TECHNICAL FIELD

The present invention relates to constructs, vectors, in particular viral vectors, relative host cells and pharmaceutical compositions for gene therapy.

In particular, it relates to constructs, vectors, relative host cells and pharmaceutical compositions for gene therapy of the gyrate atrophy of the choroid and retina.

BACKGROUND OF THE INVENTION

Gyrate atrophy of the choroid and retina (GACR, MIM#258870) is a rare inherited chorioretinal disease and a blinding disorder, first described as an atypical retinitis pigmentosa (RP) ¹ . Patients affected by GACR first experience loss of night vision and visual acuity, like in RP ² . Ocular fundoscopy in patients revealed that degeneration starts in retinal pigment epithelium (RPE) cells from the periphery of the retina; patients experience patchy vision corresponding to the areas where RPE cells are still present ^3,4 . As the disease progresses, these lesions increase in size and number and move towards the center of the retina. Cataract is also a common feature of the disease ⁴ . During the fifth-sixth decade of life, GACR subjects lose vision completely ² .

A recent study calculated the theoretical worldwide prevalence of GACR, estimating a prevalence of 1 affected individual : 1500000 ⁵ ; interestingly, GACR is much more frequent in Finland (1 individual: 50000), possibly due to a founder mutation effect, and indeed one third of all GACR cases are Finnish ² .

The causative gene of GACR, ornithine aminotransferase (OAT, MIM#613349), encodes a reversible mitochondrial enzyme involved in ornithine conversion or synthesis. Ornithine aminotransferase (OAT) monomers form an active homodimer that requires piridoxal 5’- phosphate (PLP), a derivative of vitamin B6, as a cofactor; active homodimers are able to reassemble into a mature homoexameric state ⁶ . This enzyme is widely expressed in many tissues across species and in the eye is expressed both in the RPE and in photoreceptors ^{7 9} .

Lack of OAT in adult life causes hyperornithinemia and a specific toxic effect in RPE cells. Only a few patients develop sub-clinical effects in skeletal muscle and the central nervous system ^{10 13} . Interestingly, the worst cases of GACR manifest defects of the urea cycle during the neonatal stage of life instead of increases in ornithine levels; infants experience vomiting, encephalopathy and paradoxical low levels of ornithinemia ¹⁴ . Further studies on a mouse model of GACR lacking Oat expression confirm that the enzyme reaction is shifted towards ornithine synthesis in the first stages of life to generate a source of arginine, required to form urea and correctly expel ammonia; without OAT activity, homozygous pups die of hyperammonemia a few days after birth unless they receive intra-peritoneal injections of arginine until adulthood ¹⁵ .

Clinical trials explored the use of an arginine-restricted diet after the neonatal stage of life to normalize ornithinemia levels. However, in those patients that were able to follow such a strict diet, the treatment only temporarily slowed down disease progression ¹⁶ .

Current therapies for GACR are unsatisfactory and there is a strong need for better treatment.

In GACR, dietary mediated correction of the excess of ornithine in blood can delay the development of retinal degeneration; it means that correction of the hyperornithinemia in blood is thought to be sufficient to prevent the disease phenotype. So GACR might be a target for a liver-directed genebased approach aimed at restoring the OAT enzymatic activity.

However, there is still the need for an efficient vector and system for the gene therapy of GACR.

SUMMARY OF THE INVENTION

The inventors have engineered a nucleic acid construct and derived AAV vectors for gene therapy of GACR and found that gene therapy with a single intraocular administration of adeno-associated viral (AAV) vectors has the potential to restore local expression of OAT and its metabolic pathway in the eye and to treat the disease. AAV8 vector expressing hOAT under the control of the ubiquitous CMV promoter was delivered by subretinal injection in one eye of vaOAT^ mice with the contralateral receiving excipient. Eyes administered with AAV8-h(9d 7' were found to have a significant thicker outer nuclear layer (ONL) up to 12 months of age compared to eyes administered with the excipient. The improvement in ONL thickness was observed also in distant areas from the injection site. This is mirrored by a normalized RPE morphology across the entire retinal section.

The inventors also found that systemic reduction of the blood ornithine concentrations by intravenous injection of AAV vectors delivering the OAT gene to hepatocytes, can restore enzyme activity and prevent retinal degeneration in GACR patients. Indeed following injections with AAV-OAT, Oat ^rhg mice showed sustained reductions of blood ornithine concentrations compared to control mice injected with a vector expressing the green fluorescent protein (AAV-GFP). The reduction in blood ornithine concentrations was associated with improved electroretinogram (ERG) response, suggesting preservation of retinal function. In contrast to control mice injected with AAV-GFP, Oat ^rhg mice injected with the AAV-OAT vector showed partial restoration of the retinal structure on pathology with improvements of RPE structure at 1-year post injection. In summary, hepatic OAT expression by AAV8 was effective at reducing blood ornithine concentrations and improving both the function and the structure of the retina proving the efficacy of liver-directed AAV-mediated gene therapy for GACR. GACR is not associated to hepatic inflammation or regeneration, which reduce vector persistence, and, in addition, OAT is expressed at relatively low levels in the liver, suggesting that low percentage of cell transduction are needed to prevent the hyperornithinemia for phenotypic correction. Therefore, OAT gene therapy using a liver-directed vector, an AAV vector, is particularly effective and advantageous.

The inventors also found that subretinal injection of AAV8-hOAT further improves the retinal function in vaOAT^ mouse model of GACR previously administered with AAV8-hOAT systemically. Therefore, the combination of intraocular and systemic gene therapy may be a supplementary approach in those patients where one approach or the other is not effective at treating the disease.

The present invention provides a nucleic acid construct for gene therapy of gyrate atrophy of the choroid and retina coding for an ornithine aminotransferase (OAT) enzyme comprising: a promoter sequence, a coding sequence of the ornithine aminotransferase (OAT) gene under control of said promoter.

Preferably, said nucleic acid construct is inserted in a vector.

It is an object of the invention a vector comprising the nucleic acid construct above defined.

It is therefore an object of the invention a vector for gene therapy of gyrate atrophy of the choroid and retina comprising a nucleic acid construct coding for a ornithine aminotransferase (OAT) enzyme, said construct comprising: a promoter sequence, a coding sequence of the ornithine aminotransferase (OAT) gene under control of said promoter.

Preferably said vector is a viral vector. Examples of viral vectors are adenoviral vectors, adeno- associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors vaccinia viruses, foamy viruses, cytomegaloviruses, Semliki Forest virus, poxviruses, RNA virus vector and DNA virus vector. Preferably the viral vector derives from non- pathogenic parvovirus such as adeno-associated virus (AAV), retrovirus such as gammaretrovirus, spumavirus and lentivirus, adenovirus, poxvirus and an herpes virus. More preferably, the viral vector is selected from the group consisting of: adenoviral vectors, lentiviral vectors, retroviral vectors and adeno associated viral vectors (AAV).

In another embodiment, the vector is a non-viral vector such as polymer-based, particle-based, lipid- based, peptide-based delivery vehicles or combinations thereof, such as cationic polymers, micelles, liposomes, exosomes, microparticles and nanoparticles including lipid nanoparticles (LNP).

It is a preferred object of the invention a viral vector comprising a nucleic acid construct coding for the ornithine aminotransferase (OAT) enzyme, said construct comprising: a promoter sequence, a coding sequence of the ornithine aminotransferase (OAT) gene under control of said promoter, wherein said vector is a lentiviral vector or an adeno-associated virus (AAV) vector.

Preferably, said promoter sequence is operably linked to the 5 ’end portion of said coding sequence.

The promoter can be for example a ubiquitous promoter or a tissue-specific promoter.

In an embodiment, said promoter is a cytomegalovirus (CMV) promoter.

In another embodiment said promoter is a promoter specific for liver expression, preferably a hepatocyte specific promoter, more preferably the OAT promoter or the thyroxine binding globulin (TBG) promoter. In another embodiment said promoter is a retinal-specific promoter, in particular RPE or photoreceptor specific promoter. In another embodiment said promoter is the OAT Promoter regulatory region, a sequence of 213 base pairs within the human OAT promoter, that was described to be to be sufficient for protein expression.

A further object of the invention is a viral particle containing the viral vector as defined above or herein.

It is also an object of the invention a host cell transformed with the vector according to the invention or with the viral particle of the invention.

It is also an object of the invention a pharmaceutical composition comprising the vector or the viral particle according to the invention or herein described or the host cell according to the invention or herein described and at least one pharmaceutically acceptable vehicle.

It is an object of the invention the vector or the viral particle or the host cell or the pharmaceutical composition of the invention for use as a medicament. It is an object of the invention the vector or the viral particle or the host cell or the pharmaceutical composition of the invention for use in gene therapy.

Preferably, said vector or viral particle or host cell or pharmaceutical composition is for use in the treatment of gyrate atrophy of the choroid and retina (GACR).

It is also an object of the invention the vector or the viral particle or the host cell or the pharmaceutical composition of the invention for use in ameliorating and/or contrasting and/or reducing retinal pigment epithelium (RPE) degeneration.

Preferably, the vector is systemically delivered or is delivered to the retina.

Preferably, the vector is delivered systemically and to the retina, preferably said vector is delivered systemically before the delivery to the retina or said vector is delivered systemically and at the same time to the retina.

Preferably, the vector is administered through intravenous injection or sub-retinal injection.

Preferably, said vector is administered through intravenous injection and sub-retinal injection, preferably said administration is performed at the same time or the intravenous injection is performed before the sub-retinal injection.

In an embodiment, the vector comprises a promoter specific for liver expression and is systemically administered, for example by intravenous injection. In this embodiment, the transgene is specifically expressed in the liver thereby treating GACR by liver production of the OAT enzyme. Advantageously, the increase of the OAT enzyme in the liver leads to a lowering of the circulating levels of ornithine and to a correction of the eye defects.

In another embodiment, the vector comprises a ubiquitous promoter and is delivered to the retina, for example by sub-retinal injection or intraocular administration. In this embodiment, the transgene is delivered to the target tissue, i.e. the retina, thereby treating GACR by production of the OAT enzyme in the retina.

Preferably, the vector comprising a promoter specific for liver expression which is administered systemically, for example by intravenous injection, is administered together with the vector comprising a ubiquitous promoter which delivered to the retina, for example by sub-retinal injection.

The combined use of the vectors described in the two above embodiments might be particularly advantageous in order to reduce the dose of the vector administered systemically and consequently reduce possible related toxicity effects. For combined use it is intended preferably that a vector of the invention is delivered to the retina, for example by sub-retinal injection, after or at the same time of systemically administration, for example by intravenous injection, of a vector of the invention.

It is also an object of the invention a method for treating gyrate atrophy of the choroid and retina (GACR) comprising administering to a subject in need thereof an effective amount of the vector according to the invention, a viral particle according to the invention, the host cell according to the invention or the pharmaceutical composition according to the invention.

In an embodiment, said vector is delivered systemically.

In another embodiment, said vector is delivered to the retina. Preferably, the method comprises administering systemically a first vector as defined above wherein said first vector comprises a promoter specific for liver expression and delivering to the retina a second vector as described above wherein said second vector comprises a ubiquitous promoter.

Preferably, said vector is administered by intravenous injection or sub-retinal injection.

In a preferred embodiment the vectors are delivered at the same time systemically, preferably by intravenous injection, and to the retina, by sub-retinal injection.

In a preferred embodiment firstly said vectors is delivered systemically, preferably by intravenous injection, and secondly said vector is delivered to the retina, by sub-retinal injection.

The invention also provides a method for increasing expression of ornithine aminotransferase comprising administering to a subject in need thereof the vector according to the invention, the viral particle, the host cell according to the invention or the pharmaceutical composition according to the invention. The invention also provides a method for lowering the circulating level of ornithine comprising administering to a subject in need thereof the vector, the viral particle, the host cell or the pharmaceutical composition according to the invention.

Preferably, expression of the protein is increased in the liver or in the eye, especially in the retina, of the subject.

Preferably, coding sequence of the ornithine aminotransferase (OAT) gene is a sequence coding for human ornithine aminotransferase (OAT). The coding sequence can codify for a variant of ornithine aminotransferase (OAT), for example it can comprise additions, deletions or substitutions with respect to the coding sequence of the wild type ornithine aminotransferase (OAT) gene as long as these protein variants retain substantially the same relevant functional activity as the original OAT. The coding sequence can also codify for a fragment of ornithine aminotransferase (OAT), as long as this fragment retains substantially the same relevant functional activity as the original OAT.

Suitably, the coding sequence may be codon optimized for expression in human.

Preferably the viral vector or vector comprises a 5 ’-terminal repeat (5’-TR) nucleotide sequence and a 3 ’ -terminal repeat (3 ’ -TR) nucleotide sequence, preferably the 5 ’ -TR is a 5 ’ -inverted terminal repeat (5’-ITR) nucleotide sequence and the 3’-TR is a 3 ’-inverted terminal repeat (3’-ITR) nucleotide sequence, preferably the ITRs derive from the same virus serotype or from different virus serotypes, preferably the virus is an AAV, preferably of serotype 2.

In an embodiment, said viral vector or vector further comprises one or more of: a 5’ inverted terminal repeat (ITR) sequence of AAV, preferably localized at the 5’ end of the promoter; a Kozak sequence, preferably localized at the 5’ end of the OAT-coding sequence and operably linked to said sequence; a post-transcriptional regulatory element, preferably localized at the 3’ end of the coding sequence of the ornithine aminotransferase (OAT) gene; a transcription termination sequence preferably localized at the 3’ end of the post- transcriptional regulatory element or at the 3 ’end of the coding sequence; a 3 ’ inverted terminal repeat (ITR) sequence of AAV, preferably localized at the 3 ’ end of the transcription termination sequence.

In the context of the present invention, the viral vector or vector preferably further comprises a 3XFLAG tag, more preferably at the 3’ end of the coding sequence of the ornithine aminotransferase (OAT) gene. In this case, if a post-transcriptional regulatory element is present, it is preferably localized at the 3’ end of 3XFLAG tag.

If a post-transcriptional regulatory element is present, the transcription termination sequence is preferably localized at the 3’ end of the post-transcriptional regulatory element.

Preferably the vector or viral vector comprises in a 5 ’-3’ direction:

- an AAV 5 ’-inverted terminal repeat (5 ’-ITR) sequence;

- a promoter sequence;

- a Kozak sequence;

- the coding sequence of the ornithine aminotransferase (OAT) gene under control of said promoter; - a nucleotide sequence of a post-transcriptional regulatory element;

- a nucleotide sequence of a transcription termination sequence; and

- an AAV 3 ’-inverted terminal repeat (3’-ITR) sequence.

In the context of the present invention, the viral vector or vector preferably further comprises a 3XFLAG tag, more preferably at the 3’ end of the of the coding sequence of the ornithine aminotransferase (OAT) gene. In this case, if a post-transcriptional regulatory element is present, it is preferably localized at the 3’ end of 3XFLAG tag.

If a post-transcriptional regulatory element is present, the transcription termination sequence is preferably localized at the 3’ end of the post-transcriptional regulatory element.

Preferably said post-transcriptional regulatory element is the Woodchuck hepatitis virus post- transcriptional regulatory element (WPRE).

Preferably said transcription termination sequence is a poly-adenylation signal sequence, preferably the bovine growth hormon polyA (BGH poly A).

Optionally, the vector further comprises a promoter intron, preferably selected from small T antigen intron, large T antigen intron, SV40 intron and hybrid introns made of fragments of introns, preferably said promoter intron being a chimeric promoter intron comprising or consisting of the 5’ donor site from the first intron of the human P-globin gene and the branch and 3 ’-acceptor site from the intron of an immunoglobulin gene heavy chain variable region.

Said promoter intron is preferably operably linked to the 3’ of the promoter sequence and to the 5’ of the OAT coding sequence.

Preferably said promoter intron is between the promoter and the coding sequence of the ornithine aminotransferase (OAT) gene.

In the context of the present invention, the promoter is preferably selected from a ubiquitous promoter and a tissue-specific promoter, preferably a liver-specific promoter or is preferably selected from a cytomegalovirus (CMV) promoter and a thyroxine binding globulin (TBG) promoter.

Preferably the vector is a viral vector.

Preferably the viral vector is selected from the group consisting of: adenoviral vectors, lentiviral vectors, retroviral vectors and adeno associated viral vectors (AAV). Preferably the viral vector is an adeno associated viral vector (AAV) and the adeno-associated virus is from a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, and AAV11, AAV-PhP.B and AAV-PhP.eB, preferably it is of serotype 8.

Preferably the vector is a non-viral vector, preferably selected from polymer-based, particle-based, lipid-based, peptide-based delivery vehicles or combinations thereof, such as cationic polymers, micelles, liposomes, exosomes, microparticles and nanoparticles including lipid nanoparticles (LNP).

In an embodiment the vector comprises in a 5 ’-3’ direction:

- an AAV 5 ’-inverted terminal repeat (5’-ITR) sequence;

- a promoter sequence wherein said promoter is ubiquitous, preferably a CMV promoter;

- a promoter intron;

- a Kozak sequence;

- the coding sequence of the ornithine aminotransferase (OAT) gene under control of said promoter;

- optionally a nucleotide sequence of a post-transcriptional regulatory element;

- a nucleotide sequence of a transcription termination sequence; and

- an AAV 3 ’-inverted terminal repeat (3’-ITR) sequence.

