RU2774892C2 - Vectors for treatment of friedreich's ataxia - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to biotechnology, namely to an adeno-associated viral vector (hereinafter – AAV) used in gene therapy for the treatment of Friedreich’s ataxia. The adeno-associated viral vector (AAV) of expression contains nucleic acid that includes (i) a nucleic acid sequence encoding frataxin; (ii) a promotor of phosphoglycerate kinase (PGK), consisting of SEQ ID NO:1; and (iii) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). (ii) and (iii) are functionally connected to the sequence (i) and control its expression. There is a functionally connected linker between (i) and (ii). The specified linker consists of SEQ ID NO: 6, and AAV vector is AAV vector of serotype 9. An adeno-associated viral (AAV) cloning vector is presented. A pharmaceutical composition for the treatment of Friedreich’s ataxia, containing effective amount of AAV vector is presented, as well as the use of AAV vector as a drug and in the treatment of Friedreich’s ataxia.

EFFECT: structure of AAV9 vector is capable of providing intermediate levels of frataxin in vitro and in vivo, which are more comparable with endogenous levels.

12 cl, 3 tbl, 11 dwg, 4 ex

Description

Technical field

The present invention may be included in the field of new therapeutic methods for the treatment of Friedreich's ataxia. In particular, the present application relates to new products for gene therapy. These products can be used to treat Friedreich's ataxia.

Prerequisites for the invention

Friedreich's ataxia (FRDA; OMIM # 229300) is a rare hereditary neurodegenerative disorder that causes progressive damage to the nervous system, with symptoms ranging from gait and speech disorders to heart disease or cardiomyopathy. The disease was named after the physician Nikolaus Friedreich, who first described it in the 1860s. Ataxia associated with impaired motor coordination, such as clumsy movements and instability, is known as Friedreich's ataxia, caused by degeneration of nerve tissue in the spinal cord and the nerves that control muscle movements of the arms and legs. The spinal cord shrinks along with the nerve cells, resulting in the loss of some of their myelin sheath. Friedreich's ataxia, although rare, is the most common hereditary ataxia, occurring in 1 in 30,000-50,000 newborns with a prevalence of 2-4 in 100,000. Both sexes are equally affected by this disease. Symptoms usually begin between the ages of 5 and 15, but in rare cases, they may appear as early as 18 months or even as early as 50 years of age. The first symptom detected is usually difficulty walking or gait ataxia. Ataxia gradually progresses and slowly spreads to the arms, and then to the entire body. Foot deformities such as "hollow foot", involuntary folding of the toes, or hammer toe deformity may be seen as early signs. Over time, the muscles begin to weaken and wear out, especially in the feet, lower legs and arms, and deformities develop. Other symptoms include loss of tendon reflexes, especially in the knees and ankles. There is often a gradual loss of sensation in the limbs, which can spread to other parts of the body. At the same time, dysarthria develops, as a result of which the patient quickly gets tired. Especially common are rapid, rhythmic, and involuntary eye movements called nystagmus. Most people with Friedreich's ataxia develop scoliosis, which, if severe, can affect breathing.

Other symptoms that may be observed are chest pain, shortness of breath and palpitations. These symptoms are the result of various forms of heart disease that often accompany Friedreich's ataxia, such as hypertrophic cardiomyopathy, myocardial fibrosis, or heart failure. Heart rhythm disturbances such as tachycardia and heart block are also common. Approximately 20% of people with Friedreich's ataxia develop intolerance to carbohydrates, and 30% develop diabetes. Some people with this disease lose their hearing or vision. The rate of disease progression varies from person to person. Usually, 10-20 years after the onset of the first symptoms, a person is forced to move around in a wheelchair, and in the later stages of the disease, people become completely disabled. This, presumably, may affect life expectancy. Many people with Friedreich's ataxia die in adulthood due to related heart disease, the most common cause of death. However, some people with less severe symptoms of Friedreich's ataxia sometimes live into their 60s or 70s.

Neuroimaging, such as MRI, shows the normal state of the body in the early stages of the disease, although with the progression of this disease there is variable atrophy of the cervical spinal cord and cerebellum. This often indicates atrophy of the upper pedicle. Electrophysiological studies show conduction velocities greater than 40 m/s in the absence or decrease of sensory action potentials and in the absence of the H reflex. At the neuropathological level, atrophy of the spinal cord, posterior roots, and sometimes the cerebellum, and cardiac hypertrophy are noted. Neurodegeneration is identified in peripheral sensory nerves with progressive loss of sensory neurons in the large spinal ganglions, degeneration of the dorsal column, transynaptic degeneration of neurons in Clark's spine and spinocerebellar fibers and pyramidal tracts, and atrophy of the fine fasciculus and cuneiform fasciculus nuclei. Secondary lesions may include atrophy of the dentate nucleus in the cerebellum affecting large glutamatergic neurons and atrophy of Betz cells and corticospinal tracts.

In 1996, the cause of Friedreich's ataxia was identified as a molecular defect in the FXN gene located on chromosome 9 (band 9q21). The mutation consists of an abnormal homozygous increase in the levels of the GAA triplet located within the ALU sequence in the first intron of the FXN gene. Approximately 98% of FRDA patients have 2 chromosomes with GAA repeats ranging from 201 to 1700 (most commonly 600 to 900) on each. However, 2-5% of FRDA patients have an increase in GAA levels and a point mutation in the FXN gene with combined heterozygosity (Table 1). Currently, more than 17 different mutations in the FXN gene have been described that can cause FRDA. The increase in GAA levels in FRDA is highly unstable during meiosis and interferes with transcription of the FXN gene, resulting in abnormally low levels of frataxin mRNA. Increasing GAA levels can lead to silencing of FXN gene transcription and thus abolish frataxin expression through the formation of DNA triplets or DNA-RNA hybrids, or both. More recently, it has been suggested that GAA expansion will block the transition from initiation to transcriptional upregulation due to the formation of heterochromatin-like structures near the area of GAA hyperexpansion. It has also been suggested that GAA expansion will lead to epigenetic methylation of CpG sites located in the 5' region upstream of the FXN gene and their silencing. Thus, due to a GAA mutation in the FXN gene, FRDA is deficient in the frataxin protein.

Table 1. Most common allelic variants identified in the FXN gene that cause Friedreich's ataxia OMIM ALLELIC VARIANT CAUSING DISEASE 606829.0001 Expansion of GAA in intron 1 606829.0002 Transversion in exon 3: LEU106X 606829.0003 Transition c.385-2A>G affecting splicing 606829.0004 Missense variant: Ile154Phe in exon 4 606829.0005 Missense mutation: Gly130Val 606829.0006 Missense mutation affecting start codon: Met1Ile 606829.0007 Missense mutation: Trp173Gly 606829.0008 1 nucleotide deletion at codon 75 causing protein truncation 606829.0009 Deletion of 6 nucleotides and insertion of 15 nucleotides (c.371_376del6ins15) in exon 3

The FXN gene encodes a small conserved mitochondrial protein that contains 210 amino acids (a.a.) in the form of a 23135 Da precursor called frataxin (Q16595). This form of precursor protein contains an N-terminal transfer sequence that directs it to the mitochondrial matrix, where mitochondrial peptidase converts it into various smaller isoforms (FXN42-210, FXN56-210, FXN81-210, FXN78-210) of the mature frataxin protein, which is FXN81-210 with a size of 130 a.k. and 14.2 kDa, which is the most common. In humans, frataxin is detected in the mitochondrial matrix in combination with its inner membrane in a wide range of tissues, and its highest levels are found in heart tissue, spinal cord and dividing lymphoblasts, and, notably, the lowest levels are observed in the cerebellum. To date, frataxin has not been found in the cerebral cortex. Since the gene responsible for this disease has been identified and several animal models have been generated, it has been suggested that frataxin has various functions such as mitochondrial iron homeostasis, iron storage, response to oxidative stress, Fe-S cluster biogenesis, modulation of mitochondrial aconitase activity and regulation of oxidative phosphorylation. In FRDA, frataxin deficiency caused by homozygous expansion of GAA in the FXN gene leads to insufficient biosynthesis of iron-sulfur clusters necessary for electron transport into mitochondria and aconitase assembly, and thus to dysregulation of mitochondrial function and accumulation of mitochondrial iron due to changes in its homeostasis. Frataxin also modulates the DNA-binding ability of the protein aconitase 1 (ACO1), which, in addition to its involvement in the citric acid cycle in the mitochondrial matrix, regulates cellular iron uptake and utilization.

Several animal and cell models of Friedreich's ataxia have been created through genetic manipulation. Creating a mouse AF model that mimics human disease as closely as possible has been a difficult task, and there are currently 8 different mouse FRDA models. Unfortunately, none of them has a combination of all the phenotypic symptoms of FA in the same animal, such as lesions of the spinal ganglia (DRG) and cerebellar dentate nuclei, although they may be useful for studying certain aspects of the molecular neurodegenerative process in FRDA and for evaluating different treatments for specific symptoms. In preclinical treatment, Prp-CreERT and YG8R mice are most commonly used. Prp-CreERT mice in particular develop cerebellar and progressive sensory ataxia, the most important neurological features of Friedreich's ataxia. Histological examination of these animals showed abnormalities of the spinal cord and spinal ganglions with no motor neuropathy, which is a hallmark of the disease in humans, and arborization defects in the Purkinje cells of the cerebellum. In contrast, YG8R transgenic mice in the absence of the endogenous murine frataxin protein show a slowly progressive pathology of FRDA. Unlike humans, these mice do not have heart failure.

Among viral and non-viral vectors used in gene therapy for the treatment of human pathologies, vectors derived from adeno-associated virus (AAV) have demonstrated important clinical advantages and prolonged expression in animal models of Gaucher disease, Fabry disease, Pompe disease, metachromatic leukodystrophy, Niemann disease -Peak A and mucopolysaccharidosis I, II, III A, III B, IV and VII, etc. Intravenous administration of AAV vectors to animals with models of lysosomal storage diseases results in a 16-fold increase in enzyme activity compared to their normal values in blood, liver, spleen, kidneys and muscles, which makes them very useful for the treatment of this type of pathology. Over the past 10 years, AAV vectors have been the preferred vector for the majority of clinical trials conducted for the treatment of pathologies of the central and peripheral nervous system (http://www.abedia.com/wiley/index.html). It is important to note that, to date, no side effects have been observed with the introduction of these vectors, and therefore such results are very promising. Thus, several factors make AAVs ideal vehicles for gene delivery to the central nervous system (CNS). Adeno-associated viral vectors containing the frataxin sequence have been used to treat FRDA in mice (Gérard et al., 2014. Mol Ther Methods Clin Dev. 1: 14044; Chapdelaine et al., 2016. Gene Ther. 23 (7): 606- 614 Tremblay et al., 2015 Mol Ther 23 (Suppl. 1): pS153; WO 2016/150964). However, the AAV vectors disclosed in prior work either over- or under-express frataxin and do not reach all target cells and neurons affected by FRDA, making them unsuitable for effective treatment of FRDA, and in particular its neurological symptoms. Thus, it is necessary to create an AAV vector that expresses the frataxin protein in a therapeutically effective amount.

