Cerebrum, cerebellum and spinal tissues are collected for analysis of vector genome by quantitative PCR (qPCR) and vector genome transcripts by RT-qPCR. EXAMPLE 5 - In vitro assay of ARE activation by NRF2 and NRF2 variants

Results

NRF2 protein expression analysis

All the NRF2 plasmids contain a HiBiT tag DNA sequence at the N-terminal of the NRF2 gene. The HiBiT peptide tag, a subunit of the NanoBiT® enzyme, has high affinity for the other subunit LargeBiT® of the NanoBiT® enzyme. When both subunits are incubated together, a functional NanoBiT® enzyme is formed and generates the NanoLuc® Luciferase signal.

HepG2 cells were transfected with NRF2 plasmids at 50ng, lOOng, 200ng, 500ng, or 1 pg, and analyzed at 24 hours and 48 hours post-transfection; The NRF2 protein level, expressed by NRF2 plasmids transfected, was analyzed via the N-terminal HiBiT tag of the NRF2 protein (Figure 5).

NRF2 protein, expressed by the transfected plasmids, increased with the quantity of plasmid transfected, except the WT NRF2 that slightly decreased when transfected with the highest quantity.

Transfection of I pg plasmid resulted in the highest NRF2 protein expression, except for WT NRF2. T80G NRF2 showed slightly higher expression than WT or T80L NRF2, at Ipg of plasmid transfected.

Evaluation of the transcriptional activation by NRF2, via ARE-driven Firefly luciferase detection

The activation of ARE promoter by NRF2 was analysed via the ARE-driven expression of the Firefly luciferase. The Firefly luciferase level was analysed in cells transfected with 200ng, 500ng or Ipg, at 48 hours post-transfection.

When Ipg plasmid was used in transfection, the NRF2 expressed by T80G-NRF2 was able to activate ARE-Firefly luciferase similar to the positive control Sulphoraphane. (Figure 6).

The transcriptional activation efficiency of NRF2 and NRF2 variants was evaluated as the ratio of ARE- Firefly luciferase versus HiBiT signal (Figure 7).

As shown in Figure 7, the specific activity of T80GNRF2 and T80LNRF2 in activating ARE -luciferase was higher than that of WT NRF2.

Evaluation of the transcriptional activation level of NRF2 target genes

PTGR1, HM0X1, GCLM and NQ01 are redox regulation genes that are activated by NRF2 via their ARE promoter. 48 hours after transfection into HepG2 cells with 1 pg NRF2 plasmids, total mRNA was extracted, and the transcriptional activation of these genes were analyzed by qRT-PCR. HPRT gene served as the housekeeping gene control. Untransfected condition served as negative control and Sulphoraphane stimulation condition as a positive control.

As shown in Figure 8, T80G NRF2 induced high redox gene transcription, with levels close to Sulphoraphane. T80L NRF2 showed, to a lesser extent, the transcription activation of redox genes. WT NRF2 had almost no effect on the transcriptional activation of redox genes. This is probably due to the endogenous KEAP1 expressed under the physiological HepG2 cell culture condition.

In conclusion, these results show that T80G NRF2 and to a lesser extent T80L NRF2 efficiently activate ARE-mediated transcription, with higher specific activity than WT NRF2.

Experimental procedures

1.1. ARE-Luc HepG2 cell culture

The ARE-reporter Hep G2 cell line contains a firefly luciferase gene under the control of the promoter ARE stably integrated into Hep G2 cells. This cell line is validated for the response to the stimulation of sulforaphane.

ARE-Luc HepG2 cells were maintained in Complete Medium containing EMEM supplemented with 10% heat inactivated FBS, Pen/Strep and G418 (0.4 mg/mL) at 37°C - 5%CO2.

• Medium change every 2 days

• Cells passage every 4-5 days

1.2. Transfection Mix preparation

Mix for 1 mb of cells plus transfection reagent (900 pL of cells and 100 pL of transfection mix)

• Mix A: Plasmid and P3000™ Reagent 1: 1 ratio were added in 50pl OPTIMEM

• Mix B: 1.5 pL Lipofectamine 3000 reagent in 50 pL OPTIMEM

Once ready, the two mixes were combined and incubate for 20 minutes at RT.

1.3. Assay procedure

Transfection of ARE-Luc HepG2 cells using LIPOFECTAMINE 3000

• Cells were counted using trypan blue exclusion and used when cell viability was higher than 90% and 80% confluence is reached.

• After counting, cells were resuspended in complete medium without G418 at the density of 3.2 xl05 cells/mL.

• Transfection mixes were added to cells.

• Cells were plated in different format based on the readout: o In a 384w plate (50 pL/well) for Nano-Gio® HiBiT Dual-Luciferase® and cell viability assay o In a 6 well-plate (2 ml/well) for RNA and Protein extraction

• After 24h and 48h at 37°C - 5%CO2, samples were processed

• Sulphoraphane (5 pM final concentration) was used as positive control.