In an embodiment the vector comprises in a 5 ’-3’ direction:

- an AAV 5 ’-inverted terminal repeat (5’-ITR) sequence;

- a promoter sequence wherein said promoter is liver-specific, preferably the thyroxine binding globulin (TBG) promoter;

- a promoter intron;

- a Kozak sequence;

- the coding sequence of the ornithine aminotransferase (OAT) gene operably linked to and under control of said promoter;

- a nucleotide sequence of a post-transcriptional regulatory element;

- a nucleotide sequence of a transcription termination sequence; and

- an AAV 3 ’-inverted terminal repeat (3’-ITR) sequence.

In an embodiment the vector comprises in a 5 ’-3’ direction:

- an AAV 5 ’-inverted terminal repeat (5’-ITR) sequence; - a promoter sequence wherein said promoter is liver-specific, preferably the thyroxine binding globulin (TBG) promoter;

- a Kozak sequence;

- the coding sequence of the ornithine aminotransferase (OAT) gene operably linked to and under control of said promoter;

- a nucleotide sequence of a post-transcriptional regulatory element;

- a nucleotide sequence of a transcription termination sequence; and

- an AAV 3 ’-inverted terminal repeat (3’-ITR) sequence.

A further object of the invention is a viral particle containing the viral vector as defined above or herein.

A further object of the invention is a plasmid comprising: a promoter sequence, a coding sequence of the ornithine aminotransferase (OAT) gene under control of said promoter, wherein said plasmid is for the generation of a vector. Preferably said vector is an AAV vector or a lentivirus vector. Preferably, it is a vector as described above.

Said plasmid may further comprise one or more of a 5’ inverted terminal repeat (ITR) sequence of AAV, preferably localized at the 5’ end of the promoter; a Kozak sequence, preferably localized at the 5’ end of the OAT-coding sequence and operably linked to said sequence; a post-transcriptional regulatory element, preferably localized at the 3’ end of the coding sequence of the ornithine aminotransferase (OAT) gene; a transcription termination sequence preferably localized at the 3’ end of the post- transcriptional regulatory element or at the 3 ’end of the coding sequence; a 3 ’ inverted terminal repeat (ITR) sequence of AAV, preferably localized at the 3 ’ end of the transcription termination sequence.

Preferably the plasmid comprises in a 5’-3’ direction:

- an AAV 5 ’-inverted terminal repeat (5 ’-ITR) sequence; - a promoter sequence;

- a Kozak sequence;

- the coding sequence of the ornithine aminotransferase (OAT) gene under control of said promoter;

- a nucleotide sequence of a post-transcriptional regulatory element;

- a nucleotide sequence of a transcription termination sequence; and

- an AAV 3 ’-inverted terminal repeat (3’-ITR) sequence.

The promoter can be for example a ubiquitous promoter or a tissue-specific promoter, preferably a liver-specific promoter. In an embodiment it is a cytomegalovirus (CMV) promoter. In another embodiment said promoter is a promoter specific for liver expression, preferably the thyroxine binding globulin (TBG) promoter.

In the context of the present invention, the plasmid preferably further comprises a 3XFLAGtag, more preferably at the 3’ end of the coding sequence of the ornithine aminotransferase (OAT) gene. In this case, if a post-transcriptional regulatory element is present, it is preferably localized at the 3’ end of 3XFLAG tag.

If a nucleotide sequence of a post-transcriptional regulatory element is present, a nucleotide sequence of a transcription termination sequence is preferably localized at the 3’ end of the post-transcriptional regulatory element.

Preferably said post-transcriptional regulatory element is the Woodchuck hepatitis virus post- transcriptional regulatory element (WPRE).

Preferably said transcription termination sequence is a poly-adenylation signal sequence, preferably the bovine growth hormone polyA (BGH poly A).

Preferably the plasmid further comprises a promoter intron, preferably selected from small T antigen intron, large T antigen intron, SV40 intron and hybrid introns made of fragments of introns.

Optionally, the plasmid further comprises a chimeric promoter intron comprising or consisting of the 5’ donor site from the first intron of the human P-globin gene and the branch and 3 ’-acceptor site from the intron of an immunoglobulin gene heavy chain variable region. Said promoter intron is preferably operably linked to the 3’ of the promoter sequence and to the 5’ of the OAT coding sequence. The plasmid usually further comprises backbone elements which are typically required for the large scale plasmid production in bacteria, such as bacterial origin of replication, bacterial promoter, antibiotic resistance gene.

A further object of the invention is the use of said plasmid for the generation of a vector or an AAV vector according to the invention.

Preferably the coding sequence of the ornithine aminotransferase (OAT) gene comprises or consists of a sequence having at least 95% of identity to SEQ ID N.2, SEQ ID N.7 or SEQ ID N.20.

Preferably, the promoter is a cytomegalovirus (CMV) promoter and its sequence comprises or consists of a sequence having at least 95% of identity to SEQ ID N.5.

Preferably, the promoter is a TBG promoter comprising or consisting of a sequence having at least 95% of identity to the SEQ ID N.19.

Preferably, the promoter is a OAT promoter comprising or consisting of a sequence having at least 95% of identity to the SEQ ID N.13.

Preferably the OAT promoter regulatory region comprises or consists of a sequence having at least 80% of identity to SEQ ID N.13.

Preferably, the 5’ inverted terminal repeats (ITRs) of AAV comprises or consists of a sequence having at least 95% of identity to SEQ ID N.4, SEQ ID N.I 7 or SEQ ID N.23.

Preferably, the bovine growth hormon polyA (BGH polyA) comprises or consists of a sequence having at least 95% of identity to SEQ ID N.10, SEQ ID N. I 6 or SEQ ID N.22.

Preferably, the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprises or consists of a sequence having at least 95% of identity to SEQ ID N.9 or SEQ ID N.15 or SEQ ID N.21.

Preferably, the 3xflag tag comprises or consists of a sequence having at least 80% of identity to SEQ ID NO: 8.

Preferably, the 3’ inverted terminal repeats (ITRs) of AAV comprises or consists of a sequence having at least 95% of identity to SEQ ID N.11 , SEQ ID N.18 or SEQ ID N.24

Preferably, the chimeric promoter intron comprises or consists of a sequence having at least 95% of identity to SEQ ID N.6.

Preferably, the Kozak sequence comprises or consists of a sequence having at least 95% of identity to the sequence GCGGCCGCC. Preferably, the plasmid comprises or consists of a sequence having at least 95% of identity to SEQ ID N.12.

Preferably the adeno-associated virus is from the serotype 8.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including” or “includes”; or “containing” or “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or steps. The terms “comprising”, “comprises” and “comprised of’ also include the term “consisting of’.

In the present invention “at least 80 % identity” means that the identity may be at least 80%, or 85 % or 90% or 95% or 100% sequence identity to referred sequences. This applies to all the mentioned % of identity. In the present invention “at least 95 % identity” means that the identity may be at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to referred sequences. This applies to all the mentioned % of identity. In the present invention “at least 98 % identity” means that the identity may be at least 98%, 99% or 100% sequence identity to referred sequences. This applies to all the mentioned % of identity. Preferably, the % of identity relates to the full length of the referred sequence.

Included in the present invention are also nucleic acid sequences derived from the nucleotide sequences herein mentioned, e.g. functional fragments, mutants, variants, derivatives, analogues, and sequences having a % of identity of at least 80% with the sequences herein mentioned, as far as such fragments, mutants, variants, derivatives and analogues maintain the function of the sequence from which they derive.

The term “inverted terminal repeat” means sequences which are repeated at both ends of a nucleotide sequence in the opposite orientation (reverse complementary).

For gyrate atrophy of the choroid and retina is intended a disease caused by homozygous or compound heterozygous mutation in the OAT gene (613349) on chromosome 10q26. Hyperomithinemia with gyrate atrophy of choroid and retina, ornithine aminotransferase deficiency and oat deficiency can be used as synonyms.

Description of the drawings

Figure 1 : Schematic representation of AAV-hOAT constructs used in Example 1. Black hairpins: AAV serotype 2 inverted terminal repeats (ITRs); CMV: cytomegalovirus; promoter intron: chimeric intron; OAT-3XFlag: human ornithine aminotransferase coding sequence 3XFlag-tagged; WPRE: Woodchuck hepatitis virus post-transcriptional regulatory element; BGH PA: bovine growth hormone polyA.

Figure 2: Plasmids encoding hOAT or hOAT-3XFlag significantly increase OAT activity in vitro. Quantification of OAT activity with the ninhydrin method. hARPE-19 cells were transfected with plasmids encoding for hOAT, hOAT-3XFlag or enhanced green fluorescent protein (EGFP) as a control with relatively low expression of OAT. One hundred pg of protein lysate were used per sample in the assay. Results are represented as value of each biological replicate (filled square) and as mean value for each group (column). Mean value of each group is indicated inside the corresponding bar. Statistical analysis was conducted using one-way ANOVA with Tuckey post-hoc analysis. Stars above each bar refer to the comparison between pOAT or pOAT-3XFlag vs pEGFP. **** p_ _vaiues <0.0001. P5C: pyrroline 5-carboxilate; pEGFP: plasmid encoding for EGFP; pOAT: plasmid encoding for OAT; pOAT-3XFlag: plasmid encoding for OAT-3XFlag.

Figure 3: Efficient expression of hOAT upon sub-retinal administration of AAV-hOAT-3XFlag in vivo. Western Blot analysis of C57BL/6 eyecups 4 weeks following subretinal injection of either AAV-hOAT-3XFlag or excipient. Each image shows different eyecups samples, black bars correspond to 43 and 55 KDa, arrows indicate murine Oat or human OAT-3XFlag. Fifty pg of protein were loaded in each lane. a-OAT: western blot with anti-Ornithine aminotransferase antibody; u- Calnexin: western blot with anti-Calnexin antibody, used as loading control. hOAT: human ornithine aminotransferase; mOAT : murine ornithine aminotransferase

Figure 4: AAV-hOAT-3XFlag improved outer nuclear layer thickness upon sub-retinal injection in Oat-/- mice. A) Representative retinal scannings of affected homozygous (Oat-/-) mice injected with AAV-hOAT-3XFlag or excipient. Calipers for ONL measurements are in scales of grey and show mm of retinal thickness. -I- : homozygous affected Oat mice. B) Spectral domain-Optical Coherence Tomography analysis was performed at 4-6-8-12 months of age to measure the outer nuclear layer thickness in Oat" ' mice injected sub-retina with either AAV-hOAT-3XFlag (triangles, n = 10, 9, 10 and 5 eyes per each time-point) or excipient (squares, n = 8, 7, 8 and 4 eyes per each time-point ) in the contralateral eye. Heterozygous (Oat+/-) mice injected with formulation buffer were used as unaffected controls (circles, n = 9 eyes per each time-point). For each eye, ONL thickness was measured close to the injection site, then values were averaged. Results are represented as mean value for each group± standard error of mean. Statistical analysis was conducted using two-sample t-test analysis to evaluate significant differences between Oat-/- eyes treated with AAV-hOAT-3XFlag and Oat-/- eyes receiving formulation buffer. ** p-value < 0,01. ONL: outer nuclear layer; +/- : heterozygous unaffected Oat mice; -I- : homozygous affected Oat mice.

Figure 5: Sub-retinal administration of AAV-hOAT-3XFlag results in improvements to retinal pigment epithelium degeneration (RPE) in Oat" ' mice. A) Representative pictures from montages of the entire retinal section imaged at 40X magnification and used for RPE analysis. Each picture is composed of —2 fields. Scale bar (white bar) = 20 pm. RPE, outer segments (OS) and outer nuclear layer (ONL) are indicated in white, black and white, respectively. Red arrows point at degenerared RPE. +/- : heterozygous unaffected Oat mice; -I- : homozygous affected Oat mice. RPE: retinal pigment epithelium; OS: outer segments; ONL: outer nuclear layer. B) Quantification of degenerated RPE was performed in Oat" ' mice receiving AAV-hOAT-3XFlag or excipient as a control condition. As an unaffected control, Oat ^+A mice were injected with excipient. For each eye, the length of degenerated RPE per 100 pm of RPE was measured. Results are represented as mean value for each eye (filled square) and as mean value for each treatment group (reported inside or above each column).

Figure 6: AAV-hOAT-3XFlag does not improve retinal function in subretinally injected Oat-/- mice. (A) Representative ERG waves at 20 cd/m*2 of unaffected heterozygous (Oat+/-) mice injected with formulation buffer and affected homozygous (Oat-/-) mice injected with AAV-hOAT-3XFlag or formulation buffer. Blue point indicates A-Wave, orange point indicates B-Wave. Vertical scale bar = 100 pV, horizontal scale bar = 50 ms. +/- : heterozygous unaffected Oat mice; -I- : homozygous affected Oat mice. (B-C) Electroretinogram analysis to assess A-wave (B) and B-wave (C) was performed at 4-6-8-12 months of age in Oat-/- mice injected subretinally with either AAV-hOAT- 3XFlag (triangles, n of eyes = 9, 10, 8 and 5 per each time-point) or excipient (squares, n of eyes = 9, 10, 8 and 5 per each time-point) in the contralateral eye. Heterozygous (Oat+/-) mice injected with formulation buffer (circles, n of eyes = 10, 10, 10 and 8 per each time-point) were used as unaffected controls. Mice eyes were stimulated with a luminance of 20 candelas/m ² . Data are represented as mean ± standard error of mean (SEM). Cd: candela; +/- : heterozygous unaffected Oat mice; -I- : homozygous affected Oat mice.

Figure 7: Characterization of B6Ei; AKR-rhg mouse retinal function and liver trasduction. a- and b- wave amplitudes under scotopic and photopic conditions in B6Ei; AKR-rhg pigmented mice. The amplitudes (mean ± SEM) induced by increasing light intensities under scotopic conditions in 7- month-old (a) and 11 -month-old (b) wilde type (blue circles, n = 6), heterozygous (red triangles, n = 6) and homozygous (black square, n = 6) B6Ei; AKR-rhg are shown (means ± SEM. **p<0,01; ***p<0,001, ****p<0,0001; ns: not statistically significant difference, two-way ANOVA test) (c) Liver section from C57BL/6N mice systemically injected with AAV.TBG.GFP (left panel) or AAV.OATpromoter.GFP (right panel). Representative images from experimental groups including at least n = 4 mice per group are shown. Scale bar: 20 pm. (d) Western Blot analysis of lysates from liver of C57BL/6N mice injected with TBG.GFP (n=4) or OATpromoter.GFP (n=5) vectors, (e) Representative immunofluorescence images of livers from C57BL/6N mice (n = 4) injected with AAV.TBG.GFP. OAT staining is shown in the left panel and GFP in the central panel, colocalization of the signals is shown in the right panel. Scale bar: 20 pm.

Figure 8. OAT gene transfer in Oaf ^hg pigmented mice injected at 6 weeks of age improves retinal phenotype: (a) Plasma ornithine concentrations in Oaf ^hg mice injected with AAV-OAT (n=5) or AAV-GFP (n=3) vectors. Averages ± SEM are shown; the two-way ANOVA test was used to perform a statistical comparison between groups; *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001. (b) Body weights of Oaf ^hg mice injected at 6 weeks of age; the arrow indicates the time of the injections with AAV-OAT or AAV-GFP vectors; data are shown as means ± SEM. (c) a- and b-waves amplitudes recorded in scotopic conditions plotted as a function of light intensity (log cd * s/m ² ) in eyes of 11- month-old Oaf ^hg mice injected with AAV-OAT (n=9) or AAV-GFP (n=4) vectors. Wild-type (WT) mice were used as controls (n=10). All data are shown as averages ± SEM. Two-way ANOVA test was used for comparison between groups. *p <0.05; **p<0.01; ***p<0.001, ****p<0.0001. (d) Plasma ornithine and lysine concentrations of Oaf ^hg mice at 11 months post-injection measured by HPLC. One-way ANOVA test with Tukey correction was used for statistical comparison between groups. *p <0.05; **p<0.01; ***p<0.001, ****p<0.0001. (e) Ornithine concentrations in the eyes of mice injected with AAV-OAT compared to eyes of either wild-type or AAV-GFP controls. Data are shown as z-scores. Means ± SEM are shown. One-way ANOVA with False Discovery Rate correction was used for comparison between groups. *p <0.05; **p<0.01. (f) Western blotting for OAT and GFP proteins on livers of Oaf ^hg mice 11 months after the injections of AAV-OAT or AAV-GFP vectors. A WT mouse was included as control, and Vinculin was used as loading control, (g) Morphological analysis of CW^mice retinas 11 months post-injection of AAV-GFP or AAV-OAT. An age-matched WT mouse is shown as control (left panel). Semi-thin sections (40X). Abbreviations: ns, not statistically significant difference; OS, outer segment; IS, inner segment; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer, WT, wild-type.

Fig. 9. Improvement of retinal disease of Oaf ^hg mice by liver-directed gene transfer.

(a) Plasma ornithine concentrations in Oaf ^hg mice following intravenous injections of AAV-OAT at the doses of IxlO ¹³ (n=9) and 3xl0 ¹³ genome copies (gc)/Kg (n=6), or AAV-GFP at the dose of IxlO ¹³ gc/Kg (n=9). Normal range was established on wild-type mice (n=7) and average ± 2SD was calculated. Data are shown as averages ± SEM. ***p<0.001, ****p<0.0001 (Two-way ANOVAtest). (b) Amplitude of a- and b-waves under scotopic and photopic conditions (mean ± SEM) in uninjected wild-type (WT) mice (blue circles, n=10), CW^ mice injected with AVV-OAT of IxlO ¹³ gc/kg (red circles, n=12) or 3xl0 ¹³ (gray circles, n=6) and CW^ mice injected with AVV-GFP (black circles, n=8) at 11 months of age (9-months post-injection). Two-way ANOVA test; *p <0.05; **p<0.01.