Currently, there is no effective therapy for the treatment of FRDA, and therefore, it is necessary to develop new therapeutic agents for the treatment of FRDA. The aim of the present invention is to develop a suitable therapy for the treatment of FRDA.

Blueprints

Figure 1. Evaluation of the effect of human SYN (synapsin) and FXN (frataxin; 1255 bp) promoters on frataxin (FXN) protein expression . To test the levels of expression of the human protein frataxin (FXN) under the control of the endogenous promoter fragment (phFXN) and the neuronal synapsin promoter (phSYN), compared with the expression under the control of the highly expressed constitutive CMV promoter, SH-SY5Y human neuroblastoma cells were transfected with "blank" a vector (lane 1) or constructs encoding human synapsin (phSYN) (lane 3), or a 1255 bp fragment of the frataxin promoter (phFXN) (lane 4). Constructs 1.2 and 1.3 generated using phSYN or phFXN (1255 bp) to express the human FXN coding sequence (hFXN CDS) did not result in expression of the recombinant frataxin protein (rFXN). HA=hemagglutinin label.

Figure 2. Evaluation of the effect of human SYN (synapsin), NSE (neuron-specific enolase) and FXN (frataxin; 1255 bp) promoters together with the WPRE sequence on frataxin (FXN) protein expression . The effect of the 320 bp fragment was evaluated. 5' ends of the FXN and WPRE sequences for FXN expression from the phSYN promoter previously shown in FIG. 1 (lanes 3, 4 and 8), and phNSE (lane 6). Various constructs were transfected into HEK cells and lysates were analyzed 48 hours after transfection. The effectiveness of WPRE sequences in RNA stabilization was observed using a construct expressing FXN from the CMV promoter (lane 2). However, the addition and combination of the above regulatory elements does not result in FXN expression from phSYN, hpFXN or phNSE. HA - hemagglutinin label; iFXN is an intermediate form of FXN; mFXN is the mature form of FXN; WPRE - groundhog hepatitis virus post-transcriptional regulatory element.

Figure 3. Evaluation of the effect of human SYN (synapsin), NSE (neuron-specific enolase) and FXN (frataxin; 1255 bp) promoters along with CMV and WPRE enhancer sequences on frataxin (FXN) protein expression . HEK cells and N2a mouse neuroblastoma cells were transfected with constructs containing the CMV enhancer together with SYN, NSE or FXN promoters (1255 bp). The combination of regulatory elements shown above did not result in FXN expression from the phSYN, phFXN or phNSE promoters. HA - hemagglutinin label; iFXN is an intermediate form of FXN; mFXN is the mature form of FXN; WPRE - groundhog hepatitis virus post-transcriptional regulatory element.

Figure 4. Evaluation of the effect of human promoters SYN (synapsin), NSE (neuron-specific enolase) and FXN (frataxin; 1255 bp) together with the Kozak sequence (5') and WPRE (3') on frataxin (FXN) protein expression ) . Various constructs were transfected into HEK cells and lysates were analyzed 48 hours after transfection. The effectiveness of WPRE sequences in RNA stabilization was observed using a construct expressing FXN from the CMV promoter (lane 1 and 2). The subsequent increase in FXN expression levels by adding the Kozak sequence and 105 nt sequences between the promoter and the FXN coding sequence is shown in lanes 2 and 3. However, the addition and combination of the regulatory elements shown above does not result in FXN expression from phSYN, phFXN or phNSE. HA - hemagglutinin label; iFXN is an intermediate form of FXN; mFXN is the mature form of FXN; pFXN - precursor form; WPRE - groundhog hepatitis virus post-transcriptional regulatory element.

Figure 5. Evaluation of the effect of a combination of a specific linker located between the human PGK1 (phosphoglycerate kinase 1), NSE (neuron-specific enolase), SYN (synapsin) or FXN (frataxin; 1255 bp) promoters and the FXN coding region (CDS) together with Kozak (5') and WPRE (3') sequences on frataxin (FXN) protein expression . The various constructs presented here were transfected into either N2a cells (A, B and E) or HEK cells (C and D) and the lysates were analyzed 48 hours after transfection. FXN expression was consistently detected from phPGK but not from the phNSE or phFXN promoter (1255 bp) using a 105 bp linker. between promoter and coding region (A, C and D: lane 4; B: lane 5 and E: lane 8). HA - hemagglutinin label; iFXN is an intermediate form of FXN; mFXN is the mature form of FXN; pFXN - precursor form; WPRE - groundhog hepatitis virus post-transcriptional regulatory element.

Figure 6. Expression of luciferase (LUC) under the control of the human PGK1 promoter (phosphoglycerate kinase 1) after intrathecal injection of the rAAV9 vector (4.6×10 ¹² WG/kg) in YG8R Tg/- mice and in wild-type mice. Luciferase expression was detected after intraperitoneal injection of 150 μl of mouse D-luciferin weighing 150 mg/kg 3.5 months after intrathecal injection of the vector (A). Organs were dissected and the luciferase substrate was added fresh before image fixation (relative scales are shown in left and right panels (B)).

Figure 7. Expression of frataxin mRNA in vivo in 7-month-old mice injected intrathecally with rAAV9-PGK1-FXN . Frataxin mRNA levels were quantified by qRT-PCR in liver, heart, lumbar spinal ganglions (DRG-L), lumbar spinal cord (SC-L), thoracic spinal cord (SC-T), cervical spinal cord (SC-C), cerebellum (Cb) and brain (frontal region C1) of 7-month-old hemizygous transgenic (Tg/-) or homozygous (Tg/Tg) YG8R mice. Hemizygous YG8R (Tg/-) transgenic mice were injected with either rAAV-Null (shown in black) or rAAV9-PGK1-FXN (shown in light gray), and homozygous FRDA (Tg/Tg) transgenic mice (shown in dark gray) were injected with did not enter. Unlike hemizygous YG8R (Tg/-) mice carrying two tandem copies of the human FXN gene with ~82 and ~190 GAA trinucleotide sequence repeats in one of the chromosomes, homozygous YG8R mice contained two tandem copies in each of the chromosomes. YG8R mice that are homozygous or hemizygous for human FXN did not express endogenous Fxn because they have a mouse Fxn knockout genetic background. Homozygous YG8R mice had a greater degree of regulation of human FXN mRNA expression because they contain more copies of the human transgene and, in addition, have normal functions. Mice at 2.5 months of age were intrathecally injected and primers specific for human FXN were used to quantify FXN mRNA in various tissues. Total FXN mRNA levels were normalized to levels detected in YG8R (Tg/-) hemizygous mice treated with rAAV9-Null for each of the analyzed tissues. Five months after intrathecal injection of the rAAV9-PGK1-FXN vector, FXN mRNA levels were higher (approximately 1.3 and 3.2-fold) in YG8R hemizygous mice injected with rAAV9-PGK1-FXN (Tg/-) at the tested dose. , but not higher than levels in homozygous YG8R (Tg/Tg) mice in all tissues compared to levels in Tg/− mice treated with AAV-Null. A) Relative mRNA levels of human FXN in female and male mice n=6 (3 females and 3 males). The fold differences between Friedreich's ataxia YG8R mice injected with control AAV-Null compared to YG8R mice injected with rAAV9-PGK1-FXN were statistically significant in the liver, spinal ganglia, and spinal cord (p = 0.019, p =0.004 and p=0.024; marked with an asterisk *) with an increasing trend in all other indicated tissues. Greater variability was observed in males at the dose used, but not in females. B) Relative levels of hFXN mRNA in female YG8R mice. Statistically significant fold differences in frataxin levels were found in all studied tissues, indicated by asterisks and listed in the Table below. The fold increases in frataxin levels in mice injected with rAAV9-PGK1-FXN never exceeded the levels found in Tg/Tg YG8R mice. Statistical significance, p<0.05*, p<0.005**.

Figure 8 . In vivo expression of the recombinant frataxin protein from the rAAV9-hPGK1-FXN vector after intrathecal injection. Lysates of cultured N2a cells were transfected with an empty vector or a vector expressing frataxin, which was used as a positive control (FIGS. 8A and 8B: lanes 1 and 2). Endogenous FXN protein in N2a cells and wild-type mice was detected using an anti-FXN antibody (A and B). The human FXN protein derived from the transgene in YG8R mice, as well as the human recombinant FXN protein in injected mice, were found in both liver and brain of mice (A and B). Incubation of blots with anti-FXN antibody for one hour revealed human FXN protein, and overnight incubation (o/n) revealed murine FXN in wild type mice and human FXN in hemizygous (Tg/-) and homozygous mice (Tg/ Tg). Expression levels of recombinant human FXN protein expressed after rAAV9-FXN injection were similar to those of endogenous murine FXN protein in wild-type mice. HA - hemagglutinin label; iFXN is an intermediate form of FXN; mFXN is the mature form of FXN; WPRE - groundhog hepatitis virus post-transcriptional regulatory element.

Figure 9 . Restoration of the paw reflex in YG8R mice over a period of time after injection of the rAAV9-FXN vector . Hindlimb pretension was assessed on a scale of 0 to 3 (see Methods), according to the severity of the lesion in hemizygous (Tg/-) mice and in wild-type mice injected with rAAV9-FXN or rAAV9-Null vectors. Overall results for females and males are presented (n=10, 5 males and 5 females in each group). Changes in the push reflex were detected as early as 4 months of age in hemizygous YG8R mice. Following intrathecal injection of the rAAV9-FXN vector, the push reflex appears to normalize, as indicated by the recovery of this neurological pathological phenotype over time. Asterisks (*) indicate significant differences between WT and Tg/- mice injected with rAAV9-Null, and pound signs (#) indicate significant differences between values obtained with Tg/- mice injected with rAAV9-Null. and Tg/- mice injected with rAAV9-FXN. *, #: P<0.05.