1.4. Assay detection

• Transfection efficiency: GFP control vector was included in the experiment and the number of GFP positive cells was determined by cell imaging using Incell 6000 analyser

• Western Blot: cells were lysed using RIPA buffer supplemented with protease and phosphatase inhibitors (100 pL/buffer for well). Lysates were quantified by BCA protein assay kit and 10 pg loaded on 4 - 12% Bis-Tris gel. 24h and 48h after transfection Nano-Gio® HiBiT Blotting System assay was performed as described by manufacturer. GAPDH protein amounts were used as loading control.

• For Nano-Gio® HiBiT Dual-Luciferase® studies: Nano-Gio® HiBiT Dual-Luciferase® assay was performed according to manufacturer’s recommendations at 24h and 48h after transfection. Nano- Luc and Firefly Luciferase signal were detected with Envision plate reader.

• Cells Viability assay: Cell Titer Gio or CellTiter-Blue® assay were performed according to manufacturer’s recommendations at 24h and 48h after transfection.

• Real Time: RNA was extracted according to manufacturer’s recommendations. RNA concentration was determined by OD260 absorbance. Real time was performed according to manufacturer’s recommendations using 40ng of RNA Template for each reaction.

Steps and Temperature

1.5. Data analysis

For the Firefly luciferase assay, samples were expressed as fold change over untransfected cells.

For the real time, samples were expressed as fold change over HPRT vs untransfected cells.

EXAMPLE 6 - Prediction of immunogenicity of NRF2 variants

Immunogenicity (HLA class I and II) was predicted for NRF2 peptides with Thr80 unchanged, Thr80Ala, Thr80Gly and Thr80Leu, using the methods recommended by IEDB (https://www.iedb.org/). For predicted HLA class I epitopes, the results indicated a trend for Thr80Gly to be potentially less immunogenic (worse predictions) compared to Thr80Ala. The same trend was also present for the HLA class II predictions.

1. HLA Class I predictions

31 residue peptides centered around NRF2 position 80 were subjected to HLA class I predictions using the IEDB recommended method. Predictions were performed using a subset of 16 HLA-A, 11 HLA-B and 22 HLA-C alleles as suggested on the IEDB website. Predictions were performed for peptide lengths 8 to 11.

1 11 16 21 31

EQEKAFFAQL QLDEETGEFL PIQPAQHIQS E ( SEQ ID NO : 18 )

EQEKAFFAQL QLDEEAGEFL PIQPAQHIQS E ( SEQ ID NO : 19 )

EQEKAFFAQL QLDEEGGEFL PIQPAQHIQS E ( SEQ ID NO : 20 )

EQEKAFFAQL QLDEELGEFL PIQPAQHIQS E ( SEQ ID NO : 21 ) Output from the IEDB predictions was collected for each peptide and then summarized using a custom written program. As recommended by IEDB only predictions falling into the top 1% percentile of all predictions were considered as likely binders. The results are shown in Table 1.

Lower values in columns T, A, G and C indicate stronger predicted binding to the indicated HLA class I molecule. Start and End indicate the position within the peptide used for the position (position 16 =

Thr80). G vs A indicates the percentile difference between Thr80Gly and Thr80Ala. If positive, the Thr80Gly mutant is predicted to bind weaker than Thr80Ala (Thr80Gly predicted to be potentially less immunogenic), and if negative the opposite.

Thr80Leu in principle has overall lower predicted binding compared to Thr80Ala. However, for several Class I alleles, this substitution has a relatively strong predicted epitope for the most common length 9aa, absent in the other pos 80 substitutions. For this reason, it does not appear that Thr80Leu has more favorable properties compared to Thr80Ala.

For Thr80Gly, there is a signal indicating for a number of Class I alleles a weaker binding compared to Thr80Ala.

Table 1: MHC Class I predictions (cutoff: top 1% percentile predictions). Freq indicates the percent frequency of the corresponding Class I allele (s) in the population. Columns T, A, G and L report the percentile value (lower = stronger predicted binding) for the prediction of the wt or mutated peptides.

2. HLA Class II predictions

37 residue peptides centered around NRF2 position 80 were subjected to HLA class II predictions using the IEDB recommended method. Predictions were done using a subset of 27 class II allele combinations as suggested on the IEDB website for epitope lengths 14,15 and 16.