(c) Plasma ornithine and lysine concentrations at 12 months of age (10-months post-injection) in Oaf ^hg mice injected with AAV-OAT (n=3) or AAV-GFP (n=3); WT mouse controls (n=5) are also shown; each sample was analyzed in triplicate, data are shown as averages ± SEM. One-way ANOVA test with Tukey correction; *p <0.05; ****p<0.0001.

(d) Ornithine concentrations in the eye cups of Oaf ^hg mice at 12 months of age (10-months postinjection). Data are shown as Z-scores; one-way ANOVA with False Discovery Rate correction was used for comparison between groups. Means ± SEM are shown; *p <0.05 ***p<0.001, ****p<0.0001; ns not statistically significant difference.

(e) Western blot for OAT and GFP in livers of Oaf ^hg mice injected with AAV-GFP or AAV-OAT. A WT mouse liver was included as control. Vinculin was used as loading control.

(f) OAT activity on liver lysates of Oaf ^hg mice injected with AAV-OAT or AAV-GFP vectors (n=4 for each group). Data are shown as means ± SEM. Unpaired T-test; *p<0.05.

(g) Liver immunohistochemistry showing OAT -positive cells in Oaf ^hg mice injected with AAV- OAT. Livers of WT and Oaf ^hg mice injected with AAV-GFP are shown as controls. 5x (images on the left side) and 20x (images on the right side) fields of view are shown.

(h) Representative H&E staining of the retinas of 11 -month-old Oaf ^hg mice injected with AAV-GFP or AVV-OAT at the doses of IxlO ¹³ or 3xl0 ¹³ gc/Kg; age-matched WT mice and uninjected Oaf ^hg mice were included as controls (40X). Abbreviations: OS, outer segment; IS, inner segment; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer.

Figure 10. AAV mediated OAT delivery to hepatocyte of Oaf ^hg albino mice injected.

A, B) ERG components (a- and b- waves) under scotopic and photopic conditions of 7-month-old (a) and 11-month-old (b) Oaf ^hg albino mice injected with AAV-OAT (n=10, 7-month-old; n=7, 11- month-old) and AAV-GFP vectors (n=6, 7-month-old; n=7, 11-month-old). WT mice were included as control (n=7, 7-month-old; n=3, 11 -month-old). All data are shown as averages ± SEM. Two-way ANOVA test was used for comparison between groups. **p<0.01; ***p<0.001, ****p<0.0001.

Figure 11. Ultrastructure of photoreceptor outer segment and retinal pigmented epithelium of Oaf ^hg mice after liver-directed gene transfer of OAT.

(a) Representative electron micrographs of the mouse retina of an AAV-GFP injected Oaf ^hg mouse showing retinal pigmented epithelium (RPE) and adjacent abnormal outer segment (OS) disk membranes (black arrows). Phagolysosomes containing undigested OS disc membranes are present (asterisks).

(b) Retina of AAV-OAT injected Oat ^rhg mice showing photoreceptor disk membranes (white arrows) and RPE ultrastructure comparable to age matched WT mice (c, white arrows).

(c) Control retina of WT mouse is shown as comparison.

Figure 12. Electron microscopy of basal infoldings of Bruch’s membrane of Oaf ^hg mice.

(a) AAV-GFP eyes (middle panel) displaying irregular and highly altered thickened ultrastructure of basal infoldings compared to wild-type (WT-left panel) or AAV-OAT injected mice (right panel). Basal infoldings of Bruch’s membrane is outlined by dashed line. Scale bar: 750 nm.

(b) Quantification of basal membrane thickness. Layers’ depth was measured and averaged from six fields for each group, three measurements for each image. One-way ANOVA test with Tukey correction was performed to compare experimental groups. One mouse analyzed for each group Means of six fields measurements ± SEM are shown **p<0.01; ****p<0.0001.

Fig. 13. Improvement phenotype in Oat ^A mice following AAV-mediated liver-directed OAT gene transfer.

(a) Plasma ornithine concentrations in AAV-GFP- (n=3) and AAV-OAT-injected (n=3) Oat ^A mice. Normal range of wild-type mice is also shown. Each value plotted represents mean ± SEM; Two-way ANOVA test, *p <0.05, **p<0.01.

(b) ERG at six months post-injection in Oat ^A mice injected with either AAV-GFP (n=6) or AVV- OAT (n=5); wild-type (WT) controls are also shown (n=5). Two-way ANOVA; test *p <0.05; **p<0.01; ***p<0.001. Data are shown as averages ± SEM.

(c) Retinal H&E (40X) of Oat ^A mice injected with either AAV-GFP or AAV-OAT. Representative of three mice per groups are shown; WT mice were included as control. Abbreviations: OS, outer segment; IS, inner segment; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer.

Figure 14. Schematic representation of the OAT expression cassette of Example 2.

Figure 15. Map of the plasmid AAV.TBG.OAT used in Example 2.

Figure 16: The OAT and -OAT-3XFlag plasmids express biologically-active OAT in vitro

A) Representative western blot analysis of hARPE-19 transfected with yOA T, p6M 7'-3XFlag or pEGFP as negative control. hARPE- 19 cells transfected with a plasmid encoding EGFP were used as controls. Black bars correspond to 43 and 55 KDa, arrows indicate OAT or OAT-3XFlag, 10 pg of proteins were loaded in each lane. a-OAT: western blot with anti-Ornithine aminotransferase antibody; a-Tubulin: western blot with anti-Tubulin antibody, used as loading control; pEGFP: plasmid encoding for EGFP; jpOAT: plasmid encoding for OAT; pCMT-3XFlag: plasmid encoding for 0AT-3XFlag.

(B) Quantification of OAT activity with the ninhydrin method. hARPE-19 cells were transfected with plasmids encoding for OAT, OAT-3XFlag or EGFP as a control with relatively low expression of OAT. One hundred pg of protein lysate were used per sample in the assay. Results are represented as value of each biological replicate (filled square) and as mean value for each group (column). The mean value of each group is indicated inside the corresponding bar. Statistical analysis was conducted using one-way ANOVA with Tuckey post-hoc analysis. Stars above each bar refer to the comparison between pOAT or pCMT-3XFlag vs pEGFP. **** P-values <0.0001. P5C: pyrroline 5-carboxilate; pEGFP: plasmid encoding for EGFP; pOAT: plasmid encoding for OAT; pCMTkSXFlag: plasmid encoding for OAT-3XFlag.

Figure 17: Efficient expression of OAT upon subretinal administration of AAV8-OAT in vivo

Western blot analysis of Oat^ eyecups following subretinal injection of either AAV8-CMT or excipient. Each image shows different eyecup samples, arrows indicate Oat or OAT-3XFlag. Fifty pg of protein were loaded in each lane. Measurements of OAT/Calnexin band intensity for each eye injected with AAV8-CMT is reported below each lane. a-OAT: western blot with anti-Ornithine aminotransferase antibody; u-Flag: western blot with anti-Flag antibody; u-Calnexin: western blot with anti-Calnexin antibody, used as loading control.

Figure 18: Subretinal administration of AAV8-OAT improves outer nuclear layer thickness in Oat ^A mice

SD-OCT analysis was performed at 4, 6, 8 and 12 months of age to measure the ONL thickness in Oar ~ mice injected sub-retina with either AAV8-CMT (triangles, n = 10, 9, 10 and 6 eyes per each time-point respectively) or excipient (squares, n = 8, 7, 8 and 5 eyes per each time-point respectively) in the contralateral eye. Heterozygous (Oat ) mice injected with excipient were used as unaffected controls (circles, n = 9 eyes per each time-point). For each eye, ONL thickness was measured close to the injection site, then values were averaged. Results are represented as mean value for each group ± standard error of mean. Statistical analysis was conducted using one-way analysis of variance (ANOVA) with Dunnet’s T3 post-hoc analysis for data at 4 and 8 months, and Tuckey post-hoc analysis for data at 6 and 12 months. +/- excipient vs -I- AAV8-OAT and -I- excipient vs -I- AAV8- OAT, * p-value < 0,05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001; +/- excipient vs -I- excipient. ONL: outer nuclear layer; +/-: heterozygous unaffected Oat mice; homozygous affected Oat mice.

Figure 19: Subretinal administration of AAV8-OAT improves RPE degeneration in Oat" ' mice

(A) Representative pictures from montages of the entire retinal section imaged at 40X magnification and used for RPE analysis. Each picture is composed of —2 fields. Scale bar (white bar) = 20 pm. RPE, outer segments (OS) and outer nuclear layer (ONL) are indicated. White arrows point at degenerated RPE. +/-: heterozygous unaffected Oat mice; homozygous affected Oat mice. RPE: retinal pigment epithelium; OS: outer segments; ONL: outer nuclear layer

(B) Quantification of degenerated RPE was performed in OaO~ mice receiving AAV8-CMT or excipient as a control condition. As an unaffected control, Oat ^+/ ~ mice were injected with excipient. For each eye, the length of degenerated RPE per 100 pm of RPE was measured. Results are represented as mean value for each eye (filled square) and as mean value for each treatment group (reported inside or above each column). Statistical analysis was conducted using one-way ANOVA with Tuckey post-hoc analysis. * p-value < 0.05, ** p-value < 0.01, ns (not significant) p-value > 0.05.

Figure 20: Subretinal administration of AAV8-OAT does not improve retinal function in subretinal injected Oat' ' mice.

(A-B) Electroretinogram analysis to assess A-wave (A) and B-wave (B) was performed at 4, 6, 8 and 12 months of age in OaO~ mice injected sub-retinally with either AAV8-6M 7' (triangles, n of eyes = 9, 10, 8 and 6 per each time-point) or excipient (squares, n of eyes = 9, 10, 8 and 5 per each timepoint) in the contralateral eye. Heterozygous (Oat O mice injected with excipient (circles, n of eyes = 10, 10, 10 and 9 per each time-point) were used as unaffected controls. Mouse eyes were stimulated with a luminance of 20 candelas/m ² . Data are represented as mean ± standard error of mean (SEM). Cd: candela; +/-: heterozygous unaffected Oat mice; -/-: homozygous affected Oat mice.

Figure 21 : Intraocular administration of AAV8-OAT does not alter hyperornithinaemia

Ornithinaemia measured in OaO~ mice receiving AAV8-6M 7'in one eye. Samples from Oat ^+/ ~ mice administered the excipient and OaO~ mice uninjected were used as controls of normal- and hyperornithinaemia, respectively. Age at blood collection is reported below the graph. Results are represented as mean value for each animal (filled square) and as mean value for each treatment group (reported inside each column). +/-: Oat ^+/ ' heterozygous unaffected mice; -/-: Oat' ⁷ ' affected mice; Exc: group of animals administered with excipient; NT: uninjected animal group.

Figure 22: Combination of systemic and intraocular administration of AAV8-0AT further improves retinal function in Oat' ' mice.

(A-B) Electroretinogram analysis at age 4 months to assess A-wave (A) and B-wave (B) in Oaf' mice injected systemically and subretina with either AAV8-CMZ or excipient in the contralateral eye. Heterozygous (Oat ) mice injected systemically and subretina with excipient were used as unaffected controls. Mouse eyes were stimulated with luminances from 0.0001 to 20 candela. second/m ² . Data are represented as mean ± standard error of mean (SEM), n of eyes is reported next to the 20-candela measurement of the A-WAVE. Cd: candela; s: second; +/-: heterozygous unaffected Oat mice; -/-: homozygous affected Oat mice.

(C-D) Electroretinogram analysis at age 6 months to assess A-wave (C) and B-wave (D) in Oaf mice injected systemically and subretina with either AAV8-CMZ or excipient in the contralateral eye. Heterozygous (Oaf) mice injected systemically and subretina with excipient were used as unaffected controls. Mouse eyes were stimulated with luminances from 0.0001 to 20 candela. second/m ² . Data are represented as mean ± standard error of mean (SEM), n of eyes is reported next to the 20-candela measurement of the A-WAVE. Cd: candela; s: second; +/-: heterozygous unaffected Oat mice; -/-: homozygous affected Oat mice.

Figure 23: Ornithinaemia is lower in Oat' ' treated with the combination of systemic and Intraocular AAV8-OAT

Ornithinaemia measured in Oaf mice receiving AAV8-6M /' systemically and in one eye. Samples from Oaf~ and Oaf mice administered the excipient were used as controls of normal- and hyperornithinaemia, respectively. Age at blood collection is reported below the graph. Results are represented as mean value for each animal (filled square) and as mean ± standard error of mean value for each treatment group (mean value reported inside each column). +/-: Oat ^+/ ' heterozygous mice; - /-: Oat' ⁷ ' affected mice; Exc: group of animals administered with excipient.

GENE THERAPY

During the past decade, gene therapy has been applied to the treatment of disease in hundreds of clinical trials. Various tools have been developed to deliver genes into human cells. In the present invention the vectors may be administered to a patient. A skilled worker would be able to determine an appropriate dosage range.

Gene therapy may be directed to a specific tissue in order to correct a genetic defect.

In the present invention, in order to correct the retina defect in GACR one strategy is to direct the gene delivery directly to the target tissue, e.g. to the retina, for example via subretinal injection. Alternatively, a systemic injection of a vector targets the liver via liver-specific expression of the transgene due to the presence of a liver-specific promoter.

In the first aspect, the retina defect is corrected by the retina-specific expression of the transgene. In another aspect, the retina defect is corrected by the reduction in ornithine levels achieved through liver-specific expression of the ornithine aminotransferase enzyme. In a further aspect, the retina defect is corrected by the combined reduction in ornithine levels achieved through liver-specific expression of the ornithine aminotransferase enzyme and by the retina-specific expression of the transgene.

CONSTRUCTS

The present invention provides a nucleic acid construct for gene therapy of GACR.

The nucleic acid construct of the invention comprises a nucleic acid sequence coding for a ornithine aminotransferase (OAT) enzyme comprising: a promoter sequence, a coding sequence of the ornithine aminotransferase (OAT) gene under control of said promoter.

As used herein, the term OAT indicates the enzyme ornithine aminotransferase encoded by the ornithine aminotransferase (OAT) gene.

Preferably, the nucleic acid construct further comprises a 3XFLAG tag at the 3 ’end of the coding sequence of the ornithine aminotransferase (OAT) gene.

The human OAT gene (ENSG00000065154, ensembl database, 12 April 2022 version) is located on the reverse strand of chromosome 10 (124,397,303-124,418,976) and has at least 8 annotated transcript isoforms. The canonical transcript (GenBank accession NM 000274.4) is comprised of 10 exons, the first of which is non coding, and translation initiates from an AUG signal on exon 2. The 5’UTR is 90 nucleotides long, while the 3’ UTR comprises 641 nucleotides, and contains a canonical AAUAA consensus 23 nucleotides upstream of the poly A. The open reading frame encodes for a 439 amino acid protein (NCBI Reference Sequence: NP 000265.1) which is virtually ubiquitously expressed. The other transcripts codify for shorter proteins which lack crucial functional domains and are likely to be enzymatically inactive. The relative abundance, the tissue distribution, and the potential physiological role of each of these transcripts are still unknown ¹⁷ . Exemplary sequence of the OAT gene can be found at NM_000274.4 (NCBI, last version).

The human OAT protein is a mitochondrial matrix enzyme which catalyzes the reversible interconversion of L-ornithine and 2-oxoglutarate to L-glutamate semialdehyde and L-glutamate (UniProt P04181-1). In humans, the mature enzyme is a homopolymer comprised of four or possibly six monomers. The monomers are synthesized on cytoplasmic free ribosomes as 49-kDa precursors which are cleaved to 45-kDa peptides at mitochondrial entry ¹⁸ . The OAT protein can have a sequence identified as P04181-1 or as P04181-2 in Uniprot database (last version).

It is known in the art that a method for measuring OAT activity relies on the PC5 metabolite quantification with the ninhydrin method.

In some embodiments, the OAT enzyme codified by the construct of the invention comprises or consists of an amino acid sequence that has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 1 or a fragment thereof.

In some embodiment, the OAT gene from which the OAT coding sequence is derived has sequence SEQ ID N. 3

The coding sequence can codify for a variant of ornithine aminotransferase (OAT), for example it can comprise additions, deletions or substitutions with respect to the coding sequence of the wild type ornithine aminotransferase (OAT) gene as long as these protein variants retain substantially the same relevant functional activity as the original OAT. The coding sequence can also codify for a fragment of ornithine aminotransferase (OAT), as long as this fragment retains substantially the same relevant functional activity as the original OAT.

Suitably, the coding sequence may be codon optimized for expression in human. Softwares for codon optimization are known in the art (see for example http://www.genscript:com/cgi-bin/rare codon analy si s : or http s : //eu . i dtdna. eom/ site/ aeeount/1 ogin? retumurl=%2F C odonOpt) .

Thus, a nucleic acid construct coding for a OAT enzyme may comprise a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, or 99% identity with any of sequence SEQ ID NOs: 2, 7 and 20; preferably a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, or 99% identity with any one of said sequences; more preferably a sequence having at least 95 %, 96%, 97%, 98%, or 99% identity with any one of said sequences. In some embodiments, the nucleic acid construct comprises or consists of DNA.

As used herein, the terms "nucleic acid" and "polynucleotide sequence" and “construct” refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally-occurring nucleotides. The polynucleotide sequences include both full- length sequences as well as shorter sequences derived from the full-length sequences. It is understood that a particular polynucleotide sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell. The polynucleotide sequences falling within the scope of the subject invention further include sequences which specifically hybridize with the sequences coding for a peptide of the invention. The polynucleotide includes both the sense and antisense strands as either individual strands or in the duplex.