Figure 10 . In vivo expression of the recombinant frataxin protein in mice injected with rAAV9-hPGK1-FXN leads to the restoration of electrophysiological properties at different distances from the stimulus (D1-D4) of the caudal nerve . Asterisks (*) denote significant differences between WT and FRDA Tg/- YG8R mice treated with rAAV9-Null, and a pound sign (#) denotes significant differences between rAAV9-Null treated mice and rAAV9-FXN YG8R treated mice . Intrathecal injection of 2.5-month-old mice with rAAV9-FXN vector prevents or reduces nerve conduction defects in FRDA (YG8R) mice. Wild-type mice are shown in green (n=10); FRDA Tg/-YG8R mice treated with rAAV9-Null vector are shown in gray (n=10); FRDA Tg/-YG8R mice treated with rAAV9-FXN vector are shown in blue (n=10). Measurements were taken at a distance of 1 cm, 2 cm, 3 cm and 4 cm from the tip of the tail, and these values were designated as values for regions D1, D2, D3 and D4, respectively *, #: P<0.05.

Figure 11 . Spinal cord preservation in FRDA model YG8R mice treated with rAAV9-FXN . Lumbar dorsal diameter appeared to be preserved as well as mitochondrial morphology in FRDA model mice treated with rAAV9-FXN compared to the same mice treated with rAAV9-Null virus.

The essence of the invention

The present invention relates to an adeno-associated virus (AAV) vector containing a nucleic acid, wherein the nucleic acid comprises: (i) a nucleic acid sequence encoding frataxin; (ii) a phosphoglycerate kinase (PGK) promoter; and (iii) woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The present invention also relates to a nucleic acid containing: (i) a nucleic acid sequence encoding frataxin; (ii) a PGK promoter; and (iii) WPRE; to a cloning vector containing the nucleic acid; and to a transfer vector containing the nucleic acid. In addition, the present invention encompasses the use of a nucleic acid, a cloning vector or a transfer vector in order to obtain an AAV vector according to the invention. The present invention also relates to a pharmaceutical composition and to the use of an AAV vector, nucleic acid or pharmaceutical composition as a drug, and in particular as a drug for the treatment of FRDA.

Detailed description of the invention

Definitions

The terms "treatment" and "therapy", as used in this application, refer to a set of hygienic, pharmacological, surgical and/or physical means used to treat and/or alleviate a disease and/or its symptoms in order to eliminate health problems. The terms "treatment" and "therapy" include prophylactic and therapeutic methods, as long as they are aimed at maintaining and/or restoring the health of a person or animal. Regardless of the origin of the symptoms, disease and disability, the administration of a suitable drug to alleviate the disease and/or eliminate health problems should be interpreted as a form of treatment or therapy in the context of this application.

The term "therapeutically effective amount" means the amount of a substance that has a therapeutic effect and that is able to cure FRDA.

The terms "individual", "patient" or "subject" are used in this application as synonyms and should in no way be construed as limiting the invention. An "individual", "patient", or "subject" may be of any age, gender, or physical condition.

As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable diluent" means any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known to those skilled in the art. Suitable carriers, excipients or stabilizers are non-toxic to recipients at the doses and concentrations used and include the following compounds, which do not limit the scope of the present invention, namely: additional buffering agents; preservatives; co-solvents; antioxidants including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (eg Zn-protein complexes); biodegradable polymers such as polyesters; salt-forming counterions such as sodium, polyhydric alcohols of a number of sugars; amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid and threonine; organic sugars or sugar alcohols such as lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (eg inositol), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; and hydrophilic polymers such as polyvinylpyrrolidone.

The term "frataxin" means a protein that is encoded by the FXN gene and that is normally present in mitochondria. Detailed information about the protein can be found in the UniProtKB database under accession number Q16595.

The term "promoter" means a DNA sequence to which RNA polymerase can bind to initiate transcription. The sequence may also contain binding sites for various proteins that regulate transcription, such as transcription factors. The promoter sequence may consist of different promoter fragments (different or identical fragments) that are located next to each other in the DNA sequence and can be separated by linkers or spacers. Such promoters are called chimeric promoters. In a preferred embodiment of the invention, the term "promoter" means a phosphoglycerate kinase (PGK) promoter.

The term "post-transcriptional regulatory element" means a DNA sequence which, when transcribed, creates a tertiary structure that enhances or inhibits protein expression.

The term "operably linked" refers to two or more nucleic acid sequences that are linked in such a way that one nucleic acid sequence affects the other. For example, the PGK and WPRE promoter are operably linked to the nucleic acid sequence encoding frataxin, whereby frataxin expression levels are regulated by the PGK and WPRE promoter.

The term "functional variant" means nucleic acids or amino acid sequences that differ in one or more positions from the parent nucleic acid sequence or amino acid sequence, where these differences may be additions, deletions and/or substitutions of nucleic acids or amino acid residues; and which are still functional and therefore can be used to treat FRDA. One skilled in the art can determine functional variants by searching for homology using the BLAST search program or by examining the variability of a protein or gene within a population.

The term "adeno-associated virus" means a small virus that infects humans and some other primate species. A "vector" is any vehicle that can be used to artificially transfer foreign genetic material into a cell. Thus, "AAV vector" means a recombinant AAV that transfers a nucleic acid into a cell.

AAV Vector

In its first aspect, the present invention provides an adeno-associated virus (AAV) vector comprising a nucleic acid, wherein the nucleic acid comprises: (i) a nucleic acid sequence encoding frataxin; (ii) a phosphoglycerate kinase (PGK) promoter; and (iii) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), wherein (ii) and (iii) are operably linked to sequence (i) and regulate its expression.

SEQ ID NO: 1 means the following sequence:

gaattccggggttggggttgcgccttttccaaggcagccctgggtctgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccag.

In a preferred embodiment, the PGK promoter contains SEQ ID NO: 1 or a sequence that is at least 75% identical to SEQ ID NO: 1. Preferably, the PGK promoter contains SEQ ID NO: 1 or a sequence that is at least 75% identical to SEQ ID NO: 1. %, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 1. More preferably, the PGK promoter is SEQ ID NO: 1 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 1.

The identity of two sequences can be determined by standard methods. For example, it can be determined using standard alignment algorithms known in the art, such as BLAST (Altschul et al., 1990. J. Mol. Biol. 215 (3): 403-10). In a preferred embodiment of the invention, the identity of the two sequences is determined using BLAST.

SEQ ID NO: 2 means the following sequence:

TCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG.

In a preferred embodiment, the WPRE contains SEQ ID NO: 2 or a sequence that is at least 75% identical to SEQ ID NO: 2. Preferably, the WPRE contains SEQ ID NO: 2 or a sequence that is at least 75% identical 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 2. More preferably, WPRE is SEQ ID NO: 2 or the sequence which is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 2.

SEQ ID NO: 3 means the following sequence:

ATGTGGACTCTCGGGCGCCGCGCAGTAGCCGGCCTCCTGGCGTCACCCAGCCCGGCCCAGGCCCAGACCCTCACCCGGGTCCCGCGGCCGGCAGAGTTGGCCCCACTCTGCGGCCGCCGTGGCCTGCGCACCGACATCGATGCGACCTGCACGCCCCGCCGCGCAAGTTCGAACCAGAGAGGTCTCAACCAGATTTGGAATGTCAAAAAGCAGAGTGTCTATTTGATGAATTTGAGGAAATCTGGAACTTTGGGCCACCCAGGCTCTCTAGATGAGACCACCTATGAAAGACTAGCAGAGGAAACGCTGGACTCTTTAGCAGAGTTTTTTGAAGACCTTGCAGACAAGCCATACACGTTTGAGGACTATGATGTCTCCTTTGGGAGTGGTGTCTTAACTGTCAAACTGGGTGGAGATCTAGGAACCTATGTGATCAACAAGCAGACGCCAAACAAGCAAATCTGGCTATCTTCTCCATCCAGTGGACCTAAGCGTTATGACTGGACTGGGAAAAACTGGGTGTACTCCCACGACGGCGTGTCCCTCCATGAGCTGCTGGCCGCAGAGCTCACTAAAGCCTTAAAAACCAAACTGGACTTGTCTTCCTTGGCCTATTCCGGAAAAGATGCTT.

In a preferred embodiment of the invention, the nucleic acid sequence encoding frataxin contains SEQ ID NO: 3 or a sequence that is at least 75% identical to SEQ ID NO: 3 and is a functional variant of frataxin. Preferably, the nucleic acid sequence encoding frataxin contains SEQ ID NO: 3 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 3. More preferably, the nucleic acid sequence encoding frataxin is SEQ ID NO: 3 or a sequence that is at least 75%, 80%, 85%, 89% 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 3.

In a preferred embodiment of the invention, the nucleic acid further comprises a linker between the promoter and the nucleic acid sequence encoding frataxin, where the linker consists of SEQ ID NO: 6 or includes SEQ ID NO: 6, or a sequence that is at least 75% identical to the sequence of SEQ ID NO 6. Preferably, the linker consists of SEQ ID NO: 6, or includes SEQ ID NO: 6, or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92 %, 95%, 97%, 98% or 99% identical to SEQ ID NO: 6.

SEQ ID NO: 6 means the following sequence:

TGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGCCGCCACC.

In a preferred embodiment of the invention, the nucleic acid further comprises a Kozak sequence, and this Kozak sequence is also operably linked to a nucleic acid sequence encoding frataxin and regulates its expression. The Kozak sequence is a sequence that occurs in eukaryotic mRNA and has the consensus sequence gccRccAUGG where R is a purine; lowercase letters indicate bases that occur most frequently in these positions, where, however, they may vary; and capital letters show highly conservative bases.