1 11 19 21 31 37

LQKEQEKAFF AQLQLDEETG EFLPIQPAQH IQSETSG ( SEQ ID NO : 31 )

LQKEQEKAFF AQLQLDEEAG EFLPIQPAQH IQSETSG ( SEQ ID NO : 32 )

LQKEQEKAFF AQLQLDEEGG EFLPIQPAQH IQSETSG ( SEQ ID NO : 33 )

LQKEQEKAFF AQLQLDEELG EFLPIQPAQH IQSETSG ( SEQ ID NO : 34 )

The results indicate Thr80Leu having overall decreased percentile values (stronger binding) as compared to the other three residues (Table 2). There is also a general trend for Thr80Gly having worse predictions (increased percentile values) compared to Thr80Ala, i.e. Thr80Gly is predicted to be less immunogenic (G vs A, Table 2).

Table 2: MHC Class II predictions (cutoff: top 1% percentile predictions). Freq indicates the percent frequency of the corresponding Class II allele(s) in the population. Columns T, A, G and L report the percentile value (lower = stronger predicted binding) In conclusion, T80G NRF2 is predicted to be less immunogenic than T80A NRF2.

Claims

1. A recombinant adeno-associated vims (AAV) comprising, in its genome, a polynucleotide encoding a modified NRF2 protein operably linked to a promoter, wherein the modified NRF2 protein lacks a functional Neh2 domain and wherein the modified NRF2 protein comprises an amino acid substitution T80G or T80L, by reference to SEQ ID NO: 2.

2. The recombinant AAV of claim 1, wherein the AAV has a serotype selected from the group consisting of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVrh8, AAVrhlO, AAVrh20, AAVrh39, AAVRh74, AAVRHM4-1, AAVhu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 or AAVhu68, or mixtures thereof.

3. The recombinant AAV of claim 1 or 2, wherein the promoter is a constitutive promoter; an inducible promoter; a ubiquitous promoter; or a tissue- or a cell-specific promoter, in particular a brain- , neural-, eye-, auditory system tissue-, neuronal cell-, astrocyte-, retinal cell-, or inner hair cell-specific promoter.

4. The recombinant AAV of any one of claims 1-3, wherein the modified NRF2 protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9 or 16, or is a protein having at least 90%, preferably at least 95, 96, 97, 98 or 99% identity to SEQ ID NO: 9 or 16 with the proviso that amino acid at position 80 is G or L.

5. The recombinant AAV of any one of claims 1-4, wherein the modified NRF2 protein comprises an amino acid substitution T80G by reference to SEQ ID NO: 2.

6. A method for producing a recombinant AAV comprising: a) culturing cells that have been transfected with: i) a plasmid comprising a recombinant AAV according to any one of claims 1 to 5, and a polyA; ii) a plasmid comprising polynucleotide(s) encoding Rep and Cap proteins; and iii) a plasmid comprising adenovirus; and b) recovering recombinant AAV from the transfected cells.

7. A host cell transduced with a recombinant AAV according to any one of claims 1 to 5.

8. A nucleic acid molecule comprising, consisting essentially of, or consisting of the genome of a recombinant AAV according to any one of claims 1 to 5.

9. A composition comprising a recombinant AAV according to any one of claims 1 to 5 and a pharmaceutical acceptable carrier, preferably formulated for intravenous, intraparenchymal, or cerebrospinal fluid delivery, more preferably for cerebrospinal fluid delivery.

10. A recombinant AAV according to any one of claims 1 to 5 or a composition according to claim 9 for use in the treatment of a neurodegenerative disease, preferably amyotrophic lateral sclerosis (ALS) or Huntington’s disease; a lysosomal storage disease, preferably Niemann-Pick disease or Pompe disease; or a disease or disorder that causes a total or partial loss of vision or hearing.

11. A kit comprising a recombinant AAV according to any one of claims 1 to 5, and optionally at least one pharmaceutical excipient.

12. A modified NRF2 protein comprising, consisting essentially of, or consisting of SEQ ID NO: 9 or 16.

13. A composition comprising a modified NRF2 protein of claim 12 and a pharmaceutical acceptable carrier.

14. The modified NRF2 protein of claim 12, for use as a medicament.

15. Use of a recombinant AAV according to any one of claims 1 to 5, a composition according to claim 9 or 13, or a modified NRF2 protein of claim 12 for the manufacture of a medicament for the treatment of a neurodegenerative disease, preferably amyotrophic lateral sclerosis (ALS) or Huntington’s disease; a lysosomal storage disease, preferably Niemann-Pick disease or Pompe disease; or a disease or disorder that causes a total or partial loss of vision or hearing.

16. A method for treating a neurodegenerative disease, preferably amyotrophic lateral sclerosis (ALS) or Huntington’s disease; a lysosomal storage disease, preferably Niemann -Pick disease or Pompe disease; or a disease or disorder that causes a total or partial loss of vision or hearing, in a subject, comprising administering a therapeutically efficient amount of a recombinant AAV according to any one of claims 1 to 5, a composition according to claim 9 or 13, or a modified NRF2 protein of claim 12.