The subject invention also contemplates those polynucleotide molecules having sequences which are sufficiently homologous with the polynucleotide sequences of the invention so as to permit hybridization with that sequence under standard stringent conditions and standard methods ¹⁹ .

REGULATORY ELEMENTS

The subject invention also concerns a construct that can include regulatory elements that are functional in the intended host cell in which the vector comprising the construct is to be expressed. A person of ordinary skill in the art can select regulatory elements for use in appropriate host cells, for example, mammalian or human host cells. Regulatory elements include, for example, promoters, transcription termination sequences, translation termination sequences, enhancers, signal peptides, degradation signals and polyadenylation elements.

A construct of the invention may optionally contain a transcription termination sequence, a translation termination sequence, signal peptide sequence, internal ribosome entry sites (IRES), enhancer elements, and/or post-trascriptional regulatory elements such as the Woodchuck hepatitis virus (WHV) posttranscriptional regulatory element (WPRE). Transcription termination regions can typically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences can be positioned downstream of a coding sequence to provide for efficient termination. In the system of the invention a transcription termination site is typically included.

PROMOTERS The nucleic acid construct of the invention of the invention can comprise a promoter sequence operably linked to a nucleotide sequence encoding the desired polypeptide. The term “operably linked”, means that the parts (e.g. transgene and promoter) are linked together in a manner which enables both to carry out their function substantially unhindered.

A promoter within the meaning of the present invention may be a ubiquitous promoter, meaning that it drives expression of the gene in a wide range of cells and tissues. A further promoter within the present invention is a tissue- specific promoter that shows selective activity in one or a group of tissues but is less active or not active in other tissues. The promoter may show inducible expression in response to presence of another factor, for example a factor present in a host cell.

Where the vector comprising the construct is administered for therapy, it is preferred that the promoter is functional in the target cell (e.g. retinal cell or liver cell).

In some embodiments, the promoter is a ubiquitous promoter, retina-specific promoter, preferably an RPE or photoreceptor cell specific promoter or a liver specific promoter, preferably a hepatocyte specific promoter. Promoters contemplated for use in the subject invention include, but are not limited to, native gene promoters or fragments thereof such as cytomegalovirus (CMV) promoter (see e.g. KF853603.1), thyroxine binding globulin (TBG) promoter ²⁰ , OAT promoter ²¹ , chimeric CMV/chicken beta-actin promoter (CBA) and the truncated form of CBA (smCBA) promoter (US8298818 and Light-Driven Cone Arrestin Translocation in Cones of Postnatal Guanylate Cyclase-1 Knockout Mouse Retina Treated with AAVGC1), Rhodopsin promoter (see e.g. NG 009115), Interphotoreceptor retinoid binding protein promoter (see e.g. NG 029718.1), vitelliform macular dystrophy 2 promoter (see e.g. NG 009033.1), PR-specific human G protein- coupled receptor kinase 1 (hGRKl; see e.g. AY327580.1) (Haire et al. 2006 ²² ; U.S. Patent No. 8,298,818; all the herein mentioned references are herein incorporated by reference). However any suitable promoter known in the art may be used. In a preferred embodiment, the promoter is a CMV or TBG promoter.

In preferred embodiments, the promoter is a CMV promoter for example a promoter of SEQ ID N: 5 or a TBG promoter, for example a promoter of SEQ ID N.19 ; or a fragment thereof.

Preferably, the promoter nucleic acid sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID Ns.5, 13 or 19 or a fragment thereof, preferably wherein the promoter substantially retains the natural function of the promoter of SEQ ID Ns 5, 13 or 19. Promoters can be incorporated into a construct using standard techniques known in the art. Multiple copies of promoters or multiple promoters can be used in a construct of the invention. In one embodiment, the promoter can be positioned about the same distance from the transcription start site as it is from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without a substantial decrease in promoter activity.

INTRON

Introns may be included in a nucleic acid construct. In particular, an intron placed between the promoter and the coding sequence increases mRNA stability and protein production, thereby increasing transgene expression.

Any suitable intron may be used, the selection of which may be readily made by the skilled person. Preferred introns according to the invention are small T antigen intron, large T antigen intron, SV40 intron and hybrid introns made of fragments of introns. For instance, a preferred intron of the invention is a chimeric promoter intron composed of the 5’ donor site from the first intron of the human P-globin gene and the branch and 3 ’-acceptor site from the intron of an immunoglobulin gene heavy chain variable region, for example the intron of SEQ ID N.6.

DETECTABLE MARKER

In a preferred aspect, the invention comprises at least one detectable marker. As used herein, the term “detectable marker” refers to a moiety that, when attached to the polypeptide, confers detectability upon that polypeptide or another molecule to which the polypeptide binds. Preferably, the detectable marker comprises an affinity tag. Non-limiting examples of affinity tags include Strep-tags, chitin binding proteins (CBP) , maltose binding proteins (MBP) , glutathione-S-transferase (GST) , FLAG- tags, HA-tags, Myc-tags, poly (His) -tags as well as derivatives thereof. In some embodiments of any of the aspects, the detectable marker is 3xFLAG (i.e., the FLAG motif repeated three times).

In some embodiments of any of the aspects, the detectable marker comprises or is encoded by SEQ ID NO: 8, or comprises or is encoded by a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 8, that maintains the same functions as SEQ ID NO: 8 or by the sequence encoded by SEQ ID NO: 8 (e.g., detection of the polypeptide) . Fluorescent moieties can be used as detectable markers, but detectable markers also include, for example, isotopes, fluorescent proteins and peptides, enzymes, components of a specific binding pair, chromophores, affinity tags as described herein or known in the art, antibodies, colloidal metals (i.e.g old) and quantum dots. Detectable markers can be either directly or indirectly detectable. Directly detectable markers do not require additional reagents or substrates in order to generate detectable signal. Examples include isotopes and fluorophores. Indirectly detectable markers require the presence or action of one or more co-factors or substrates. Examples include enzymes such as P- galactosidase which is detectable by generation of colored reaction products upon cleavage of substrates such as the chromogen X-gal (5-bromo-4-chloro-3-indoyl-P-D-galactopyranoside) , horseradish peroxidase which is detectable by generation of a colored reaction product in the presence of the substrate diaminobenzidine and alkaline phosphatase which is detectable by generation of colored reaction product in the presence of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.

POLYADENYLATION SEQUENCE

The nucleic acid construct of the present invention may comprise a polyadenylation sequence. Suitably, the transgene is operably linked to a polyadenylation sequence. A polyadenylation sequence may be inserted downstream of the transgene to improve transgene expression.

A polyadenylation sequence typically comprises a polyadenylation signal, a polyadenylation site and a downstream element: the polyadenylation signal comprises the sequence motif recognised by the RNA cleavage complex; the polyadenylation site is the site of cleavage at which a poly-A tails is added to the mRNA; the downstream element is a GT-rich region which usually lies just downstream of the polyadenylation site, which is important for efficient processing.

In some embodiments, the polyadenylation sequence is a bovine growth hormone (bGH) polyadenylation sequence or an SV40 polyadenylation sequence; or a fragment thereof that retains the natural function of the polyadenylation sequence.

In preferred embodiments, the polyadenylation sequence is a bovine growth hormone (bGH) polyadenylation sequence.

A preferred polyadenylation sequence of the invention is SEQ ID N. 10 or SEQ ID N.16 or SEQ ID N.22. In some embodiments, the polyadenylation sequence comprises or consists of a nucleic acid sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID N.10, 16 or 22, preferably wherein the polyadenylation sequence substantially retains the natural function of the polyadenylation sequence of SEQ ID N.10, 16 or 22.

POST-TRANSCRIPTIONAL REGULATORY ELEMENTS

The nucleic acid constructs of the present invention may comprise post-transcriptional regulatory elements. Suitably, the protein-coding sequence is operably linked to one or more further post- transcriptional regulatory elements that may improve gene expression.

The construct of the present invention may comprise a Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element (WPRE). Suitably, the OAT coding sequence is operably linked to a WPRE.

Suitable WPRE sequences will be well known to those of skill in the art (see, for example, Zufferey et al. ²³ ; Zanta-Boussif et al. ²⁴ ). Suitably, the WPRE is a wild-type WPRE or is a mutant WPRE. For example, the WPRE may be mutated to abrogate translation of the woodchuck hepatitis virus X protein (WHX), for example by mutating the WHX ORF translation start codon.

In some embodiments, the WPRE comprises or consists of the nucleotide sequence SEQ ID NOV, 15 or 21 or a fragment thereof.

KOZAK SEQUENCE

The nucleic acid construct of the present invention may comprise a Kozak sequence. Suitably, the OAT-coding sequence is operably linked to a Kozak sequence. A Kozak sequence may be inserted before the start codon to improve the initiation of translation.

Suitable Kozak sequences will be well known to the skilled person (see, for example, Kozak ²⁵ ).

In some embodiments, the Kozak sequence comprises or consists of the nucleotide sequence GCGGCCGCC or a fragment thereof.

VECTORS

The present invention also relates to a vector comprising the nucleic acid construct as described herein. Such vector may therefore contain any of the elements above described in relation to the construct. In particular, it can comprise, besides the OAT coding sequence, one or more regulatory elements including, for example, promoters, transcription termination sequences, translation termination sequences, enhancers, signal peptides, degradation signals and polyadenylation elements, in particular as above defined.

Vectors suitable for the delivery and expression of nucleic acids into cells for gene therapy are encompassed by the present invention.

Vectors of the invention include viral and non-viral vectors.

Non-viral vectors include non-viral agents commonly used to introduce or maintain nucleic acid into cells. Said agents include in particular polymer-based, particle-based, lipid-based, peptide-based delivery vehicles or combinations thereof, such as cationic polymers, micelles, liposomes, exosomes, microparticles and nanoparticles including lipid nanoparticles (LNP).

Among viral delivery, genetically engineered viruses, including adeno-associated viruses, are currently amongst the most popular tools for gene delivery. The concept of virus-based gene delivery is to engineer the virus so that it can express the gene(s) of interest or regulatory sequences such as promoters and introns. Depending on the specific application and the type of virus, most viral vectors contain mutations that hamper their ability to replicate freely as wild-type viruses in the host. Viruses from several different families have been modified to generate viral vectors for gene delivery. These viruses include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, baculoviruses, picomaviruses, and alphaviruses.

Viral vectors of the invention may be derived from non-pathogenic parvovirus such as adeno- associated virus (AAV), retrovirus such as gammaretrovirus, spumavirus and lentivirus, adenovirus, poxvirus and an herpes virus.

Particularly preferred viruses according to the present invention are lentivirus and adeno-associated virus.

Viral vectors are by nature capable of penetrating into cells and delivering nucleic acids of interest into cells, according to a process known as viral transduction.

As used herein, the term “viral vector” refers to a non-replicating, non-pathogenic virus engineered for the delivery of genetic material into cells. Viral genes essential for replication and virulence are replaced with an expression cassette for the transgene of interest. Thus, the viral vector genome comprises the transgene expression cassette flanked by the viral sequences required for viral vector production. The term “virus particle” or “viral particle” is intended to mean the extracellular form of a non- pathogenic virus, in particular a viral vector, composed of genetic material made from either DNA or RNA surrounded by a protein coat, called capsid, and in some cases an envelope derived from portions of host cell membranes and including viral glycoproteins.

As used herein, a viral vector refers also to a viral vector particle.

Viral vectors encompassed by the present invention are suitable for gene therapy.

Viral particles can be for example obtained using vectors that are capable of accommodating genes of interest and helper cells that can provide the viral structural proteins and enzymes to allow for the generation of vector-containing infectious viral particles.

Adeno-associated virus (AAV)

Adeno-associated virus is a family of viruses that differs in nucleotide and amino acid sequence, genome structure, pathogenicity, and host range. This diversity provides opportunities to use viruses with different biological characteristics to develop different therapeutic applications.

An ideal adeno-associated virus-based vector for gene delivery must be efficient, cell-specific, regulated, and safe. The efficiency of delivery may determine the efficacy of the therapy. Current efforts are aimed at achieving cell-type-specific infection and gene expression with adeno-associated viral vectors. In addition, adeno-associated viral vectors are being developed to regulate the expression of the gene of interest, since the therapy may require long-lasting or regulated expression.

Adeno-associated virus (AAV) is a small virus which infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response. Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell. These features make AAV a very attractive candidate for creating viral vectors for gene therapy, and for the creation of isogenic human disease models.

Wild-type AAV has attracted considerable interest from gene therapy researchers due to a number of features. Chief amongst these is the virus's apparent lack of pathogenicity. It can also infect nondividing cells and has the ability to stably integrate into the host cell genome at a specific site (designated AAVS1) in the human chromosome 19. Development of AAVs as gene therapy vectors, however, has eliminated this integrative capacity by removal of the rep and cap from the DNA of the vector. The desired gene together with a promoter to drive transcription of the gene is inserted between the ITRs that aid in concatemer formation in the nucleus after the single-stranded vector DNA is converted by host cell DNA polymerase complexes into double-stranded DNA. AAV-based gene therapy vectors form episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers remain intact for the life of the host cell. In dividing cells, AAV DNA is lost through cell division, since the episomal DNA is not replicated along with the host cell DNA. Random integration of AAV DNA into the host genome is detectable but occurs at very low frequency. AAVs also present very low immunogenicity, seemingly restricted to generation of neutralizing antibodies, while they induce no clearly defined cytotoxic response. This feature, along with the ability to infect quiescent cells make AAV particularly suitable for human gene therapy.

The AAV genome is built of single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed, which is about 4.7 kilobase long. The genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. The former is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry.

The Inverted Terminal Repeat (ITR) sequences received their name because of their symmetry, which was shown to be required for efficient multiplication of the AAV genome. Another property of these sequences is their ability to form a hairpin, which contributes to so-called self-priming that allows primase-independent synthesis of the second DNA strand. The ITRs were also shown to be required for efficient encapsidation of the AAV DNA combined with generation of a fully assembled, deoxyribonuclease-resistant AAV particles.

With regard to gene therapy, ITRs seem to be the only sequences required in cis next to the therapeutic gene: structural (cap) and packaging (rep) genes can be delivered in trans. With this assumption many methods were established for efficient production of recombinant AAV (rAAV) vectors containing a reporter or therapeutic gene.

The AAV vector comprises an AAV capsid able to transduce the target cells of interest. The AAV capsid may be from one or more AAV natural or artificial serotypes.

AAV may be referred to in terms of their serotype. A serotype corresponds to a variant subspecies of AAV which, owing to its profile of expression of capsid surface antigens, has a distinctive reactivity which can be used to distinguish it from other variant subspecies. Typically, an AAV vector particle having a particular AAV serotype does not efficiently cross-react with neutralising antibodies specific for any other AAV serotype. All of the known serotypes can infect cells from multiple diverse tissue types. Tissue specificity is determined by the capsid serotype and pseudotyping of AAV vectors to alter their tropism range affects their use in therapy.

The inverted terminal repeat (ITR) sequences used in an AAV vector system of the present invention can be any AAV ITR. The ITRs used in an AAV vector can be the same or different. For example, a vector may comprise an ITR of AAV serotype 2 and an ITR of AAV serotype 5. In one embodiment of a vector of the invention, an ITR is from AAV serotype 2, 4, 5, or 8. In the present invention ITRs of AVV serotype 2 are preferred. AAV ITR sequences are well known in the art (for example, see for ITR2, GenBank Accession Nos. AF043303.1 ; NC_001401.2; J01901.1 ; JN898962.1; see for ITR5, GenBank Accession No. NC_006152.1).

Serotype 2 (AAV2) has been the most extensively examined so far. AAV2 presents natural tropism towards skeletal muscles, neurons, vascular smooth muscle cells and hepatocytes.

Three cell receptors have been described for AAV2: heparan sulfate proteoglycan (HSPG), aVp5 integrin and fibroblast growth factor receptor 1 (FGFR-1). The first functions as a primary receptor, while the latter two have a co-receptor activity and enable AAV to enter the cell by receptor-mediated endocytosis. HSPG functions as the primary receptor, though its abundance in the extracellular matrix can scavenge AAV particles and impair the infection efficiency.

Although AAV2 is the most popular serotype in various AAV-based research, it has been shown that other serotypes can be effective as gene delivery vectors. For instance AAV6 appears much better in infecting airway epithelial cells, AAV7 presents very high transduction rate of murine skeletal muscle cells (similarly to AAV1 and AAV5), AAV8 is superb in transducing hepatocytes and photoreceptors and AAV1 and 5 were shown to be very efficient in gene delivery to vascular endothelial cells. In the brain, most AAV serotypes show neuronal tropism, while AAV5 also transduces astrocytes. AAV6, a hybrid of AAV1 and AAV2, also shows lower immunogenicity than AAV2.

Serotypes can differ with respect to the receptors they are bound to. For example AAV4 and AAV5 transduction can be inhibited by soluble sialic acids (of different form for each of these serotypes), and AAV5 was shown to enter cells via the platelet-derived growth factor receptor.

Methods for preparing viruses and virions comprising a heterologous polynucleotide or construct are known in the art. In the case of AAV, cells can be coinfected or transfected with adenovirus or polynucleotide constructs comprising adenovirus genes suitable for AAV helper function. Examples of materials and methods are described, for example, in U.S. Patent Nos. 8,137,962 and 6,967,018. An AAV virus or AAV vector of the invention can be of any AAV serotype, including, but not limited to, serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV1 1, AAV-PhP.B and AAV-PhP.eB.