SEQ ID NO: 4 means the following sequence:

GAATTCCGGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTCTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAGTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGCCGCCACCATGTGGACTCTCGGGCGCCGCGCAGTAGCCGGCCTCCTGGCGTCACCCAGCCCGGCCCAGGCCCAGACCCTCACCCGGGTCCCGCGGCCGGCAGAGTTGGCCCCACTCTGCGGCCGCCGTGGCCTGCGCACCGACATCGATGCGACCTGCACGCCCCGCCGCGCAAGTTCGAACCAGAGAGGTCTCAACCAGATTTGGAATGTCAAAAAGCAGAGTGTCTATTTGATGAATTTGAGGAAATCTGGAACTTTGGGCCACCCAGGCTCTCTAGATGAGACCACCTATGAAAGACTAGCAGAGGAAACGCTGGACTCTTTAGCAGAGTTTTTTGAAGACCTTGCAGACAAGCCATACACGTTTGAGGACTATGATGTCTC CTTTGGGAGTGGTGTCTTAACTGTCAAACTGGGTGGAGATCTAGGAACCTATGTGATCAACAAGCAGACGCCAAACAAGCAAATCTGGCTATCTTCTCCATCCAGTGGACCTAAGCGTTATGACTGGACTGGGAAAAACTGGGTGTACTCCCACGACGGCGTGTCCCTCCATGAGCTGCTGGCCGCAGAGCTCACTAAAGCCTTAAAAACCAAACTGGACTTGTCTTCCTTGGCCTATTCCGGAAAAGATGCTTTGCCCACCTAGGGATCGGATCCCCGGGTACCGAGCTCGAATTCTGCAGATATCCAGCACACTTTGCCTTTCTCTCCACAGGTGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG.

In a preferred embodiment of the invention, the nucleic acid sequence that contains (i), (ii) and (iii) is SEQ ID NO: 4 or a sequence that is at least 75% identical to SEQ ID NO: 4. Preferably, a nucleic acid that contains (i), (ii) and (iii) is SEQ ID NO: 4 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91% 92%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 4.

In a preferred embodiment of the invention, the nucleic acid further comprises a PolyA signal. Preferably, the PolyA signal is at least 50, 100, 150, or 228 bp long. More preferably, the PolyA signal is at least 228 bp long. Most preferably, the PolyA signal is 228 bp long.

In a preferred embodiment of the invention, the nucleic acid further comprises one or more inverted terminal repeat (ITR) sequences. Preferably, the nucleic acid contains two ITR sequences. More preferably, the ITR sequences flank the rest of the nucleic acid components.

SEQ ID NO: 5 means the following sequence:

CTGGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGAATTCCGGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTCTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAGTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGCCGCCACCATGTGGACTCTCGGGCGCCGCGCAGTAGCCGGCCTCCTGGCGTCACCCAGCCCGGCCCAGGCCCAGACCCTCACCCGGGTCCCGCGGCCGGCAGAGTTGGCCCCACTCTGCGGCCGCCGTGGCCTGCGCACCGACATCGATGCGACCTGCACGCCCCGCCGCGCAAGTTCGAACCAGAGAGGTCTCAACCAGATTTGGAATGTCAAAAAGCAGAGTGTCTATTTGATGAATTTGAGGAAATCTGGA ACTTTGGGCCACCCAGGCTCTCTAGATGAGACCACCTATGAAAGACTAGCAGAGGAAACGCTGGACTCTTTAGCAGAGTTTTTTGAAGACCTTGCAGACAAGCCATACACGTTTGAGGACTATGATGTCTCCTTTGGGAGTGGTGTCTTAACTGTCAAACTGGGTGGAGATCTAGGAACCTATGTGATCAACAAGCAGACGCCAAACAAGCAAATCTGGCTATCTTCTCCATCCAGTGGACCTAAGCGTTATGACTGGACTGGGAAAAACTGGGTGTACTCCCACGACGGCGTGTCCCTCCATGAGCTGCTGGCCGCAGAGCTCACTAAAGCCTTAAAAACCAAACTGGACTTGTCTTCCTTGGCCTATTCCGGAAAAGATGCTTTGCCCACCTAGGGATCGGATCCCCGGGTACCGAGCTCGAATTCTGCAGATATCCAGCACACTTTGCCTTTCTCTCCACAGGTGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCC TCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACGATCAGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTGGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT.

In a preferred embodiment, the nucleic acid is SEQ ID NO: 5, or a sequence that is at least 75% identical to SEQ ID NO: 5. Preferably, the nucleic acid is SEQ ID NO: 5, or a sequence that is at least 75% identical to SEQ ID NO: 5. 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 5.

In a preferred embodiment of the invention, the vector allows the expression of a therapeutically effective amount of frataxin in a patient suffering from Friedreich's ataxia. The vector thus delivers the nucleic acid to the patient's cells, where the nucleic acid then expresses frataxin in a therapeutically effective amount.

In a preferred embodiment of the invention, the AAV vector is a serotype 9 AAV vector, ie the AAV-9 vector.

Nucleic acid

In its second aspect, the present invention relates to a nucleic acid containing: (i) a nucleic acid sequence encoding frataxin; (ii) a PGK promoter; and (iii) WPRE; where (ii) and (iii) are operably linked to sequence (i) and regulate its expression.

In a preferred embodiment, the PGK promoter contains SEQ ID NO: 1 or a sequence that is at least 75% identical to SEQ ID NO: 1. Preferably, the PGK promoter contains SEQ ID NO: 1 or a sequence that is at least 75% identical to SEQ ID NO: 1. %, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 1. More preferably, the PGK promoter is SEQ ID NO: 1 or a sequence that is at least 75%, 80%, 85%, 89%, 905, 915, 92%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 1.

In a preferred embodiment, the WPRE contains SEQ ID NO: 2, or a sequence that is at least 75% identical to SEQ ID NO: 2. Preferably, the WPRE contains SEQ ID NO: 2, or a sequence that is at least 75%, 80 %, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 2. More preferably, the WPRE is SEQ ID NO: 2 or a sequence that at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 2.

In a preferred embodiment of the invention, the nucleic acid sequence encoding frataxin contains SEQ ID NO: 3 or a sequence that is at least 75% identical to SEQ ID NO: 3 and is a functional variant of frataxin. Preferably, the nucleic acid sequence encoding frataxin contains SEQ ID NO: 3 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 3. More preferably, the nucleic acid sequence encoding frataxin is SEQ ID NO: 3 or a sequence that is at least 75%, 80%, 85%, 89% 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 3.

In a preferred embodiment of the invention, the nucleic acid further comprises a linker between the promoter and the nucleic acid sequence encoding frataxin, where the linker consists of SEQ ID NO: 6 or contains SEQ ID NO: 6, or a sequence that is at least 75% identical to the sequence of SEQ ID NO 6. Preferably, the linker consists of SEQ ID NO: 6 or contains SEQ ID NO: 6 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 6.

In a preferred embodiment of the invention, the nucleic acid further comprises a Kozak sequence, and this Kozak sequence is also operably linked to a nucleic acid sequence encoding frataxin and regulates its expression.

In a preferred embodiment of the invention, the nucleic acid sequence that contains (i), (ii) and (iii) is SEQ ID NO: 4 or a sequence that is at least 75% identical to SEQ ID NO: 4. Preferably, the nucleic acid the acid that contains (i), (ii) and (iii) is SEQ ID NO: 4 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92 %, 95%, 97%, 98% or 99% identical to SEQ ID NO: 4.

In a preferred embodiment of the invention, the nucleic acid further comprises a PolyA signal. Preferably, the PolyA signal is at least 50, 100, 150, or 228 bp long. More preferably, the PolyA signal is at least 228 bp long. Most preferably, the PolyA signal is 228 bp long.

In a preferred embodiment of the invention, the nucleic acid further comprises one or more inverted terminal repeat (ITR) sequences. Preferably, the nucleic acid contains two ITR sequences. More preferably, the ITR sequences flank the rest of the nucleic acid components.

In a preferred embodiment, the nucleic acid is SEQ ID NO: 5, or a sequence that is at least 75% identical to SEQ ID NO: 5. Preferably, the nucleic acid is SEQ ID NO: 5, or a sequence that is at least 75% identical to SEQ ID NO: 5. 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 5.

Cloning vector

In its third aspect, the present invention relates to a cloning vector that contains a nucleic acid in accordance with any of the previously described embodiments of the invention, and additional nucleic acid elements for promoting replication of the cloning vector in a bacterial cell.

The term "cloning vector" means any vector that is suitable for cloning, and which typically includes restriction sites, an origin of replication for bacterial propagation, and a selectable marker.

The cloning vector according to the invention contains the nucleic acid according to the invention and can be preferably used to obtain a transfer vector or an AAV vector according to the invention.

Transfer Vector

In its fourth aspect, the present invention relates to a transfer vector that comprises a nucleic acid according to any of the previously described embodiments of the invention, and additional nucleic acid elements to promote integration or transposition of the transfer vector into an AAV vector, preferably the AAV vector. 9.

The term " transfer vector " means a vector that is suitable for integration or transposition into an AAV vector. Thus, the transfer vector essentially allows the insertion of genetic information into the AAV vector.

The transfer vector of the invention comprises the nucleic acid of the invention and can be used to produce an AAV vector of the invention.

Use of Nucleic Acid, Cloning Vector and Transfer Vector

In its fifth aspect, the present invention relates to the use of a nucleic acid according to the invention, a cloning vector according to the invention or a transfer vector according to the invention in order to obtain an AAV vector according to any of the previously described embodiments of the invention.

pharmaceutical composition

In its sixth aspect, the present invention relates to a pharmaceutical composition comprising an AAV vector according to the invention or a nucleic acid according to the invention and a pharmaceutically acceptable carrier or diluent.

The pharmaceutical composition described herein may also contain other substances. Such substances include, but are not limited to, cryoprotectants, lyoprotectants, surfactants, fillers, antioxidants, and stabilizers. In some embodiments of the invention, the pharmaceutical composition may be lyophilized.

The term "cryoprotectant" as used herein includes agents that render the AAV vector resistant to freezing stress, predominantly by removing the AAV vector from the surface. Cryoprotectants can also provide protection during primary and secondary drying and long-term storage of the product. Non-limiting examples of cryoprotectants are sugars such as sucrose, glucose, trehalose, mannitol, mannose, and lactose; polymers such as dextran, hydroxyethyl starch and polyethylene glycol; surfactants such as polysorbates (eg PS-20 or PS-80); and amino acids such as glycine, arginine, leucine and serine. A cryoprotectant with low toxicity in biological systems is usually used.

In one embodiment of the invention, the lyoprotectant is added to the pharmaceutical composition described here. As used herein, the term "lyoprotectant" includes agents that provide stability to the AAV vector during freeze-drying or during dehydration (in primary and secondary freeze-drying cycles) by creating an amorphous glassy matrix and binding to the surface of the AAV vector via hydrogen bonding followed by replacement water molecules that are removed during the drying process. This minimizes product degradation during the lyophilization cycle and imparts long-term stability to the product. Non-limiting examples of lyoprotectants are sugars such as sucrose or trehalose; an amino acid such as sodium monoglutamate, non-crystalline glycine or histidine; methylamine such as betaine; a lyotropic salt such as magnesium sulfate; a polyol such as trihydric alcohols or polyhydric alcohols of a number of sugars, for example, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol and mannitol; propylene glycol; polyethylene glycol; pluronics; and their combinations. The amount of lyoprotectant added to a pharmaceutical composition is usually an amount that does not result in an unacceptable level of degradation of the strain if the pharmaceutical composition is lyophilized.

In some embodiments of the invention, the pharmaceutical composition includes a filler. As used herein, the term "filler" includes agents that provide the desired structure to the lyophilized product without direct interaction with the pharmaceutical product. Excipients, in addition to imparting a pharmaceutically elegant appearance to the product, can also confer beneficial properties to the product in terms of compression temperature change, freeze-thaw protection, and increased strain stability over a long storage period. Non-limiting examples of fillers are mannitol, glycine, lactose and sucrose. The excipients may be crystalline (eg glycine, mannitol or sodium chloride) or amorphous (eg dextran, hydroxyethyl starch) and are typically used in the compositions in amounts of 0.5% to 10%.

Other pharmaceutically acceptable carriers, excipients or stabilizers as described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), provided that they do not adversely affect the desired properties of the pharmaceutical composition. As used herein, the term " pharmaceutically acceptable carrier " means any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, and absorption retardants that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known to those skilled in the art. Suitable carriers, excipients or stabilizers are non-toxic to recipients at the doses and concentrations used and include the following compounds, which do not limit the scope of the present invention, namely: additional buffering agents; preservatives; co-solvents; antioxidants including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (eg Zn-protein complexes); biodegradable polymers such as polyesters; salt-forming counterions such as sodium, polyhydric alcohols of a number of sugars; amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid and threonine; organic sugars or sugar alcohols such as lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (eg inositol), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; and hydrophilic polymers such as polyvinylpyrrolidone.

The pharmaceutical composition may be formulated for oral, sublingual, buccal, intravenous, intramuscular, subcutaneous, intraperitoneal, conjunctival, rectal, transdermal, intrathecal and topical administration and/or administration by inhalation. In a preferred embodiment of the invention, the pharmaceutical composition may be a solution which is suitable for intravenous, intramuscular, conjunctival, transdermal, intraperitoneal and/or subcutaneous administration. In another embodiment of the invention, the pharmaceutical composition may be a solution that is suitable for sublingual and buccal administration and/or administration by inhalation. In an alternative embodiment of the invention, the pharmaceutical composition may be a gel or solution which is suitable for intrathecal administration. In an alternative embodiment of the invention, the pharmaceutical composition may be an aerosol suitable for administration by inhalation. In a preferred embodiment of the invention, the pharmaceutical composition may be formulated for intrathecal administration.

The pharmaceutical composition may further contain conventional excipients and carriers that are known to those skilled in the art. For solid pharmaceutical compositions, standard non-toxic solid carriers can be used, which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. In order to prepare a solution for injection, the pharmaceutical composition may additionally contain cryoprotectants, lyoprotectants, surfactants, excipients, antioxidants, stabilizers and pharmaceutically acceptable carriers. For aerosol administration, pharmaceutical compositions are usually supplied in finely divided form along with a surfactant and a propellant. It goes without saying that the surfactant must be non-toxic and substantially soluble in the propellant. Such representative agents are esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters such as mixed or natural glycerides can be used. If desired, a carrier may also be included, such as, for example, lecithin for intranasal delivery. In the case of suppositories, conventional binders and carriers may be included, such as polyalkylene glycols or triglycerides.

Application in medicine

In its seventh aspect, the present invention relates to an AAV vector according to the invention, to a nucleic acid according to the invention, or to a pharmaceutical composition according to the invention for their use as a medicine. In its eighth aspect, the present invention provides an AAV vector according to the invention, a nucleic acid according to the invention, or a pharmaceutical composition according to the invention for their use in the treatment of Friedreich's ataxia.

In a preferred embodiment of the invention, the AAV vector according to the invention, the nucleic acid according to the invention or the pharmaceutical composition according to the invention are administered intrathecally, intramuscularly, intracerebrally or intracerebroventricularly, preferably intrathecally or intramuscularly.

In a preferred embodiment of the invention, the AAV vector according to the invention, the nucleic acid according to the invention or the pharmaceutical composition according to the invention are administered at a dose of at least 1×10 ⁹ vector genomes/kg body weight, preferably 1×10 ¹⁰ , 1×10 ¹¹ or 1× 10 ¹² vector genomes/kg body weight. More preferably, at least or approximately 4.6×10 ¹² vector genomes/kg of body weight are administered.

Examples

Example 1 Construction of a Plasmid

The human frataxin isoform 1 (hFXN) coding sequence was attached to a hemagglutinin tag (HA) and cloned into the pcDNA3.1 expression vector using the In-Fusion® HD cloning kit (Clontech). This hybrid system was used in all stages of cloning. Several constructs were generated by attaching CMV promoters or human (h) promoters (p) synapsin (phSYN), neuron-specific enolase (phNSE), 1255 bp. FXN promoter (phFXN1255) or human phosphoglycerate kinase isoform 1 (phPGK1) to the coding region of the FXN gene. Additional regulatory elements such as the CMV enhancer and Kozak sequences were added at the 5' end, and the woodchuck hepatitis virus response element (WPRE) sequence was added at the 3' end. All of these expression vector constructs are listed in Table 2.

Plasmids containing the same combination of regulatory elements were also generated to initiate luciferase expression by replacing the FXN coding sequence with the firefly luciferase (LUC) coding sequence, which was amplified from the pGL3-LUC vector (Promega), also using the In cloning kit. -Fusion® HD (Clontech). These plasmids are listed in Table 3.

Example 2 Construction and production of a recombinant adeno-associated viral vector.

The expression cassettes of plasmid pcDNA3.1-phPGK-kFXN-HA-WPRE and plasmid pcDNA3.1-phPGK-kLUC-HA-WPRE were cloned into the SnaBI-MfeI sites of the recombinant AAV-9 vector (vector rAAV9-phPGK1-FXN-HA-WPRE and vector rAAV9-phPGK-LUC-WPRE). In addition, a control null vector without the FXN coding sequence (AAV2/9-null) was generated. All three constructs and viral particles were obtained in the vector production laboratory at the Center for Biotechnology and Animal Gene Therapy (Universitat Autònoma de Barcelona). The final titers obtained were 1.4×10 ¹³ VG/ml for AAV9-phPGK-FXN-HA-WPRE, 9.8×10 ¹² VG/ml for AAV9-phPGK-LUC-WPRE and 6×10 ¹² VG/ml for AAV2/9-null.

Example 3 Optimization of Frataxin Expression

1. Cell culture, expression in vitro and assay for the luciferase reporter gene.

Mouse neuroblastoma cells (N2a), human neuroblastoma cells (SH-SY5Y), and human embryonic kidney cells (HEK 293) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (Sigma), 2 mM glutamine, 50 µg/ml penicillin/streptomycin (Life technologies) at 70% confluence in 10 cm culture dishes. Transfection was performed using Lipofectamine 2000 (Life technologies) for N2a and SH-SY5Y cells and calcium phosphate for HEK 293, using only 4 μg of plasmid DNA in addition to 0.25 μg of EGFP. After transfection, medium was replaced with fresh DMEM culture medium for HEK cells and medium containing Neurobasal, B27 supplement, 10 μM retinoic acid, 2 mM glutamine, 50 μg/ml penicillin/streptomycin for N2a and SH-SY5Y cells. 48 hours after changing the cell culture medium, cells transfected with the expression plasmids listed in Table 2 were collected and stored at -80°C until further use. For the purposes of the luciferase reporter gene assay, the plasmids listed in Table 3 were transfected as above and the cells were replated in 384-well plates, and luciferase activity was determined using the luciferase two reporter assay kit according to the instructions. manufacturer (Promega).

2. Electrophoresis in SDS-PAGE and immunoblotting

Proteins were extracted from cultured cells by homogenization in RIPA lysis buffer: 10 mM Tris-HCl, pH 7.4, 140 mM NaCl, 0.1% sodium deoxycholate, 1% Triton X-100, 1% SDS, 2 mM EDTA (EDTA), 25 mM sodium fluoride (NaF), 2.5 mM NaVO ₃ and protein inhibitor cocktail (Roche). Protein concentration was determined using the DC-BioRad protein assay (BioRad). The total protein extract was mixed with 4× Protein Sample Loading Buffer (Li-Cor Biosciences) containing 1 mM DTT and separated by 15% acrylamide gel electrophoresis at a constant current of 20 mA prior to transfer to PVDF membranes. Anti-FXN antibody PAC 2518 (courtesy of Dr. Grazia Isaya, Mayo Clinic, Rochester MN), anti-FXN antibody (1G2, Merck Millipore), anti-HA tag antibody (clone 16B12, MMS-101P , Covance) and an anti-beta actin antibody (AC15, Sigma). The infrared dye conjugated "second" antibodies were anti-mouse IRDye-800CW and anti-rabbit IRDye 700CW (Li-Cor Biosciences), and immunoreactivity was determined using the Odyssey v2.1 analyzer software (Li-Cor Biosciences).

3. Results

As can be seen in Figures 1-5 and Tables 2-3, the generation of a construct containing the hPGK1 promoter, linker sequences (of defined length and composition) and WPRE allowed frataxin to be expressed in cells. The human PGK1 promoter is a metabolically regulated promoter expressed in physiologically relevant tissues, making it a good candidate for a frataxin promoter. However, it was not clear whether regulatory elements or linkers with a specific sequence/length could express frataxin encoding sequences. The results obtained by the authors using various combinations of elements and sequences showed that protein expression from human frataxin coding sequences requires the inclusion of an operably linked linker between the hPGK1 promoter and FXN coding sequences, since constructs of approximately 105 nucleotides in length allow the expression of FXN, and linkers of approximately 35 nucleotides in length do not perform this function even with the addition of the WPRE RNA stabilizer sequence. It is interesting to note that the linker was only required to do so when using the hPGK1 promoter and not the other promoters tested (ie, hSYN). In addition, levels of frataxin protein expressed from this vector were much lower than those obtained from vectors inducible by the CMV or CAG promoter, even in the absence of WPRE sequences, and thus these levels were more comparable to endogenous levels. One of the drawbacks of previous approaches is that highly inducible promoters such as CAG or CMV promoters were used to achieve frataxin expression, which gave very high levels of protein expression capable of inducing cellular stress and overriding some of the protective effects. The vector presented here can induce the expression of the frataxin protein in the appropriate nervous system and in peripheral tissues in amounts within the physiologically functional range.