In a specific embodiment, an AAV2 or an AAV5 or an AAV7 or an AAV8 or an AAV9 serotype is utilized. Preferably, the AAV8 is used.

Suitably, the AAV genome is derivatized for the purpose of administration to patients. Such derivatization is standard in the art and the invention encompasses the use of any known derivative of an AAV genome, and derivatives which could be generated by applying techniques known in the art. The AAV genome may be a derivative of any naturally occurring AAV. Suitably, the AAV genome is a derivative of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11.

Derivatives of an AAV genome include any truncated or modified forms of an AAV genome which allow for expression of a transgene from an AAV vector of the invention in vivo. In one embodiment, the AAV serotype provides for one or more tyrosine to phenylalanine (Y-F) mutations on the capsid surface.

The plasmid described above can be used to generate the AAV vector of the invention. The AAV vector can be for example produced by triple transfection of producer cells, such as HEK293 cells, a method known in the field wherein the plasmid comprising the gene of interest, OAT in the present case, is transfected along with two additional plasmids into a producer cell wherein the viral particles will then be produced.

HOST CELL

The subject invention also concerns a host cell comprising the viral vector of the invention. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for instance, DH5a, E. coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like. The cell can be a human cell or from another animal. In one embodiment, the cell is a retina cell, particularly a photoreceptor cell, an RPE cell or a cone cell. The cell may also be liver cell, particularly a hepatocyte. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein. Preferably, said host cell is an animal cell, and most preferably a human cell. The cell can express a nucleotide sequence provided in the viral vector of the invention. The man skilled in the art is well aware of the standard methods for incorporation of a polynucleotide or vector into a host cell, for example transfection, lipofection, electroporation, microinjection, viral infection, thermal shock, transformation after chemical permeabilization of the membrane or cell fusion.

As used herein, the term "host cell or host cell genetically engineered" relates to host cells which have been transduced, transformed or transfected with the viral vector of the invention.

COMPOSITIONS

The present invention also provides a pharmaceutical composition for treating an individual by gene therapy, wherein the composition comprises a therapeutically effective amount of the vector of the present invention comprising the therapeutic transgenes or a viral particle produced by or obtained from the same.

Pharmaceutical compositions within the meaning of the present invention comprise the vector or the host cell of the invention optionally in combination with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s), and other carrier agents that may aid or increase the viral entry into the target site (such as for example a lipid delivery system). The vector can be administered in vivo or ex vivo.

Pharmaceutical compositions adapted for parenteral or ocular (e.g. retinal) administration, comprising an amount of a compound, constitute a preferred embodiment of the invention. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. The vector or the pharmaceutical composition of the present invention may be delivered to the retina preferentially via the subretinal injection or it can also be prepared in the form of injectable suspension, eye lotion or ophthalmic ointment that can be delivered to the retina with a non-invasive procedure. In another preferred embodiment the vector or the pharmaceutical composition is systemically delivered, for example by intravenous injection.

In a further preferred embodiment, the vector or the pharmaceutical composition of the present invention may be delivered to the retina, preferentially via the subretinal injection or in the form of injectable suspension, eye lotion or ophthalmic ointment, and it may be systemically delivered, for example by intravenous injection, at the same time of the delivery to retina or before the delivery to the retina.

The methods of the present invention can be used with humans and other animals. As used herein, the terms "patient" and "subject" are used interchangeably and are intended to include such human and non-human species. Likewise, in vitro methods of the present invention can be earned out on cells of such human and non- human species.

KITS

The subject invention also concerns kits comprising the viral vector or the host cells of the invention in one or more containers. Kits of the invention can optionally include pharmaceutically acceptable carriers and/or diluents. In one embodiment, a kit of the invention includes one or more other components, adjuncts, or adjuvants as described herein. In one embodiment, a kit of the invention includes instructions or packaging materials that describe how to administer a vector system of the kit. Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, the viral vector or the host cell of the invention is provided in the kit as a solid. In another embodiment, the viral vector or the host cell of the invention is provided in the kit as a liquid or solution. In one embodiment, the kit comprises an ampoule or syringe containing the viral vector or the host cell of the invention in liquid or solution form.

MEDICAL USES AND METHODS OF TREATMENT

In one aspect the invention provides the vector, cell, kit or composition of the invention for use in therapy.

In another aspect the invention provides the vector, cell, kit or composition of the invention for use in treatment of GACR. Typically, an ordinary skilled clinician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular individual and administration route. A dose range between IxlOe ⁹ and IxlOe ¹⁵ genome copies of each vector/kg, preferentially between IxlOe ¹¹ and IxlOe ¹³ genome copies of each vector/kg are expected to be effective in humans. A dose range between IxlOe ⁹ and IxlOe ¹⁵ genome copies of each vector/eye, preferentially between IxlOe ¹⁰ and IxlOe ¹³ are expected to be effective for ocular administration.

Dosage regimes and effective amounts to be administered can be determined by ordinarily skilled clinicians. Administration may be in the form of a single dose or multiple doses, preferably as a single dose. General methods for performing gene therapy using polynucleotides, expression constructs, and vectors are known in the art (see, for example, Gene Therapy: Principles and Applications, Springer Verlag 1999 ²⁶ ; and U.S. Patent Nos. 6,461 ,606; 6,204,251 and 6,106,826).

The vector for the use according to the present invention may be used alone or in combination with other treatments or components of the treatment. For example, it can be used together with other treatments which might be helpful for GACR, for example together with an arginine-restricted diet.

In an embodiment, the vector for the use of the invention is delivered to the retina, for example by sub-retinal injection, and it is used in combination with an arginine-restricted diet.

In another embodiment, the vector for the use of the invention is delivered to the liver, for example by intra-venous injection, and it is used in combination with an arginine-restricted diet.

In another embodiment, a first vector according to the invention comprising a promoter specific for liver expression is used in combination with a second vector according to the invention delivered to the retina. Preferably, said first vector is systemically administered and said second vector is delivered to the retina. The combined use of said first and second vectors according to the invention might advantageously allow to reduce the dose of the systemically administered vector with subsequent toxicity reduction.

The subject invention also concerns methods for expressing the OAT polypeptide in a cell. In one embodiment, the method comprises incorporating in the cell the vector system of the invention, that comprises polynucleotide sequences encoding the OAT polypeptide, and expressing the polynucleotide sequences in the cell. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a photoreceptor or a RPE cell or a liver cell or hepatocyte.

VARIANTS, DERIVATIVES, ANALOGUES, AND FRAGMENTS

As those skilled in the art can readily appreciate, there can be a number of variant sequences of a protein found in nature, such as the OAT, in addition to those variants that can be artificially created by the skilled artisan in the lab. The polynucleotides and polypeptides of the subject invention encompasses those specifically exemplified herein, as well as any natural variants thereof, as well as any variants which can be created artificially, so long as those variants retain the desired functional activity. Also within the scope of the subject invention are polypeptides which have the same amino acid sequences of a polypeptide exemplified herein, such as the OAT polypeptide, except for amino acid substitutions, additions, or deletions within the sequence of the polypeptide, as long as these variant polypeptides retain substantially the same relevant functional activity as the polypeptides specifically exemplified herein. For example, conservative amino acid substitutions within a polypeptide which do not affect the function of the polypeptide would be within the scope of the subject invention. Thus, the polypeptides disclosed herein should be understood to include variants and fragments, as discussed above, of the specifically exemplified sequences. The subject invention further includes nucleotide sequences which encode the polypeptides disclosed herein. These nucleotide sequences can be readily constructed by those skilled in the art having the knowledge of the protein and amino acid sequences which are presented herein. As would be appreciated by one skilled in the art, the degeneracy of the genetic code enables the artisan to construct a variety of nucleotide sequences that encode a particular polypeptide or protein. The choice of a particular nucleotide sequence could depend, for example, upon the codon usage of a particular expression system or host cell. Polypeptides having substitution of amino acids other than those specifically exemplified in the subject polypeptides are also contemplated within the scope of the present invention. For example, non-natural amino acids can be substituted for the amino acids of a polypeptide of the invention, so long as the polypeptide having substituted amino acids retains substantially the same activity as the polypeptide in which amino acids have not been substituted. Examples of non-natural amino acids include, but are not limited to, ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, 2- amino butyric acid, y-amino butyric acid, s-amino hexanoic acid, 6-amino hexanoic acid, 2-amino isobutyiic acid, 3 -amino propionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, r-butylglycine, r-butylalanine, phenylglycine, cyclohexylalanine, P-alanine, fluoro-amino acids, designer amino acids such as P-methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogues in general. Non- natural amino acids also include amino acids having derivatized side groups. Furthermore, any of the amino acids in the protein can be of the D (dextrorotary) form or L (levorotary) form. Amino acids can be generally categorized in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby a polypeptide having an amino acid of one class is replaced with another amino acid of the same class fall within the scope of the subject invention so long as the polypeptide having the substitution still retains substantially the same biological activity as a polypeptide that does not have the substitution. Table 1 provides a listing of examples of amino acids belonging to each class.

Also within the scope of the subject invention are polynucleotides which have the same nucleotide sequences of a polynucleotide exemplified herein except for nucleotide substitutions, additions, or deletions within the sequence of the polynucleotide, as long as these variant polynucleotides retain substantially the same relevant functional activity as the polynucleotides specifically exemplified herein (e.g., they encode a protein having the same amino acid sequence or the same functional activity as encoded by the exemplified polynucleotide). Thus, the polynucleotides disclosed herein should be understood to include variants and fragments, as discussed above, of the specifically exemplified sequences.

The subject invention also contemplates those polynucleotide molecules having sequences which are sufficiently homologous with the polynucleotide sequences of the invention so as to permit hybridization with that sequence under standard stringent conditions and standard methods (Maniatis, T. et al, 1982). Polynucleotides described herein can also be defined in terms of more particular identity and/or similarity ranges with those exemplified herein. The sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%. The identity and/or similarity of a sequence can be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% or greater as compared to a sequence exemplified herein. Unless otherwise specified, as used herein percent sequence identity and/or similarity of two sequences can be determined using the algorithm of Karlin and Altschul ²⁷ , modified as in Karlin and Altschul ²⁸ . Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. ²⁹ . BLAST searches can be performed with the NBLAST program, score = 100, wordlength = 12, to obtain sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. ³⁰ . When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used. See NCBI/N1H website. The invention will be now illustrated by the following examples.

Examples

Example 1

Intraocular Gene Delivery Ameliorates Retinal Structure in a Mouse Model of Gyrate Atrophy of the Choroid and Retina

MATERIALS AND METHODS

Generation of adeno-associated viral vector plasmids

The plasmids used for adeno-associated viral (AAV) vector production contain the inverted terminal repeats (ITRs) of AAV serotype 2.

The AAV vector plasmid required to generate AAV-human ornithine aminotransferase (hOA T) contains a cytomegalovirus (CMV) promoter and a chimeric promoter intron composed of the 5 donor site from the first intron of the human P-globin gene and the branch and 3 ’-acceptor site from the intron of an immunoglobulin gene heavy chain variable region followed by the Kozak sequence and the human OAT coding sequence (CDS, NM_000274.4) with or without a 3XFlag tag at the C- terminal of the protein; the expression cassette is completed with the Woodchuck hepatitis virus post- transcriptional regulatory element (WPRE) and the bovine growth hormon polyA (BGH poly A). The enhanced green fluorescent protein (EGFP) plasmid used in some experiments is as follows. The CMV promoter described above was coupled with the chimeric intron followed by the EGFP CDS. The expression cassette is completed with the WPRE sequence and the BGH polyA.

AAV vector production and characterization

The AAV-hCMZ-3XFlag vector was produced by InnovaVector. Vectors were produced by triple transfection of HEK293 cells followed by two rounds of CsC12 purification. AAV-hCM 7'-3XFlag excipient is composed of phosphate buffer saline (PBS, Thermo Fisher Scientific, Waltham, Massachusetts) + 5% glycerol. Physical titers [genome copies (GC)/ml] were determined by TaqMan quantitative PCR (Applied Biosystems, Carlsbad, CA, USA). Primers and probes were designed to anneal on BGH pA. The titer of AAV-hCMZwas achieved by averaging the Taqman PCR on BGHpA with a dot-blot analysis on CMV promoter.

Cell culture and transfection hARPE- 19 cells were maintained in F12 medium supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific). Cells were plated in 10 cm dishes at 4,8E+6 cells/dish. To transfect cells, medium was replaced 24 hours later with 3 mL of fresh pre-heated medium + 10 pg of plasmids diluted in lipofectamine LTX (Thermo Fisher Scientific). After an over-night incubation, cells received 7 mL of fresh pre- heated medium and were harvested 72 hours after transfection.

Animal models

Mice were housed at the TIGEM animal house (Pozzuoli, Italy) and maintained under a 12-h light/dark cycle (10-50 lux exposure during the light phase). Oat tmlDVa/tmlDVa mice (referred to as Oat' ') were bom by breeding heterozygous females with heterozygous males. Oat mice used in this study were either affected (Oat' ') or unaffected (Oat ^+/ ‘). Oat tmlDVa/tmlDVa mice were generated by inserting the neomycin cassette in exon 3 of the murine Oat which leads to a frameshift and an early stop codon thus murine Oat is not expressed. The genotype for Oat tmlDVa allele was performed by PCR analysis of genomic DNA extracted from mouse fingertip. The primers used for the PCR amplification are as follows: Fw (5’- AACTAGCAAGTCTGCAGACC-3’, SEQ ID N.25) and Rev (5’-TCCACAAGGCATTCAGTGCG-3’, SEQ ID N.26), which generate a product of 290 bp for the wild type allele and a product of 1658 bp for the tmlDVa allele. Oat' ' pups die because of hypoargininemia-hyperammonemia; for this reason, pups were intraperitoneally injected twice a day from post-natal day (p) 1 to pl5 with a solution 2: 1 of IM hydrochloride arginine and IM arginine base (arginine final concentration IM pH —9); mice were injected with 10-15 pmol/gr of body weight and volume of injection was maintained constant so that final dose tapered to —2-5 pmol/gr of body weight during mice growth.

Subretinal injection of AAV vectors in mice

This study was carried out in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and with the Italian Ministry of Health regulation for animal procedures (Oat mice authorization n° 860/2020-PR). Surgery was performed under anesthesia and all efforts were made to minimize suffering. Four- weeks old mice were anesthetized with an intraperitoneal injection of 2 mL/100 g body weight of ketamine/medetomidine. An equal volume (1 pL) of either vector solution or excipient were delivered subretinally via a posterior trans-scleral trans-choroidal approach.

Western blot analysis

Eyecups (cups + retinas) for Western blot (WB) analysis were lysed in a custom buffer (50 mM Tris- HC1 pH 7.4, 100 mM NaCl, 0,2 mM EDTA, 0,5% Triton) for OAT protein. Lysis buffer was supplemented with 0,5% phenylmethyl sulfonyl fluoride (PSMF) (Sigma-Aldrich) and 1% complete EDTA-free protease inhibitor cocktail (Roche, Milan, Italy). Protein concentration was determined using Pierce BCA protein assay kit (Thermo-Scientific). After lysis, samples were denatured at 99°C for 5 min in 4X Laemmli sample buffer (Bio-rad, Milan, Italy) supplemented with 13- mercaptoethanol (Sigma-Aldrich) diluted 1 : 10. Samples for OAT analysis were separated on 12% SDS-polyacrilamide electrophoresis home-made gel. The following antibodies were used for immuno-blotting: anti-0 AT (1 : 1000, polyclonal, abl37679; Abeam, Cambridge, UK) that recognizes a peptide corresponding to aminoacids 30-395 of the hOAT protein

(TKKTVOGPPTSDDIFEREYKYGAHNYHPLPVALERGKGIYLWDVEGRKYFDFLSSY SAVN QGHCHPI<IVNALI<SQVDI<LTLTSRAFYNNVLGEYEEYITI<LFNY HI<VLPMNTGVEAGETA CKLARKWGYTVKGIQKYKAKIVFAAGNFWGRTLSAISSSTDPTSYDGFGPFMPGFDIIPY N DLPALERALQDPNVAAFMVEPIQGEAGVVVPDPGYLMGVRELCTRHQVLFIADEIQTGLA RTGRWLAVDYENVRPDIVLLGKALSGGLYPVSAVLCDDDIMLTIKPGEHGSTYGGNPLGC RVAIAALEVLEEENLAENADKLGIILRNELMKLPSDVVTAVRGKGLLNAIVIKETKDWDA WKVCL [SEQ ID N.27]; underlined aminoacids are different (8,2%) in murine Oat); anti-Calnexin.

Spectral-Domain Optical Coherence Tomography

Spectral domain optical coherence tomography (SD-OCT) images were obtained using the Bioptigen Spectral Domain Ophthalmic Imaging System (SDOIS; Envisu R2200, Bioptigen, Morrisville, NC, USA). Mice were anesthetized and pupils were dilated by applying 1-2 drops of topical 0.5% tropicamide (Visufarma, Rome, Italy). To prevent corneal desiccation during the procedure, topical lubricant eye drops (Recugel; Bausch & Lomb, Rochester, NY, USA) were applied bilaterally with a small brush. Mice were positioned into the animal imaging mount and rodent alignment stage (AIM- RAS; Bioptigen, Morrisville, NC, USA); the laser source was placed in front of the mouse, and images were acquired by the InVivoVue Clinic software (Bioptigen, Morrisville, NC, USA). Three images, one central, one superior, and one inferior to the optic nerve, were taken from the temporal side and the nasal side of each eye. ONL thickness was manually measured three times from each OCT scan image and averaged.