Table 2: Summary of results. Relative levels of FXN after transfection with various constructs Plasmids expressing frataxin cell type Antibody Immunoreactivity 1.1 pcDNA3.1-pCMV-FXN-HA N2A/HEK293/SHSY Anti-HA tag (Covance)/anti-FXN 1G2 (Merck Millipore) ++ 1.2 pcDNA3.1-phSYN-FXN-HA SHSY Antibody against HA-tag (Covance) = 1.3 pcDNA3.1-phFXN-FXN-HA HEK293/SHSY Antibody against HA-tag (Covance) - 1.4 pcDNA3.1-prNSE-FXN-HA N2A Antibody against HA-tag (Covance) - 1.5 pcDNA3.1-phNSE-FXN-HA HEK293 Antibody against HA-tag (Covance) - Adding WPRE 2.1 pcDNA3.1-pCMV-FXN-HA-WPRE N2A/HEK293 Anti-HA tag (Covance)/anti-FXN 1G2 (Merck Millipore) +++ 2.2 pcDNA3.1-phSYN-FXN-HA-WPRE N2A/HEK293 Antibody against HA-tag (Covance) - 2.3 pcDNA3.1-phSYN-5'UTR-FXN-FXN-HA-WPRE N2A/HEK293 Antibody against HA-tag (Covance) - 2.4 pcDNA3.1-phFXN-FXN-HA-WPRE N2A/HEK293 Antibody against HA-tag (Covance) - 2.5 pcDNA3.1-prNSE-FXN-HA-WPRE N2A Antibody against HA-tag (Covance) - Addition of CMV enhancer 3.1 pcDNA3.1-E-phFXN-FXN-HA-WPRE HEK293 Antibody against the HA-tag (Covance - 3.2 pcDNA3.1-E-phFXN-FXN-HA N2A/HEK293 Antibody against the HA-tag (Covance - 3.3 pcDNA3.1-E-phSYN-FXN-HA-WPRE N2A Antibody against the HA-tag (Covance - Addition of Kozak and WPRE sequences 4.1 pcDNA3.1-pCMV-105nt-kFXN-HA-WPRE N2A/HEK293 Anti-HA tag (Covance)/anti-FXN 1G2 (Merck Millipore) ++++ 4.2 pcDNA3.1-phSYN-kFXN-HA-WPRE HEK293/N2A Antibody against HA-tag (Covance) -/+ 4.3 pcDNA3.1-prNSE-kFXN-HA-WPRE HEK293/N2A Antibody against HA-tag (Covance) -/+ 4.4 pcDNA3.1-phFXN-kFXN-HA-WPRE HEK293 Antibody against HA-tag (Covance) - 4.5 pcDNA3.1-E-phFXN-kFXN-HA-WPRE HEK293 Antibody against HA-tag (Covance) - Application of specific linker, Kozak sequence and WPRE 5.1 pcDNA3.1-phPGK-105nt-kFXN-HA-WPRE N2a/HEK293 Anti-HA tag (Covance)/anti-FXN 1G2 (Merck Millipore) ++ 5.2 pcDNA3.1-phNSE-105nt-kFXN-HA-WPRE N2a/HEK293 Antibody against HA-tag (Covance) + 5.3 pcDNA3.1-phSYN-105nt-kFXN-HA-WPRE N2a/HEK293 Antibody against HA-tag (Covance) - 5.4 pcDNA3.1-phFXN-105nt-kFXN-HA-WPRE N2A/HEK293 Antibody against HA-tag (Covance) -

Table 3: Analysis for frataxin and PGK1 promoter reporter constructs Plasmids expressing luciferase cell type Expression pcDNA3.1-pCMV-135nt-LUC-WPRE N2a/HEK293 ++++ pcDNA3.1-phPGK-135nt-LUC-WPRE N2a/HEK293 ++ pcDNA3.1-E-phPGK-135nt-LUC-WPRE N2a ++ pcDNA3.1-phFXN1255-135nt-LUC-WPRE HEK293 - pcDNA3.1-phFXN1255-35nt-LUC-WPRE N2a/HEK293 - pcDNA3.1-phFXN220-35nt-LUC-WPRE N2a/HEK293 + pcDNA3.1-phFXN1255OCT-35nt-LUC-WPRE N2a/HEK293 -

Example 4: Data in vivo o

1. Animals

Friedreich ataxia model YG8R mice obtained by Pook et al. (Pook et al. 2001. Neurogenetics. 3(4):185-93; Virmouni et al. in this study were taken from the collections of the Jackson Laboratory (accession number 012253). These human FXN model transgenic mice were knocked out at the endogenous mouse frataxin gene (Fxn -/-) and contained the human YAC transgene from the original YG8 (carrying two tandem copies of the human FXN gene with approximately 82 and 190 GAA trinucleotide repeat sequences). Mice were housed under SPF-like conditions in NexGen IVC Mouse Cages (Allentown, NJ) with a 12-hour day-night cycle and controlled negative pressure, temperature, and humidity, as well as free access to water and irradiated food. for rodents Teklad with a total protein content of 18% (Envigo). A group of YG8R mice was obtained from the Germans Trias i Pujol Research Institute for Health (IGTP). In all experiments, hemizygous female and male YG8R (Tg/-) and C57Bl/6 wild-type mice were used.

Mice hemizygous for the mutated human FXN gene (YG8R) were genotyped as previously reported (29,36). Briefly, genomic DNA was extracted from the tail of a YG8R mouse using a DNA purification kit taken from the tail of a Maxwell® 16 mouse (Promega). The copy number of the transgenes was determined for each mouse by quantitative real-time PCR (qPCR) on a LightCycler® 480 device (Roche) using the SYBR Premix Ex Taq II enzyme (RNase Tli H Plus) (Takara) and the following primers for the human transgene. frataxin: hFXN_Tg_FW: 5'-GAACTTCAAATTAGTTCCCCTTTCTTC-3' (SEQ ID NO: 7), hFXN_Tg_RV: 5'-CACAGCCATTCTTTGGGTTTC-3' (SEQ ID NO: 8); and internal control, apolipoprotein B-100 isoform X1: IC_FW: 5'-CACGTGGGCTCCAGCATT-3' (SEQ ID NO: 9), IC_RV: TCACCAGTCATTTCTGCCTTTG (SEQ ID NO: 10). The analysis was carried out by conducting thermal cycles under the following conditions: 95°C for 5 minutes and 40 cycles at 95°C for 20 seconds and 60°C for 30 seconds, 72°C for 30 seconds and finally 1 cycle at 72°C for 2 minutes. Samples were analyzed in triplicate for each gene of interest, and transgene levels were determined by the Ct (ΔΔCt) method. The number of copies of the transgenics was estimated in comparison with the Ct data for the control sample with a known number of copies. GAA repeat length was determined by PCR amplification of GAA using LA taq (Invitrogen) and the following primers: GAA-F: 5'-GGGATTGGTT-GCCAGTGCTT-AAAAGTTAG-3' (SEQ ID NO: 11) and GAA-R: 5' -GATCTAAGGACCATCATGG-CCACACTTGCC-3' (SEQ ID NO: 12). The PCR products were separated on 1.5% agarose gels by electrophoresis at 100 V for 3 hours and the band sizes were analyzed. The number of GAA repeats was then determined by subtracting 451 bp. (flanking non-repeated DNA) from the size of the PCR product and dividing the resulting base pair repeat size by 3.

All animal procedures were carried out in accordance with EU approved guidelines and approved by local Regulatory Authorities and relevant local Ethics Committees.

2. Introduction of AAV

Hemizygous YG8R mice and 10-week-old wild-type mice were anesthetized by intraperitoneal injection of ketamine (10 mg/kg bw; Imalgene 500; Rhône-Merieux, Lyon, France) and xylazine (1 mg/kg bw; Rompun; Bayer). Intrathecal administration of AAV9-phPGK-FXN-HA-WPRE, AAV9-phPGK-LUC-WPRE or AAV2/9-null was performed in the lumbar region. After opening the lateral spine by excision of the paravertebral muscles, the viral vectors were slowly injected into the CSF with a 33 gauge needle and a Hamilton syringe between the lumbar vertebrae L3 and L4. Appropriate access to the intrathecal space was confirmed by movement of the animal's tail. After that, the muscles and skin were sutured. The number of different viral vectors administered to each mouse was 4.6×10 ^-12 VG/kg mouse weight.

3. Biodistribution of the vector using luciferase imaging.

To assess the in vivo biodistribution of the AAV9-phPGK-LUC-WPRE vector, mice at the age of 10 weeks (2.5 months) were intrathecally injected with the vector, and after another 3.5 months, mice were injected with one intraperitoneal injection of a solution of the substrate D-luciferin in an amount of 150 mg /kg mouse weight. Mice were anesthetized 15 minutes after substrate administration by inhalation anesthesia (isoflurane 4% for induction and 2% for maintenance). The anesthetized mice were kept in a dark chamber Perkin Elmer Ivis Lumina II (Caliper Life Sciences, Germany) for registration of photon emission. Images were analyzed with Living Imaging software (Xenogen Corporation, CA, EUA) where integration time was 1 min and field of view = 12.5 cm. For ex vivo evaluation, mouse tissues and organs were harvested 15 minutes after substrate injection. Tissues and organs were placed in transparent dishes and images were captured using the Living Imaging imaging software (Xenogen Corporation, CA, EUA) as described above. The results can be seen in figure 6.

4. number of RT-PCR

RNA was extracted from 30 mg fresh frozen mouse tissue using the RNeasy Mini kit (Qiagen). RIN analysis for RNA and RNA quantification were performed on an Agilent 2200 TapeStation (Agilent). RNA was reverse transcribed into cDNA (25 ng/µl) using the PrimeScriptTM RT reagent kit (Takara) under thermal conditions: 37°C for 15 minutes, 85°C for 5 seconds. col.RT-PCR was performed using cDNA taken from mouse tissues to assess frataxin expression levels in a multiwell plate on a LightCycler480 device (Roche Diagnostics). Reaction mixtures contained a total volume of 10 μl, consisting of 0.1 mM of each primer, 10 ng of cDNA, and 5 μl of TaqMan Universal Master Mix II, without UNG (Thermofisher Scientific). The human frataxin detection primer probe was pre-engineered by Bio-rad (dHsaCPE5031641, Bio-rad) and the beta-2-microglobulin (B2m) housekeeping gene primers for mouse samples were pre-engineered by IDT (Mm.PT.39a .22214835;IDT). The analysis was carried out by conducting thermal cycles under the following conditions: 50°C for 2 minutes, 95°C for 10 minutes and 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Samples were analyzed in triplicate for each gene of interest, and transgene levels were determined by the Ct (ΔΔCt) method. The results can be seen in Figure 7.

5. Electrophoresis in SDS-PAGE and immunoblotting

Proteins were extracted from mouse tissue by homogenization in RIPA lysis buffer: 10 mM Tris-HCl, pH 7.4, 140 mM NaCl, 0.1% sodium deoxycholate, 1% Triton X-100, 1% SDS, 2 mM EDTA (EDTA), 25 mM sodium fluoride (NaF), 2.5 mM NaVO₃ and a cocktail of protein inhibitors (Roche). Protein concentration was determined using the DC-BioRad protein assay (BioRad). The total protein extract was mixed with 4× Protein Sample Loading Buffer (Li-Cor Biosciences) containing 1 mM DTT and separated by 15% acrylamide gel electrophoresis at a constant current of 20 mA prior to transfer to PVDF membranes. Anti-FXN antibody PAC 2518 (courtesy of Dr. Grazia Isaya, Mayo Clinic, Rochester MN), anti-FXN antibody (1G2, Merck Millipore), anti-HA tag antibody (clone 16B12, MMS-101P , Covance) and anti-beta actin antibody (AC15, Sigma). The infrared dye-conjugated "second" antibodies were anti-mouse IRDye-800CW and anti-rabbit IRDye 700CW (Li-Cor Biosciences), and immunoreactivity was determined using the Odyssey v2.1 analyzer program (Li-Cor Biosciences). The results can be seen in figure 8.

6. Pressing the paws

Hindleg tuck has been shown to be an indicator of disease progression in a range of mouse models of neurodegeneration. Each mouse was lifted by the tail above any surrounding objects. The position of the hind limbs was observed for 10 seconds and was evaluated as follows: if the hind limbs were consistently extended outward from the peritoneum, then a score of 0 was assigned. If one hind limb was drawn to the stomach with more than 50% time delay, then a score of 1 was assigned A score of 2 was assigned if both hind limbs were partially tucked in to the abdomen with more than 50% time lag, then a score of 3 was assigned. limb curls were assessed every 2 months in mice aged 4 months or older. The results can be seen in figure 9.

7. Electrophysiology

Amplitude (μV) and nerve conduction velocity (m/s) were measured in the caudal nerve of the mouse tail. In animals under inhalation anesthesia (2% isoflurane), needles with a stimulus electrode were placed subcutaneously at 4 different stimulation points (1 cm, 2 cm, 3 cm, 4 cm from the tip of the tail), while the tip of the recording needle was fixed at a distance 6 cm from the tip of the tail. Values were recorded using 2-channel EMG/PE N-EP on Medelec Synergy (Viasys, EUA). During electrophysiological tests, the temperature of the skin of the animals was maintained above 32°C. Mice were assessed every 2 months from 4 months of age. The results can be seen in figure 10.

8. Results

The authors suggest that treatment with the AAV vector according to the invention will be effective for FRDA, since AAV can be expressed in physiologically relevant tissues, and at levels showing a process similar to that for an endogenous protein, and can significantly improve the electrophysiological properties of affected neurons and neurological symptoms in mice with the FRDA model (YG8R). Figure 6 shows that the luciferase gene in the AAV9 vector, under the control of the same regulatory elements as FXN in the AAV9-hPGK1-FXN vector, when administered intrathecally, is expressed throughout the spinal cord, brain, and also in peripheral tissues. In addition, the expression of luciferase under the control of the same vector and regulatory elements remains constant for at least about 7 months after injection (data not shown). Since reduced frataxin levels underlie most, if not all, FRDA symptoms, and individual carrier individuals with a mutation expressing frataxin levels ≥25% do not have FRDA symptoms, a small increase in FXN expression should prevent or greatly alleviate symptoms in patients with FRDA. However, very high levels of expression of FXN or any other protein can induce cellular effects and ultimately reduce protective effects or even exacerbate symptoms. Therefore, it is of particular interest to develop an FXN expression vector that produces levels nearly the same as those of endogenous FXN protein. Therefore, the authors developed a vector (AAV9-hPGK1-FXN) that allows FXN to be expressed under the control of a metabolically regulated promoter at physiologically significant levels and delivered intrathecally to 10-week-old hemizygous YG8R (Tg/-) mice. Higher levels of FXN mRNA were detected in hemizygous YG8R (Tg/-) mice 3.5 months after intrathecal injection of the AAV9-PGK1-FXN vector compared to uninjected mice, both in the liver and in the thoracic spinal cord (at 0 , 5 and 2 times, respectively; Figure 7). These results were the same or lower than homozygous YG8R mice, which had spinal cord levels 0.5 and 3 times higher, respectively, compared to hemizygous YG8R mice. This indicates that the FXN-coding vector injected into the L3-L4 intrathecal region was taken up and expressed in neurons of the thoracic spinal cord, either by reverse transport or by diffusion through the cerebrospinal fluid. The authors also noted that the intrathecally administered AAV9 vector is also able to cross the blood-brain barrier and reach peripheral tissues such as the liver, heart, and the like. (Figure 6) and is able to express hFXN mRNA after injection of this vector (Figure 7).

The expression levels of the FXN protein from the resulting recombinant vector were also analyzed. Although the antibody used has a higher affinity for human FXN protein, as indicated by the protein structure observed after incubating the blot with the "first" anti-FXN antibody for 1 hour compared to overnight incubation, however, Figure 8 shows that FXN protein levels expressed from the rAAV9-PGK1-FXN vector appear to be similar to mouse FXN levels in wild-type mice (Figure 8A, lanes 3 and 4 and Figure 8B, second panel, lane 3 versus lane 4 and lane 5 compared to lanes 6 and 7). Notably, the authors were able to detect recombinant FXN protein in the motor neuron region of the cerebral cortex (C1) in mice (Fig. 8B, lane 7). This indicates that the delivery of the vector obtained by the authors by intrathecal injection into the lumbar spinal cord leads to the distribution of the vector in the areas of the CNS related to the motor system. In addition, the recombinant protein appears to be processed in a manner similar to that of the wild-type FXN protein, with its intermediate and mature forms being the most common processed forms.

The authors also analyzed the phenotype of YG8R (Tg/-) hemizygous mice treated with the rAAV9-PGK1-FXN vector, compared with the phenotype of mice treated with the rAAV9-null vector, in terms of neurological paw tuck reflex and electrophysiological properties of the caudal nerve. The push reflex has been shown to be observed in several neurodegenerative disorders. This reflex appears to involve sensory as well as spinal motor pathways that regulate forelimb and hindlimb movements. In particular, the involvement of the cortico-cerebellar-reticular pathways has been shown. Therefore, to determine the effect of rAAV9-hPGK1-FXN vector treatment on these pathways in YG8R mice, the authors quantified the push reflex at various time points after intrathecal vector injection (see Methods). Figure 9 shows that YG8R mice treated with AAV9-null and AAV9-FXN vectors show defects in paw tuck as early as four months of age (1.5 months after intrathecal injection). However, over time, this paw-paw reflex normalizes in AAV-FXN-treated mice, and is more similar to the effect in wild-type mice, even at an older age of over 10 months (6.5 months after injection). This suggests that abnormalities in the sensory and cortico-cerebellar-reticular systems in FRDA model mice are observed early and are largely reduced or eliminated by treatment with the AAV-FXN vector.

To more accurately determine the effect of treatment on sensory neurons, the authors evaluated the electrophysiological properties (amplitude and velocity) of the caudal nerve in YG8R mice treated with the AAV-FXN vector, compared with those in mice treated with the control AAV9-null vest. Figure 10 shows that at all distances from the tip of the tail (1-4 cm) there were no significant differences between wild-type mice treated with null vector or hemizygous YG8R mice treated with null vector and AAV-FXN vector at the age of four months (1.5 months after treatment). However, starting at 6 months of age, YG8R (Tg/-) mice treated with the AAV9-null vector showed a significant decrease in amplitude, and mice treated with AAV9-FXN were more similar to wild-type mice even at a later age. namely, 13 months (9.5 months after treatment). No change in speed was detected (data not shown). These data indicate that treatment with the AAV-hPGK1-FXN vector preserved caudal nerve function in YG8R mice, possibly by preventing loss of axons from affected neurons.