Retinal pigment epithelium analysis

Eyes from Oat mice (+/- or -/-) were enucleated at 12 months of age and marked on the temporal side of the cornea with a surgical needle. Fixation was performed using Davidson fixative overnight, then kept in 70% ethanol prior to processing and embedding in paraffin performed by Tigem Histology Core. Sections were cut at 6 pm thickness on a Leica Microtome RM2245 (Leica Microsystems, Bannockburn, IL, USA), mounted on slides and stained with toluidine blue and borace staining. Length of denegerated retinal pigment epithelium (RPE) was measured by a masked operator in a montage of the entire retinal section obtained through acquisition of overlapping fields using a Zeiss Apotome (Carl Zeiss, Oberkochen, Germany) with 40X magnification; then, the entire retinal section was reconstituted on Photoshop software (Adobe, San Jose, California). RPE measurements were performed using ImageJ software. Degenerated RPE length was normalized over the length of the RPE divided by 100 pm.

Statistical analysis

One-way analysis of variance (ANOVA) followed by Tuckey post-hoc analysis was used to perform multi pairwise comparisons between groups in Figure 2. The ANOVA p-values are the following. hARPE-19 pEGFP Vs either hARPE-19 pOAT (pANOVA < 0,0001) or hARPE-19 pOAT-3XFlag (pANOVA < 0,0001); hARPE-19 pOAT Vs hARPE-19 pOAT-3XFlag (pANOVA = 0,506). The Student’s t-test was used to compare data depicted in Figure 4.. The p-values are the following: Oat-/- treated with AAV-hCMZ-3XFlag Vs Oat-/- injected with excipient at 4 months of age = 0,0071; Oat-/- treated with AAV-hCMZ-3XFlag Vs Oat-/- injected with excipient at 6 months of age = 0,0016; Oat-/- treated with AAV-hCMZ-3XFlag Vs Oat-/- injected with excipient at 8 months of age = 0,0023; Oat-/- treated with AAV-hCM 7'-3XFlag Vs Oat-/- injected with excipient at 12 months of age = 0,0028.

Results

Eye gene therapy Approach

Activity of human ornithine aminotransferase in vitro

The hCMZ-3XFlag plasmid (hereafter referred to as pCMZ-3XFlag) was tested for enzymatic activity upon transfection into a cell line with relatively low expression of hOAT, hARPEl 9-cell s. Additionally, we performed a comparison between the hOAT plasmid (pOAT) and pCMZ-3XFlag tagged to ensure that tag addition to the protein does not alter enzymatic activity. Cell lysates were tested in an OAT activity assay based on the use of ninhydrin, which forms complexes with the OAT reaction product, pyrroline 5-carboxilate (P5C) ³¹ (Fig. 2). Ninhydrin complexes form a red pigment in hot acidic conditions that precipitates in water, thus allowing absorbance to be measured at 490 nm wavelength. From the results, we detected a 4-5X significant increase of hOAT activity compared to the negative control, pEGFP, in hARPE-19 cells (Fig. 2, pOAT or pCMZ-3XFlag vs pEGFP, p-value < 0,0001) upon transfection of either pOAT or pCMZ-3XFlag. No significant difference in hOAT enzymatic activity was measured between pOAT and pCMZ-3XFlag transfected cells (p-value = 0,506) (Figure 2).

Sub-retinal administration of AAV-hO 17-3XI Iag results in efficient transgene expression in mouse retina Adult C57BL/6 mice were subretinally injected with AAV-CMV-hCM 7-3XFlag-WPRE-BGHpA (AAV-hCMZ-3XFlag) [dose 3.0E+9 genome copies (GC)/eye] to assess vector-induced transgene expression in the murine eye. Four weeks post-injection, western blot (WB) analysis was performed on whole eyecups and confirmed efficient expression of h6M 7'-3XFlag in 100% of eyes (8/8 eyes) injected with AAV-hCM 7- 3XFlag (Figure 3).

Intraocular gene delivery ameliorates retinal structure but not function in a mouse model of gyrate atrophy of the choroid and retina

To assess retinal improvements of AAV-hCMr-SXFlag, we selected a GA mouse model lacking exon 3 of murine Oat resulting in the absence of murine Oat expression. Oat~ ~ mice die in the neonatal stage of life due to hypoargininemia- hyperammonemia. To save knockout pups, we performed intraperitoneal injection of arginine (from pndl to pndl4, injections every 12 ± 3 hours). Electroretinogram (ERG) and histology analysis published in a previous study ⁸ showed that both retinal function and structure are preserved in Oaf^~ mice up to 2 months of age, therefore we subretinally injected Oaf^~ at 1 month of age. Each animal was injected with AAV-hCM 7'-3XFlag (dose 5.4E+9 GC/eye) in one eye while the contralateral eye received formulation buffer as negative control. Oat ^+,L mice were injected with formulation buffer as unaffected controls. Spectral domain- Optical coherence tomography (SD-OCT) analysis shows that AAV-hO^Z-SXFlag significantly increased outer nuclear layer (ONL) thickness at the temporal side of the eye (close to the injection site) up to 12 months of age in Oaf^~ eyes compared to contralateral control eyes, which received the formulation buffer (Figure 4A-B).

This result is mirrored by a normalized RPE morphology across the entire retinal section, as shown by histology analysis of the retina performed at 12 months of age (figure 5A-B).

Conversely, ERG analysis (Figure 6A-B-C) did not show retinal functional improvement, measured both with the A-Wave (Fig. 6B) and the B-Wave (Fig. 6C) in OaC ~ eyes injected with AAV-hCM 7- 3XFlag compared to contralateral eyes injected with the formulation buffer. This data suggests that systemical hyperornithinemia, presumably unchanged by subretinal administration of AAV, impairs retinal function and that its reduction, in combination or not with local OAT delivery, is required for proper photoreceptor function.

Example 2

AAV-mediated liver-directed gene therapy for gyrate atrophy of the choroid and retina

Methods Generation of AAV.TBG.OAT plasmid, and AAV vector production

The human OAT coding sequence was obtained by OriGene Technologies Inc. (Rockville) and amplified by PCR with the following primers:

Notl Forward: 5'-ATAAGAATGCGGCCGCATGTTTTCCAAACTAGC-3' [SEQ ID N.28]

Hindlll - Reverse: 5'-CCCAAGCTTTCAGAAAGACAAGATGG-3'. [SEQ ID N.29]

PCR was performed using Phusion High-Fidelity DNA Polymerase (NEB, M0530), PCR product was subsequently digested with Notl and Hindlll, and subcloned into a pAAV2/8.TBG.GFP vector ¹⁹ by removing the GFP coding sequence (Notl-HindllY). The same pAAV2/8.TBG.GFP was used for control vector production.

AAV vectors were produced by the TIGEM AAV vector core, by triple transfection in HEK 293 cells, purified by CsCh ultracentrifugation, and titered (in genome copies/milliliter) using a real-time PCR-based assay and a dot blot analysis.

Animal model and procedures

The study was carried out in accordance with the regulations and were authorized by the Italian Ministry of Health. B6Ei; AKR-rhg mice were purchased from the Jackson Laboratory (J AX stock #003544) and housed at TIGEM animal facility (Naples). Animals were inbred and maintained under normal animal house conditions: mice were stored in ventilated cages in a 12-hour light-dark cycle environment, receiving a standard chow diet and water ad libitum.

Injections of AAV.TBG.OAT and AAV.TBG.GFP were performed in a final volume of 200 pl into retro-orbital plexus of 6 or 10 weeks-old male and female B6Ei; AKR-rhg mice. To monitor ornithine levels, blood samples were collected by submandibular bleedings at baseline, and at 1, 2, 3, 4,5 6-, 9- and 12-months post-injection. Body weight was evaluated at the same time points. Treated animals were sacrificed by sub-lethal injection of ketamine/medetomidine (300 mg/kg and lOmg/kg, respectively) and perfused with ice-cold phosphate-buff ered saline (PBS) at 12 months of age; liver and eyes samples were harvested for further analyses. Wild type littermates were used as controls, and for all experiments, WT and affected mice were housed in the same condition.

Immunofluorescence staining

IF staining was performed on 5-pm thick paraffin sections of livers. Liver samples were fixed in 4% PFA overnight, washed three times with PBS and stored in 70% Ethanol. Finally, were embedded into paraffin blocks and cut into 5 pm sections. The sections were rehydrated and permeabilized for 20 minutes in PBS containing 0.2% Triton® X-100. Blocking solution containing 5% donkey serum was applied for 1 hour. Primary antibodies were diluted in blocking solution and incubated overnight at 4°C. Anti-GFP (Abeam, Cat# abl3970) was used at 1 :900 dilution and rabbit anti-OAT (Abeam; Cat# abl37679) was used at 1 :200 dilution. Next, sections were washed three times in PBS for 5 min each and then incubated with fluorescently labeled secondary antibodies. The secondary antibody Anti-chicken Fluor® 488, and anti-rabbit Alexa Fluor® 594 were used at 1 :400 dilutions. Sections were washed three times in PBS for 5 min each and mounted on glass slides with Vectashield (Vector Laboratories, Burlingame, CA) mounting reagent. Images were obtained using ZEISS Axioscan 7 Microscope Slide Scanner at 20X magnification.

Electrophysiological Testing

For ERG analysis, B6Ei; AKR-rhg mice were dark adapted for 180 minutes and anesthetized with an intraperitoneal injection of ketamine / medetomidine (100 mg/kg and 0.25 mg/kg, respectively); eyes were stimulated at regular time intervals (0, 5, 15, 30, 45 and 60 minutes) using a flash light and the amplitudes of a- and b-waves were recorded and plotted as a function of increasing light intensities, ranging from 1 x 10 ⁴ to 20.0 cd s/m ² . Body temperature was monitored over time with a rectal thermometer and kept constantly at 37.5 ° C. To allow dilation of the pupils, 1% tropicamide was used. The electrophysiological signals were recorded by gold-plate electrodes positioned on the cornea of each eye, referred to a needle electrode inserted subcutaneously in the frontal region. The different electrodes were connected to a two-channel amplifier. After testing the dark-adapted conditions (scotopic), the cone pathway (photopic) was evaluated by a single flash of 20.0 cd s/m ² in the presence of a continuous background illumination set at 50 cd/m ² .

Statistical Analyses

Data were statistically analyzed using GraphPad Prism software, two-way ANOVA was used to show significance difference for mean comparisons. Experimental group sizes are reported in the figure legends. Data are shown as average ± SEM.

Metabolite measurements. Plasma ornithine concentrations were monitored on plasma samples collected at various times post-vector administration using an ornithine fluorometric assay kit (Abeam; Cat# ab252903) according to the manufacturer’s instructions. Briefly, in this enzyme-based assay, ornithine is converted into a series of intermediates, reacting with a probe producing a stable fluorometric signal (Ex/Em = 535/587nm). At sacrifice plasma amino acid concentrations were measured by HPLC using an Agilent Technologies 1200 Series LC System with an Agilent Zorbax Eclipse XDB-C18 analytical column (5pm, 4.6 x 150mm) and Agilent Eclipse XDB-C18 analytical guard column (5pm, 4.6 x 12.5mm). Aliquots of 500pl of plasma were analyzed. Automated and online derivatization using o-phthalaldehyde (OP A) for and 9-fluorenylmethyl chloroformate (FMOC) was carried out for primary and secondary amino acids, respectively. Amino acids were separated by elution at a flow rate of 1.3 ml/min at 40°C with a linear gradient of solvent B (CH3CN/CH3OHH2O, 40/40/20) in solvent A (40 mM Phosphate buffer pH 7.8) from 10% to 20% in 6 minutes, from 20% to 27% in 6 minutes, from 27% to 60% in 10 minutes, from 60% to 100% in 2 minutes, followed by isocratic step to 100% for 6 minutes. Amino acids were identified by their retention time and quantified by absorption ratio by comparison with the ratio of authentic compounds in the calibration solution, composed by a mixture of amino acids in final concentration of 200pM. Murine eye cups for ornithine determination were harvested upon mice sacrifice and perfusion, dissected under a light microscope to isolate the eyecups from lens and immediately frozen in liquid nitrogen. Collected samples were processed as previously described (Audano et al, 2021) and ornithine concentrations were obtained by liquid chromatography coupled to tandem mass spectrometry.

Retina pathology and ultrastructure. Eyes from 12-month-old Oat ^rhg and 8-month-old Oat ^A mice were harvested and fixed by immersion in Davidson's Fixative (acetic acid, Ethanol, and formaldehyde). Before harvesting, the temporal aspect of the sclera was marked with a suture stitch (Vicryl 7-0, ethicon) to allow eye orientation in paraffin. After 24 hours, samples were dehydrated with 70% Ethanol and paraffin embedded. Seven pm-thick sections were transversally cut on a Leica RM2125 RTS microtome and progressively distributed on 10 slides to have 15-21 sections representative of the whole eye at different levels in each slide. Sections were stained with hematoxylin and eosin and stained section were examined by light microscopy. Images were captured using ZEISS Axioscan 7 Microscope Slide Scanner.

For TEM, murine eyes were collected upon mice sacrifice by sub-lethal injection of ketamine/medetomidine (300 mg/kg and lOmg/kg, respectively) and perfusion with a mixture of 2% paraformaldehyde and 1% glutaraldehyde prepared in 0.2M HEPES buffer (pH 7.4). The eye was gently secured in place and lens and iris were removed though the opening made by cornea excision. The optic cup was centered at the base of the eye and bisected parallel to the median plane; then this half-sphere was bisected again with each of the two cuts. Samples were placed on a slow rotator to circulate the fixative for 30 minutes and stored in PBS at 2°C to 8°C. Next, samples were postfixed, dehydrated and embedded in epoxy resin, as described previously (Polishchuk & Polishchuk, 2019). 60-nm thin sections were cut on a Leica EM UC7 microtome. EM images were acquired from thin sections using a FEI Tecnai-12 electron microscope equipped with a VELETTA CCD digital camera (FEI, Eindhoven, the Netherlands). For basal infoldings of Bruch’s membrane quantification of six images from each experimental group were acquired and analyzed at the same magnification. From each image, three measurements of basal infoldings of Bruch’s membrane in RPE were taken. Each measurement was considered as n=l so a total of n=18 was collected. ITEM software was used for thickness quantification. Results are shown as means with SEM. Statistical analysis was performed using Ordinary one-way ANOVA test.

Immunohistochemistry. Mouse liver samples were fixed in 4% paraformaldehyde overnight, stored in 70% ethanol after washing in PBS IX, and finally embedded into paraffin blocks. 5-pm thick sections were rehydrated and permeabilized in PBS with 0.5% Triton (Sigma-Aldrich) for 20 minutes. Antigen unmasking was performed in 0.01 M citrate buffer pH 6.0 in a microwave oven. Next, sections underwent blocking of endogenous peroxidase activity in methanol/1.5% H2O2 (Sigma- Aldrich) for 30 minutes and were incubated in blocking solution (3% BSA [Sigma-Aldrich], 5% donkey serum [Millipore], 1.5% horse serum [Vector Laboratories] 20 mM MgCh, 0.3% Triton [Sigma- Aldrich] in PBS) for 1 h. Sections were stained with primary antibody rabbit anti-OAT (Abeam; Cat# abl37679, dilution 1 : 100) overnight at 4°C and with universal biotinylated horse antimouse IgG secondary antibody (dilution 1/200) (Vector Laboratories, PK-620) for 1 h RT. Biotin/avidin-HRP signal amplification was achieved using ABC Elite Kit (Vector Laboratories, PK- 6200) according to the manufacturer’ s instructions. 3,3 '-diaminobenzidine (Vector Laboratories) was used as peroxidase substrate. Mayer’s hematoxylin (Bio-Optica) was used as counter-staining. Sections were de-hydrated and mounted in VECTASHIELD® (Vector Laboratories). Image capture was performed using an Axioscan microscope (Zeiss).

Western blot and enzyme activity. Liver specimens were mechanically homogenized using metal beads and lysed in RIPA buffer, supplemented with protease inhibitor cocktail (Sigma). Samples were incubated for 30 minutes on ice, vortexed every 10 minutes, and centrifuged at 13000 rpm for 20 minutes. Pellets were discarded and lysates were used for Western blot analysis. After lysis protein concentration was determined by Bradford Reagent (Bio-Rad), samples were denatured at 100°C for 5 minutes in IX Laemmli sample buffer supplemented with 1 M DTT. Next protein extracts were resolved on SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Thirty micrograms of liver protein were loaded for each specimen into a 4-15% SDS-PAGE; after transfer to PVDF membrane, blots were blocked with TBS-Tween-20 containing 5% non-fat milk for 1 h at room temperature followed by incubation with primary antibody overnight at 4°C. The primary antibodies used for immuno-blotting were: rabbit anti-OAT (Abeam; Cat# abl37679; dilution: 1/1,000), mouse anti-Calnexin (Santa Cruz Biotechnology; Cat# sc23954; dilution: 1/1000), mouse anti-Vinculin (Santa Cruz Biotechnology; Cat# sc73614; dilution: 1/1000) and rabbit anti-GFP (Novus Biologicals; Cat# nb 600-308; dilution: 1/1,000). Proteins of interest were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG antibody (GE Healthcare). Peroxidase substrate was provided by ECL Western Blotting Substrate kit (Pierce). To measure OAT activity, liver specimens were lysed in PBS with protease inhibitor cocktail (Roche) by sonication (4 x5 seconds, 10 second pause) on ice. Enzyme activity was determined by a spectrophotometric assay measuring the dihydroquinazolium derivative of P5C after incubation with 2-aminobenzaldehyde as previously described (Montioli et al, 2017). To measure OAT enzymatic activity in liver, lOOpg of the soluble crude lysate were incubated in 50mM Hepes, pH 8.0, 150mM NaCl and lOOpM PLP at 25 °C with saturating concentration of L-Om (lOOmM) and a-KG (50mM) for 45 min. Reactions were stopped by 10% (v/v) trichloroacetic acid and samples incubated for 40 minutes at 25°C with a fresh solution of 15mM 2-aminobenzaldehyde to allow color development. After centrifugation (12,000 g for 2 minutes), the absorbance at 440nm of the supernatant was measured and a molar extinction coefficient of 2.71 x 103 M ¹ cm ¹ was used to calculate the amount of P5C formed.