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SEQUENCE LIST

<110> FUNDACIÓ INST D'INVESTIGACIÓ EN CIÈNCIES DE LA SALUT GERMANS TRIAS I

PUJOL

<120> Vectors for the treatment of Friedreich's ataxia

<130> 903 551

<160> 12

<170> BiSSAP 1.3.6

<210> 1

<211> 518

<212> DNA

<213> Artificial sequence

<220>

<223> PGK promoter

<400> 1

gaattccggg gttggggttg cgccttttcc aaggcagccc tgggtctgcg cagggacgcg 60

gctgctctgg gcgtggttcc gggaaacgca gcggcgccga ccctgggtct cgcacattct 120

tcacgtccgt tcgcagcgtc acccggatct tcgccgctac ccttgtgggc cccccggcga 180

cgcttcctgc tccgccccta agtcgggaag gttccttgcg gttcgcggcg tgccggacgt 240

gacaaacgga agccgcacgt ctcactagta ccctcgcaga cggacagcgc cagggagcaa 300

tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg 360

agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg tggggcggta gtgtgggccc 420

tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg 480

gctccctcgt tgaccgaatc accgacctct ctccccag 518

<210> 2

<211> 597

<212> DNA

<213> Artificial sequence

<220>

<223>WPRE

<400> 2

tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg 60

ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt 120

180

agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc 240

ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc 300

tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc 360

ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc 420

tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg 480

ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc 540

gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctg 597

<210> 3

<211> 631

<212> DNA

<213> Artificial sequence

<220>

<223> Frataxin

<400> 3

atgtggactc tcgggcgccg cgcagtagcc ggcctcctgg cgtcacccag cccggcccag 60

gccgaccc tcacccgggt cccgcggccg gcagagttgg ccccactctg cggccgccgt 120

ggcctgcgca ccgacatcga tgcgacctgc acgccccgcc gcgcaagttc gaaccagaga 180

ggtctcaacc agatttggaa tgtcaaaaag cagagtgtct atttgatgaa tttgaggaaa 240

tctggaactt tgggccaccc aggctctcta gatgagacca cctatgaaag actagcagag 300

gaaacgctgg actctttagc agagtttttt gaagaccttg cagacaagcc atacacgttt 360

gaggactatg atgtctcctt tgggagtggt gtcttaactg tcaaactggg tggagatcta 420

ggaacctatg tgatcaacaa gcagacgcca aacaagcaaa tctggctatc ttctccatcc 480

agtggaccta agcgttatga ctggactggg aaaaactggg tgtactccca cgacggcgtg 540

tccctccatg agctgctggc cgcagagctc actaaagcct taaaaaccaa actggacttg 600

tcttccttgg cctattccgg aaaagatgct t 631

<210> 4

<211> 1934

<212> DNA

<213> Artificial sequence

<220>

<223> Frataxin, PGK and WPRE cluster

<400> 4

gaattccggg gttggggttg cgccttttcc aaggcagccc tgggtctgcg cagggacgcg 60

gctgctctgg gcgtggttcc gggaaacgca gcggcgccga ccctgggtct cgcacattct 120

tcacgtccgt tcgcagcgtc acccggatct tcgccgctac ccttgtgggc cccccggcga 180

cgcttcctgc tccgccccta agtcgggaag gttccttgcg gttcgcggcg tgccggacgt 240

gacaaacgga agccgcacgt ctcactagta ccctcgcaga cggacagcgc cagggagcaa 300

tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg 360

agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg tggggcggta gtgtgggccc 420

tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg 480

gctccctcgt tgaccgaatc accgacctct ctccccagtg gctaactaga gaacccactg 540

cttactggct tatcgaaatt aatacgactc actataggga gacccaagct ggctagcgtt 600

taaacttaag cttggccgcc accatgtgga ctctcgggcg ccgcgcagta gccggcctcc 660

tggcgtcacc cagcccggcc caggcccaga ccctcacccg ggtcccgcgg ccggcagagt 720

tggccccact ctgcggccgc cgtggcctgc gcaccgacat cgatgcgacc tgcacgcccc 780

gccgcgcaag ttcgaaccag agaggtctca accagatttg gaatgtcaaa aagcagagtg 840

tctatttgat gaatttgagg aaatctggaa ctttgggcca cccaggctct ctagatgaga 900

ccacctatga aagactagca gaggaaacgc tggactcttt agcagagttt tttgaagacc 960

ttgcagacaa gccatacacg tttgaggact atgatgtctc ctttgggagt ggtgtcttaa 1020

ctgtcaaact gggtggagat ctaggaacct atgtgatcaa caagcagacg ccaaacaagc 1080

aaatctggct atcttctcca tccagtggac ctaagcgtta tgactggact gggaaaaact 1140

gggtgtactc ccacgacggc gtgtccctcc atgagctgct ggccgcagag ctcactaaag 1200

ccttaaaaac caaactggac ttgtcttcct tggcctattc cggaaaagat gctttgccca 1260

cctagggatc ggatccccgg gtaccgagct cgaattctgc agatatccag cacactttgc 1320

ctttctctcc acaggtgtcg acaatcaacc tctggattac aaaatttgtg aaagattgac 1380

tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt 1440

gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt 1500

1560

gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg 1620

1680

ctgctggaca ggggctcggc tgtggggcac tgacaattcc gtggtgttgt cggggaagct 1740

gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt 1800

ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc 1860

tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc 1920

cgcctccccg cctg 1934

<210> 5

<211> 2458

<212> DNA

<213> Artificial sequence

<220>

<223> Frataxin ITR, PGK, polyA-ITR WPRE

<400> 5

ctggcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt 60

tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac 120

taggggttcc tgaattccgg ggttggggtt gcgccttttc caaggcagcc ctgggtctgc 180

gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg accctgggtc 240

tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg 300

ccccccggcg acgcttcctg ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc 360

gtgccggacg tgacaaacgg aagccgcacg tctcactagt accctcgcag acggacagcg 420

ccagggagca atggcagcgc gccgaccgcg atgggctgtg gccaatagcg gctgctcagc 480

agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt 540

agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac 600

gtcggcagtc ggctccctcg ttgaccgaat caccgacctc tctccccagt ggctaactag 660

agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc 720

tggctagcgt ttaaacttaa gcttggccgc caccatgtgg actctcgggc gccgcgcagt 780

agccggcctc ctggcgtcac ccagcccggc ccaggcccag accctcaccc gggtcccgcg 840

gccggcagag ttggccccac tctgcggccg ccgtggcctg cgcaccgaca tcgatgcgac 900

ctgcacgccc cgccgcgcaa gttcgaacca gagaggtctc aaccagattt ggaatgtcaa 960

aaagcagagt gtctatttga tgaatttgag gaaatctgga actttgggcc acccaggctc 1020

tctagatgag accacctatg aaagactagc agaggaaacg ctggactctt tagcagagtt 1080

ttttgaagac cttgcagaca agccatacac gtttgaggac tatgatgtct cctttgggag 1140

tggtgtctta actgtcaaac tgggtggaga tctaggaacc tatgtgatca acaagcagac 1200

gccaaacaag caaatctggc tatcttctcc atccagtgga cctaagcgtt atgactggac 1260

tgggaaaaac tgggtgtact cccacgacgg cgtgtccctc catgagctgc tggccgcaga 1320

1380

tgctttgccc acctagggat cggatccccg ggtaccgagc tcgaattctg cagatatcca 1440

gcacactttg cctttctctc cacaggtgtc gacaatcaac ctctggatta caaaatttgt 1500

gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct 1560

1620

aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg 1680

gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag 1740

ctcctttccg ggactttcgc ttttccccctc cctattgcca cggcggaact catcgccgcc 1800

tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg 1860

tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg gattctgcgc 1920

gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc 1980

ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc 2040

tccctttggg ccgcctcccc gcctggaatt cgagctcggt acgatcagct gatcagcctc 2100

2160 gactgtgcct tctagttgcc

cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg 2220

tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga 2280

ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctg gcgcgctcgc 2340

2400

tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcct 2458

<210> 6

<211> 105

<212> DNA

<213> Artificial sequence

<220>

<223> Linker

<400> 6

tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg 60

gagacccaag ctggctagcg tttaaactta agcttggccg ccacc 105

<210> 7

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> hFXN_Tg_FW

<400> 7

gaacttcaaa ttagttcccc ttttcttc 27

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> hFXN_Tg_RV

<400> 8

cacagccatt ctttggggttt c 21

<210> 9

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> IC_FW

<400> 9

cacgtgggct ccagcatt 18

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> IC_RV

<400> 10

tcaccagtca tttctgcctt tg 22

<210> 11

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> GAA-F

<400> 11

gggattggtt gccagtgctt aaaagttag 29

<210> 12

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> GAA-R

<400> 12

gatctaagga ccatcatggc cacacttgcc 30

<---

Claims (20)

1. An adeno-associated viral (AAV) expression vector suitable for the treatment of Friedreich's ataxia, containing a nucleic acid, where the nucleic acid includes: (i) a nucleic acid sequence encoding frataxin; (ii) a phosphoglycerate kinase (PGK) promoter consisting of SEQ ID NO:1; and (iii) woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), where (ii) and (iii) are operably linked to sequence (i) and regulate its expression, where between (i) and (ii) is an operably linked linker, and where said linker consists of SEQ ID NO: 6, and where AAV- the vector is an AAV serotype 9 vector. 2. The vector according to claim 1, where the nucleic acid sequence encoding frataxin contains SEQ ID NO: 3 or a sequence that is at least 90% identical to the sequence of SEQ ID NO: 3 and is a functional variant of frataxin. 3. The vector according to claim 1, where the nucleic acid sequence encoding frataxin consists of SEQ ID NO: 3 or a sequence that is at least 98% identical to the sequence of SEQ ID NO: 3 and is a functional variant of frataxin. 4. Vector according to any one of paragraphs. 1-3, where the WPRE consists of SEQ ID NO: 2 or a sequence that is at least 90% identical to the sequence of SEQ ID NO: 2. 5. Vector according to any one of paragraphs. 1-3, where the WPRE consists of SEQ ID NO: 2 or a sequence that is at least 99% identical to the sequence of SEQ ID NO: 2. 6. The vector according to claim 1, where the nucleic acid sequence encoding frataxin consists of SEQ ID NO: 3, and WPRE consists of SEQ ID NO: 2 or a sequence that is at least 90% identical to SEQ ID NO: 2. 7. The vector according to claim 1, where the nucleic acid sequence encoding frataxin consists of SEQ ID NO: 3, and WPRE consists of SEQ ID NO: 2 or a sequence that is at least 99% identical to SEQ ID NO: 2. 8. The vector according to claim 1, wherein the nucleic acid sequence that includes (i), (ii), (iii) and the linker consists of SEQ ID NO: 4 and where the AAV vector is a serotype 9 AAV vector. 9. A cloning vector for transfer, which includes a nucleic acid containing: (i) a nucleic acid sequence encoding frataxin; (ii) a PGK promoter consisting of SEQ ID NO:1, and (iii) WPRE, where (ii) and (iii) are operably linked to sequence (i) and regulate its expression, where between (i) and (ii) is an operably linked linker, and where said linker consists of SEQ ID NO: 6, and where the vector for The transfer vector also contains additional nucleic acid elements to promote integration or transposition of the transfer vector into a serotype 9 AAV vector. 10. Pharmaceutical composition for the treatment of Friedreich's ataxia, containing an effective amount of the AAV vector according to any one of paragraphs. 1-8 and a pharmaceutically acceptable carrier or diluent. 11. The use of AAV-vector according to any one of paragraphs. 1-8 as a medicine. 12. The use of AAV-vector according to any one of paragraphs. 1-8 in the treatment of Friedreich's ataxia.

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