Results

Gene delivery to hepatocyte

To investigate the efficacy of OAT transfer to hepatocyte, an animal model of OAT deficiency named B6Ei; AKR-rhg was used. In these mice, a spontaneous missense mutation in the exon 9 of OAT gene, known to mutate a Gly in position 353 into alanine, results in a strong reduction of OAT enzyme activity. This model was generated in a mixed background of pigmented (C57BL/6JEi) and albino (AK/J) mice and recapitulates features typical of GACR patients such as high ornithine blood levels and the early onset of cataracts ³² .

To evaluate retinal degeneration in this model over time, we performed electroretinogram (ERG) response of B6Ei; AKR-rhg pigmented mice, compared to heterozygous or wild type controls at 7 and 12 months of age. Despite at 7 months of age ERG in B6Ei; AKR-rhg mice (n=3) is comparable to heterozygous (n=3) or WT animals (n=3) (Fig. 7 a), aged mutants (12 months of age) showed a significant decrease in the reaction to light stimulus, suggesting a retinal degeneration (Figure 7 b).

To establish the best strategy to drive OAT gene transfer, we in vivo tested two promoter sequences: thyroxine binding globulin (TBG) promoter, that has been previously used in clinical trials, and a 230 base pairs regulatory sequence identified within the human OAT endogenous promoter (OATpromoter.GFP), that was described sufficient for protein expression ²¹ .

We injected C57BL/6N mice with AAV vectors expressing green fluorescent protein (GFP) under the control of either the TBG (n=4) or the OAT promoter regulatory sequence (n=5) at the dose of 1 x 10 ¹³ vp/kg, to compare the reporter gene expression mediated by both promoters in liver. Mice were sacrificed 4 weeks after the injection and GFP expression was evaluated. At 4 weeks post- injection higher levels of the GFP protein were detected in livers of mice injected with TBG.GFP vector if compared to OATpromoter.GFP injected animals, by immunofluorescence (Fig.7 c) and western blot analysis (Figure 7 d), respectively.

Next, to ensure that the TBG promoter was effective in delivering the OAT gene to the hepatocytes physiologically expressing OAT, z.e., at pericentral hepatocytes, we evaluated by immunofluorescence staining the co-localization of GFP with the endogenous OAT in liver specimens of C57BL/6N mice injected with TBG.GFP. The OAT and GFP proteins showed a completely overlapping pericentral expression pattern in mice liver (Fig.7 e).

AAV mediated correction of OAT deficiency in B6Ei; AKR rhg mice

We injected i.v. 6-week-old Oaf ^hs pigmented mice with an AAV vector expressing the human OAT under the control of the TBG promoter (AAV-OAT) at the dose of IxlO ¹³ gc/kg (n=5) whereas age- matched control Oaf ^hs mice (n=3) were injected with IxlO ¹³ gc/kg of AAV-TBG-GFP (AAV-GFP) vector. Following vector administrations, mice injected with AAV-OAT showed significantly lower plasma ornithine concentrations compared to mice injected with the control vector expressing GFP (Fig. 8a) Oaf "" mice had a significant weight gain between 6 and 10 weeks of life and thus, vector loss due to liver growth might explain the lack of normalization of plasma ornithine concentrations (Fig. 8b). Compared to the eyes of control Oaf ^h " mice injected i.v. with the AAV-GFP vector, retinal electrical activity by ERG showed that both the a- and b-wave amplitudes significantly improved in the eyes of mice injected with AAV-OAT vector that were comparable to WT mice, suggesting that both photoreceptor (A wave) and bipolar cell (B wave) functions were preserved (Fig. 8c), because the A wave reflects hyperpolarization of photoreceptors whereas the B wave arises from depolarization of bipolar cells and Muller cells. At 12-month post-injection, plasma ornithine was reduced, and lysine was increased in AAV-OAT-injected Oaf ^hs mice compared to AAV-GFP - injected controls (Fig. 8d) but no other changes in plasma amino acid concentrations were detected, except for a mild and clinically not relevant increase in glutamine (Table 2). Moreover, ornithine concentrations were reduced in the eye cups of 6-week-old Oaf "" mice injected with AAV-OAT compared to controls (Fig. 8e), thus supporting the hypothesis that retinal degeneration of GACR is dependent upon the systemic increase of blood ornithine concentration. Western blot on livers harvested at 12 months post-injection showed greater OAT expression in mice injected with AAV- OAT compared to mice injected with the control vector (Fig. 8f). Consistent with the ERG results, the retinal structure evaluated by H&E at 12 months post-injection in Oaf ^il " mice injected with AAV- OAT was comparable to unaffected wild-type controls and strikingly different from Oaf ^il " mice injected with AAV-GFP vector that showed abnormal retinal pigment epithelium (RPE) cells with irregular size and shape and disorganized photoreceptors of the outer segment layer (Fig. 8g).

Table 2

Table 2. Plasma amino acids (pM) in Oat ^rhg mice injected with IxlO ¹³ gc/kg AAV-OAT or AAV- GFP at 12-month post-injection. Wild-type (WT) control mice are also shown. §Mean value significantly different from WT (p<0.05). ¥AAV-GFP mean value significantly different from AAV- OAT (p<0.05).

AAV mediated OAT expression improves the phenotype in adult B6Ei; AKR rhg mice

To overcome the vector dilution due to the delayed growth, Oaf "" mice were injected at 10 weeks of age when the mouse weight reached the adult size, and they showed a greater approximately 60% reduction of baseline plasma ornithine concentrations after the injection of IxlO ¹³ gc/kg of AAV- OAT vector (Fig. 9a). Further reduction in plasma ornithine concentrations was observed in mice injected with the higher dose of 3xl0 ¹³ gc/kg (Fig. 9a). By ERG, compared to the eyes of mice injected with AAV-GFP, eyes of Oaf ^h " mice treated with both doses of AAV-OAT showed a significant improvement of both a- and b-wave amplitudes at high luminance intensities in flash dark- adapted scotopic and photopic condition at 11 months post-injection (Fig. 9b). At the time of sacrifice 12-months after gene therapy, Oaf ^hg mice injected with the AAV-OAT vector were confirmed to have reduced plasma ornithine concentrations and increased plasma concentrations of lysine (Fig. 9c), but no other significant changes in plasma amino acid concentrations compared to Oaf ^hg mice injected with AAV-GFP or wild-type control mice (Table 3). Moreover, ornithine concentrations were also reduced in the eye cups (Fig. 9d). OAT protein expression and activity were increased in livers of Oaf ^hg mice injected with AAV-OAT compared to control mice (Fig. 9 e-f) and OAT-positive hepatocytes were detected by liver immunohistochemistry (Fig. 9g). At 12 months post-injection, the retinal structure of Oaf ^hg mice injected with AAV-GFP vector showed retinal degeneration with irregular swelling and frequent doming in the RPE (Fig. 9h). In contrast, the RPE of Oaf ^hg mice injected with AAV-OAT at both doses showed a compacted appearance similar to wild-type mice (Fig. 9h)

Table 3

Table 3. Plasma amino acids (pM) in Oaf ^hg mice injected with AAV-OAT or AAV-GFP at 12-month post-injection. Wild-type (WT) control mice are also shown. ^§ Mean value significantly different from WT (p<0.05). ^¥ AAV-GFP mean value significantly different from AAV-OAT (p<0.05). Retinal function recovery in B6Ei; AKR rhg albino mice

The rhg mutation arose spontaneously in the AKR/J genetic background, that were later outcrossed to C57BL/6JEi for line maintenance. As AKR/J mice are an albino strain, by crossing Oaf ^hg mice, mice with white fur are also obtained. As expected, these Oat ^rhg white mice showed signs of ERG abnormalities at earlier age compared to the black mice and showed an improved ERG response following the i.v. injections performed at 10-weeks of age with AAV-OAT compared to control mice injected with AAV-GFP (Fig. 10).

Analysis of the retinal ultrastructure in B6Ei; AKR rhg after AAV mediated OAT delivery

By transmission electron microscopy (TEM), Oaf ^hg mice injected with AAV-GFP showed disorientated and swollen photoreceptors with disorganized disc membranes and frequent phagolysosomes containing undigested material (Fig. Ila) whereas mice injected with AAV-OAT exhibited near normal ultrastructure (Fig. 11b) similar to wild-type controls (Fig. 1c). Moreover, basal infoldings of the Bruch’s membrane Oat ^rhg mice injected with AAV-GFP was irregularly thickened with discontinuous layers compared to wild-type mice (Fig. 12a). In contrast, layer thickness was reduced in Oat ^rhg mice injected with the AAV-OAT vector (Fig. 12b).

AAV mediated correction of OAT deficiency correction in Oat null mice

Oat null mice (Oat ^A ) have increased perinatal mortality and die 24-48 hours after birth if not supplemented by intraperitoneal injections of arginine to prevent hyperammonemia (Wang et al, 1995; Wang et al, 1996). Compared to Oaf ^hg mice, Oat ^A mice have earlier onset of retinal degeneration and show signs of loss of retinal function by 4 months of age (Wang et al., 2000). Consistent with the results in the Oat ^rhg mice, 6-weeks-old Oat ^A mice injected with 3xl0 ¹³ gc/kg of AAV-OAT showed sustained reduction of plasma ornithine concentrations (Fig. 13a) and improvement in ERG response and retinal pathology (Fig. 13b-c).

Example 3

Materials and methods

Generation of adeno-associated viral vector plasmids

The plasmids used for AAV vector production contain the ITRs of AAV serotype 2. The AAV vector plasmid required to generate AAV8-CMZ administered subretina contains a cytomegalovirus (CMV) promoter and a chimeric promoter intron composed of the 5’ donor site from the first intron of the human P-globin gene and the branch and 3 ’-acceptor site from the intron of an immunoglobulin gene heavy chain variable region followed by the human OAT coding sequence [CDS (NM_000274.4)] with the addition of the 3XFlag at the 3’ end; the expression cassette is completed with the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and the bovine growth hormone polyA (BGH poly A). The EGFP plasmid used in vitro contains the same genetic elements as the OAT plasmid. The AAV vector plasmid required to generate AAV8-CMZ administered systemically is similar to the one described above for the exception of the promoter and promoter’s intron, substituted by the thyroxine binding globulin (TBG) promoter.

AAV vector production and characterization

The AAV8-CMZ vector was produced by InnovaVector S.R.L. Vector was produced by triple transfection of HEK293 cells followed by two rounds of CsC12 purification. AAV8-6M 7' formulation buffer is composed of phosphate buffer saline (PBS, Thermo Fisher Scientific, Waltham, Massachusetts) + 5% glycerol. Physical titers [genome copies (GC)/mL] were determined by TaqMan quantitative PCR (Applied Biosystems, Carlsbad, CA, USA); primers and probes were designed to anneal the BGH pA. The titer of AAV8-CMZ was achieved by averaging the Taqman PCR on BGHpA with a dot-blot analysis on CMV promoter.

Animal models

Mice were housed at the TIGEM animal house (Pozzuoli, Italy) and maintained under a 12- hr light/dark cycle (10-50 lux exposure during the light phase). Oat ^{tmlDVa/tmlDVa} (referred to as Oaf ) mice were born by breeding heterozygous females with heterozygous males. Oat mice used in this study were either affected (CkzU) or unaffected (Oat ^+/ ~). Oaf mice were generated by inserting the neomycine cassette in exon 3 of the Oat gene which leads to a frameshift and an early stop codon ²⁴ thus Oat is not expressed. Genotype analysis for the Oat ^tmIDVa allele was performed by PCR analysis of genomic DNA extracted from mouse fingertip. Oaf' pups die because of hypoargininemia- hyperammonemia; for this reason, pups were intraperitoneally injected twice a day from post-natal day (p) 1 to pl5 with a solution 2: 1 of IM hydrochloride arginine and IM arginine base (arginine final concentration IM pH —9); mice were injected with 10-15 pmol/gr of body weight and volume of injection was maintained constant so that final dose tapered to —2-5 pmol/gr of body weight during growth. Subr etinal and systemic injection of AAV vectors in mice

This study was carried out in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and with the Italian Ministry of Health regulations for animal procedures (Oat mice authorization n° 860/2020- PR). Surgery was performed under anesthesia and all efforts were made to minimize suffering. Adult mice (6 weeks of age) were anesthetized with an intraperitoneal injection of 2 mL/100 g body weight of ketamine/medetomidine. An equal volume (150 pL) of either vector solution or excipient were delivered systemically via the retro-orbital plexus. An equal volume (1 pL) of either vector solution or excipient were delivered subretinally via a posterior trans-scleral trans-choroidal approach.

Retinal electrophysiology

For electroretinographic analysis, mice were dark-adapted for 3 hours, anesthetized and positioned in a stereotaxic apparatus under dim red light. Their pupils were dilated with a drop of 0.5% tropicamide (Visufarma, Rome, Italy), and body temperature was maintained at 37°C. Light flashes were generated by a Ganzfeld stimulator (CSO, Costruzione Strumenti Oftalmici, Florence, Italy). The electrophysiological signals were recorded through gold-plate electrodes inserted under the lower eyelids in contact with the cornea. Subcutaneous needles used as negative references were inserted at the level of the corresponding frontal region. The different electrodes were connected to a two- channel amplifier. After completion of responses obtained in dark-adapted conditions (scotopic), the recording session continued with the purpose of dissecting the cone pathway mediating the light response (photopic). To minimize the noise, different responses evoked by light were averaged for each luminance step. The maximal scotopic response of rods and cones was measured in dark conditions with two flashes of 0.7 Hz and a light intensity of 20 cd.s/m ² , photopic cone responses were isolated in light conditions with a continuous background white light of 50 cd.s/m ² , with 10 flashes of 0.7 Hz and a light intensity of 20 cd.s/m ² .

Systemic ornithine measurement

Blood was withdrawn from the retroorbital plex of Oaf ⁷ ' mice administered with AAV8.6M /' at 12 months of age, prior to sacrifice. Blood samples from adult Oat ^+/ ~ mice administered the excipient and Oaf ⁷ ~ mice uninjected were used as controls of normal ornithine levels and hyperornithinaemia, respectively. Samples were mixed 1 : 10 in buffered trisodium citrate 0.109 M (5T31.363048; BD, Franklin Lakes, New Jersey, USA) to separate plasma from the whole blood. Blood plasma was collected after sample centrifugation at 13000 rpm at 4°C for 15 minutes. Plasma samples were diluted 1:10 - 1:17 and ornithine was measured using the Abeam fluorimetric Ornithine Assay Kit (Abeam) following the provided protocol.

Results

Combination of intraocular and systemic administration of AA V8-0A T further improves OaC~ retinal function

We tested the combination of intraocular and systemic gene therapies in Oat ^A mouse model of GACR. Preliminary results show mice administered at 6 weeks of age with AAV8-CMZ systemically (dose 3E+13 GC/Kg) have improved retinal electrical activity in eyes treated with intraocular AAV8- OAT (dose 5.4E+9 GC/eye) compared to contralateral untreated eyes at 4 and 6 months of age (Fig.22); we also detected improvements of retinal electrical activity in animals treated systemically with AAV8-CMZ compared to animals that received the excipient at 4 months of age (Fig.22). Accordingly, ornithinaemia levels were lower in animals treated systemically with AAV8-CMZ compared to untreated animals at 4 and 6 months of age (Fig. 23).

SEQUENCES

Sequence of OAT HUMAN Ornithine aminotransferase

>sp|P04181|OAT_HUMAN Ornithine aminotransferase, mitochondrial OS=Homo sapiens OX=9606 GN=OAT PE=1 SV=1

OAT coding sequence

Sequence of OAT gene

>NM_000274.4 Homo sapiens ornithine aminotransferase (OAT), transcript variant 1, mRNA; nuclear gene for mitochondrial product

Sequences Example 1

Construct AAV-htZ4T-3XFlag

Cytomegalovirus (CMV) promoter

Human ornithine aminotransferase coding sequence

WPRE: AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAG

GCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGC G

AGCGAGCGCGCAG [SEQ ID N.I 1]

Sequences example 2 and 3

Construct AAV-hO 17-3XI la«

5’-ITR from AAV serotype 2

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACC T TTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCAT CACTAGGGGTTCCT [SEQ ID N.4]

Cytomegalovirus (CMV) promoter

TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTT CC

GCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCC C

ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG AC

GTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC AT

ATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTAT G

CCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA TC

GCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTT GA

CTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCA CC AAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGG CGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGT [SEQ ID N.5]

Chimeric promoter’s intron

GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCG A GACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTT GCCTTTCTCTCCACAG [SEQ ID N.6]

Optimized kozak sequence gcggccgcC

Human ornithine aminotransferase coding sequence

ATGTTTTCCAAACTAGCACATTTGCAGAGGTTTGCTGTACTTAGTCGCGGAGTTCAT TC

TTCAGTGGCTTCTGCTACATCTGTTGCAACTAAAAAAACAGTCCAAGGCCCTCCAAC CT

CTGATGACATTTTTGAAAGGGAATATAAGTATGGTGCACACAACTACCATCCTTTAC CT GTAGCCCTGGAGAGAGGAAAAGGTATTTACTTATGGGATGTAGAAGGCAGAAAATATT

TTGACTTCCTGAGTTCTTACAGTGCTGTCAACCAAGGGCATTGTCACCCCAAGATTG TG

AATGCTCTGAAGAGTCAAGTGGACAAATTGACCTTAACATCTAGAGCTTTCTATAAT A

ACGTACTTGGTGAATATGAGGAGTATATTACTAAACTTTTCAACTACCACAAAGTTC TT

CCTATGAATACAGGAGTGGAGGCTGGAGAGACTGCCTGTAAACTAGCTCGTAAGTGG G

GCTATACCGTGAAGGGCATTCAGAAATACAAAGCAAAGATTGTTTTTGCAGCTGGGA A

CTTCTGGGGTAGGACGTTGTCTGCTATCTCCAGTTCCACAGACCCAACCAGTTACGA TG

GTTTTGGACCATTTATGCCGGGATTCGACATCATTCCCTATAATGATCTGCCCGCAC TG

GAGCGTGCTCTTCAGGATCCAAATGTGGCTGCGTTCATGGTAGAACCAATTCAGGGT G

AAGCAGGCGTTGTTGTTCCGGATCCAGGTTACCTAATGGGAGTGCGAGAGCTCTGCA C

CAGGCACCAGGTTCTCTTTATTGCTGATGAAATACAGACAGGATTGGCCAGAACTGG T

AGATGGCTGGCTGTTGATTATGAAAATGTCAGACCTGATATAGTCCTCCTTGGAAAG G

CCCTTTCTGGGGGCTTATACCCTGTGTCTGCAGTGCTGTGTGATGATGACATCATGC TG

ACCATTAAGCCAGGGGAGCATGGGTCCACATACGGTGGCAATCCACTAGGCTGCCGA G

TGGCCATCGCAGCCCTTGAGGTTTTAGAAGAAGAAAACCTTGCTGAAAATGCAGACA A

ATTGGGCATTATCTTGAGAAATGAACTCATGAAGCTACCTTCTGATGTTGTAACTGC CG

TAAGAGGAAAAGGATTATTAAATGCTATTGTCATTAAAGAAACCAAAGATTGGGATG C

TTGGAAGGTGTGTCTACGACTTCGAGATAATGGACTTCTGGCCAAGCCAACCCATGG C

GACATTATCAGGTTTGCGCCTCCGCTGGTGATCAAGGAGGATGAGCTTCGAGAGTCC A

TTGAAATTATTAACAAGACCATCTTGTCTTTC [SEQ ID N.7]

3XFlag tag:

GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGAC G

ATGACAAGTGA [SEQ ID N.8]

WPRE:

AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTT GC

TCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTC CCG

TATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGA GTT

GTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCC CA

CTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCC TCC

CTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTC G

GCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTG GC

TGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTT CG GCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCC

GCGTCTTCG [SEQ ID N.9]

BGH pA

CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGA CCC

TGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATT GT

CTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAG

GATTGGGAAGACAATAGCAGGCATGCTGGGGA [SEQ ID N.I 0]

3’ITR from AAV serotype 2

AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTG AG

GCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGC G

AGCGAGCGCGCAG [SEQ ID N.I 1]

AAV.TBG.OAT plasmid sequence (OAT CDS in uppercase)

GGCCGCATGTTTTCCAAACTAGCACATTTGCAGAGGTTTGCTGTACTTAGTCGCGGA GT

TCATTCTTCAGTGGCTTCTGCTACATCTGTTGCAACTAAAAAAACAGTCCAAGGCCC TC

CAACCTCTGATGACATTTTTGAAAGGGAATATAAGTATGGTGCACACAACTACCATC CT

TTACCTGTAGCCCTGGAGAGAGGAAAAGGTATTTACTTATGGGATGTAGAAGGCAGA A

AATATTTTGACTTCCTGAGTTCTTACAGTGCTGTCAACCAAGGGCATTGTCACCCCA AG

ATTGTGAATGCTCTGAAGAGTCAAGTGGACAAATTGACCTTAACATCTAGAGCTTTC TA

TAATAACGTACTTGGTGAATATGAGGAGTATATTACTAAACTTTTCAACTACCACAA AG

TTCTTCCTATGAATACAGGAGTGGAGGCTGGAGAGACTGCCTGTAAACTAGCTCGTA A

GTGGGGCTATACCGTGAAGGGCATTCAGAAATACAAAGCAAAGATTGTTTTTGCAGC T

GGGAACTTCTGGGGTAGGACGTTGTCTGCTATCTCCAGTTCCACAGACCCAACCAGT TA

CGATGGTTTTGGACCATTTATGCCGGGATTCGACATCATTCCCTATAATGATCTGCC CG

CACTGGAGCGTGCTCTTCAGGATCCAAATGTGGCTGCGTTCATGGTAGAACCAATTC A

GGGTGAAGCAGGCGTTGTTGTTCCGGATCCAGGTTACCTAATGGGAGTGCGAGAGCT C

TGCACCAGGCACCAGGTTCTCTTTATTGCTGATGAAATACAGACAGGATTGGCCAGA A

CTGGTAGATGGCTGGCTGTTGATTATGAAAATGTCAGACCTGATATAGTCCTCCTTG GA

AAGGCCCTTTCTGGGGGCTTATACCCTGTGTCTGCAGTGCTGTGTGATGATGACATC AT

GCTGACCATTAAGCCAGGGGAGCATGGGTCCACATACGGTGGCAATCCACTAGGCTG C

CGAGTGGCCATCGCAGCCCTTGAGGTTTTAGAAGAAGAAAACCTTGCTGAAAATGCA G

ACAAATTGGGCATTATCTTGAGAAATGAACTCATGAAGCTACCTTCTGATGTTGTAA CT

GCCGTAAGAGGAAAAGGATTATTAAACGCTATTGTCATTAAAGAAACCAAAGATTGG G ATGCTTGGAAGGTGTGTCTACGACTTCGAGATAATGGACTTCTGGCCAAGCCAACCCA TGGCGACATTATCAGGTTTGCGCCTCCGCTGGTGATCAAGGAGGATGAGCTTCGAGAG TCCATTGAAATTATTAACAAGACCATCTTGTCTTTCTGAAagcttggatccaatcaacct ctggattacaaa atttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatac gctgctttaatgcctttgtatcatgctattgcttcccgtatgg ctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggc ccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgct gacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttc gctttccccctccctattgccacggcggaactcatc gccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtg gtgttgtcggggaagctgacgtcctttccatggct gctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggc cctcaatccagcggaccttccttcccgcggcctgct gccggctctgcggcctcttccgcgtcttcgagatctgcctcgactgtgccttctagttgc cagccatctgttgtttgcccctcccccgtgccttccttg accctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcat tgtctgagtaggtgtcattctattctggggggtgggg tggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggactcga gttaagggcgaattcccgattaggatcttcct agagcatggctacgtagataagtagcatggcgggttaatcattaactacaaggaacccct agtgatggagttggccactccctctctgcgcgctc gctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcgg cctcagtgagcgagcgagcgcgcagcctta attaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgtta cccaacttaatcgccttgcagcacatccccctttcgcc agctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctg aatggcgaatgggacgcgccctgtagcggcg cattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccc tagcgcccgctcctttcgctttcttcccttcctttct cgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccg atttagtgctttacggcacctcgaccccaaaaaactt gattagggtgatggttcacgtagtgggccatcgccccgatagacggtttttcgccctttg acgctggagttcacgttcctcaatagtggactcttgtt ccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggatttt tccgatttcggcctattggttaaaaaatgagctgatttaac aaaaatttaacgcgaattttaacaaaatattaacgtttataatttcaggtggcatctttc ggggaaatgtgcgcggaacccctatttgtttatttttctaaa tacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatatt gaaaaaggaagagtatgagtattcaacatttccgtgtcg cccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctgg tgaaagtaaaagatgctgaagatcagttgggtgcacgag tgggttacatcgaactggatctcaatagtggtaagatccttgagagttttcgccccgaag aacgttttccaatgatgagcacttttaaagttctgctat gtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacact attctcagaatgacttggttgagtactcaccagtc acagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataacc atgagtgataacactgcggccaacttacttctgac aacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaac tcgccttgatcgttgggaaccggagctgaatgaa gccataccaaacgacgagcgtgacaccacgatgcctgtagtaatggtaacaacgttgcgc aaactattaactggcgaactacttactctagcttc ccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctc ggcccttccggctggctggtttattgctgataaatc tggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagcc ctcccgtatcgtagttatctacacgacggggagt caggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaag cattggtaactgtcagaccaagtttactcatatata ctttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatccttttt gataatctcatgaccaaaatcccttaacgtgagttttcgttcca ctgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcg cgtaatctgctgcttgcaaacaaaaaaaccaccgcta ccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggc ttcagcagagcgcagataccaaatactgtccttctag tgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctc tgctaatcctgttaccagtggctgctgccagtggcg ataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggt cgggctgaacggggggttcgtgcacacagccca gcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcg ccacgcttcccgaagggagaaaggcggacag gtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaa cgcctggtatctttatagtcctgtcgggtttcg ccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaa aaacgccagcaacgcggcctttttacggttcctgg ccttttgctgcggttttgctcacatgttctttcctgcgttatcccctgattctgtggata accgtattaccgcctttgagtgagctgataccgctcgccgc agccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgc aaaccgcctctccccgcgcgttggccgatt cattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgca attaatgtgagttagctcactcattaggcacccc aggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaat ttcacacaggaaacagctatgaccatgattacgccaga tttaattaaggctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtc gggcgacctttggtcgcccggcctcagtgagc gagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatg attaacccgccatgctacttatctacgtagccatg ctctaggaagatcggaattcgcccttaagctagcaggttaatttttaaaaagcagtcaaa agtccaagtggcccttggcagcatttactctctctgttt gctctggttaataatctcaggagcacaaacattccagatccaggttaatttttaaaaagc agtcaaaagtccaagtggcccttggcagcatttactct ctctgtttgctctggttaataatctcaggagcacaaacattccagatccggcgcgccagg gctggaagctacctttgacatcatttcctctgcgaat gcatgtataatttctacagaacctattagaaaggatcacccagcctctgcttttgtacaa ctttcccttaaaaaactgccaattccactgctgtttggcc caatagtgagaactttttcctgctgcctcttggtgcttttgcctatggcccctattctgc ctgctgaagacactcttgccagcatggacttaaacccctc cagctctgacaatcctctttctcttttgttttacatgaagggtctggcagccaaagcaat cactcaaagttcaaaccttatcattttttgctttgttcctctt ggccttggttttgtacatcagctttgaaaataccatcccagggttaatgctggggttaat ttataactaagagtgctctagttttgcaatacaggacat gctataaaaatggaaagatgttgctttctgagagactgcagaagttggtcgtgaggcact gggcaggtaagtatcaaggttacaagacaggttta aggagaccaatagaaactgggcttgtcgagacagagaagactcttgcgtttctgataggc acctattggtcttactgacatccactttgcctttctct ccacaggtgtccaggc [SEQ ID N.12]

AAV.OATpromoter.GFP plasmid sequences

OAT promoter sequence

TTAAGCAAAGATCCTTGATGCGCGGCCGGAGGGCGGGGCGGAGGACGGGACCCACGC GATTGGTATCCTGCCCTCCGCCCCAGCCAATGAGCGGCGAGGGTGTCTTGGGGGCGGG GCAGAATCAGCCTTTAAGTTGCAGTGACTCTCCGGCGTCACTGTTGCGCTTCATAGACG CCGCGTGTACCCGGTTGTCCTCAGGCGCTGTCAGCTGCA [SEQ ID N.13]

Optimized kozak sequence gcggccgcC eGFP CDS atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggac ggcgacgtaaacggccacaagttcagcgtgtc cggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccac cggcaagctgcccgtgccctggcccaccctc gtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcag cacgacttcttcaagtccgccatgcccgaaggc tacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgag gtgaagttcgagggcgacaccctggtgaacc gcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctgg agtacaactacaacagccacaacgtctatatc atggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgag gacggcagcgtgcagctcgccgaccactacc agcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagca cccagtccgccctgagcaaagaccccaacg agaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggca tggacgagctgtacaag [SEQ ID N. M]

WPRE

Aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgtt gctccttttacgctatgtggatacgctgctttaatgcctttgtat catgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctg tctctttatgaggagttgtggcccgttgtcaggcaacgtggc gtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgt cagctcctttccgggactttcgctttccccctccctat tgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgtt gggcactgacaattccgtggtgttgtcggggaa gctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtc cttctgctacgtcccttcggccctcaatccagcggac cttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcg [SEQ ID N.I 5]

BGHpA gcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttcc ttgaccctggaaggtgccactcccactgtcctttcctaa taaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggg gtggggcaggacagcaagggggaggattgggaa gacaatagcaggcatgctgggga [SEQ ID N. I 6]

5’-ITR

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACC T TTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCAT CACTAGGGGTTCCT [SEQ ID N.17]

3’ITR

AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTG AG GCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCG AGCGAGCGCGCAG [SEQ ID N.I 8]

AAV.TBG.OAT plasmid sequences

TBG promoter sequence

Gctagcaggttaatttttaaaaagcagtcaaaagtccaagtggcccttggcagcatt tactctctctgtttgctctggttaataatctcaggagcaca aacattccagatccaggttaatttttaaaaagcagtcaaaagtccaagtggcccttggca gcatttactctctctgtttgctctggttaataatctcagg agcacaaacattccagatccggcgcgccagggctggaagctacctttgacatcatttcct ctgcgaatgcatgtataatttctacagaacctattag aaaggatcacccagcctctgcttttgtacaactttcccttaaaaaactgccaattccact gctgtttggcccaatagtgagaactttttcctgctgcctc ttggtgcttttgcctatggcccctattctgcctgctgaagacactcttgccagcatggac ttaaacccctccagctctgacaatcctctttctcttttgttt tacatgaagggtctggcagccaaagcaatcactcaaagttcaaaccttatcattttttgc tttgttcctcttggccttggttttgtacatcagctttgaaa ataccatcccagggttaatgctggggttaatttataactaagagtgctctagttttgcaa tacaggacatgctataaaaatggaaagatgttgctttct gagagactgca [SEQ ID N.I 9]

Optimized kozak sequence gcggccgcC

OAT CDS

GGCCGCATGTTTTCCAAACTAGCACATTTGCAGAGGTTTGCTGTACTTAGTCGCGGA GT TCATTCTTCAGTGGCTTCTGCTACATCTGTTGCAACTAAAAAAACAGTCCAAGGCCCTC CAACCTCTGATGACATTTTTGAAAGGGAATATAAGTATGGTGCACACAACTACCATCCT

TTACCTGTAGCCCTGGAGAGAGGAAAAGGTATTTACTTATGGGATGTAGAAGGCAGA A AATATTTTGACTTCCTGAGTTCTTACAGTGCTGTCAACCAAGGGCATTGTCACCCCAAG ATTGTGAATGCTCTGAAGAGTCAAGTGGACAAATTGACCTTAACATCTAGAGCTTTCTA

TAATAACGTACTTGGTGAATATGAGGAGTATATTACTAAACTTTTCAACTACCACAA AG

TTCTTCCTATGAATACAGGAGTGGAGGCTGGAGAGACTGCCTGTAAACTAGCTCGTA A GTGGGGCTATACCGTGAAGGGCATTCAGAAATACAAAGCAAAGATTGTTTTTGCAGCT GGGAACTTCTGGGGTAGGACGTTGTCTGCTATCTCCAGTTCCACAGACCCAACCAGTTA

CGATGGTTTTGGACCATTTATGCCGGGATTCGACATCATTCCCTATAATGATCTGCC CG

CACTGGAGCGTGCTCTTCAGGATCCAAATGTGGCTGCGTTCATGGTAGAACCAATTC A

GGGTGAAGCAGGCGTTGTTGTTCCGGATCCAGGTTACCTAATGGGAGTGCGAGAGCT C TGCACCAGGCACCAGGTTCTCTTTATTGCTGATGAAATACAGACAGGATTGGCCAGAA

CTGGTAGATGGCTGGCTGTTGATTATGAAAATGTCAGACCTGATATAGTCCTCCTTG GA AAGGCCCTTTCTGGGGGCTTATACCCTGTGTCTGCAGTGCTGTGTGATGATGACATCAT GCTGACCATTAAGCCAGGGGAGCATGGGTCCACATACGGTGGCAATCCACTAGGCTGC

CGAGTGGCCATCGCAGCCCTTGAGGTTTTAGAAGAAGAAAACCTTGCTGAAAATGCA G

ACAAATTGGGCATTATCTTGAGAAATGAACTCATGAAGCTACCTTCTGATGTTGTAA CT GCCGTAAGAGGAAAAGGATTATTAAACGCTATTGTCATTAAAGAAACCAAAGATTGGG ATGCTTGGAAGGTGTGTCTACGACTTCGAGATAATGGACTTCTGGCCAAGCCAACCCA

TGGCGACATTATCAGGTTTGCGCCTCCGCTGGTGATCAAGGAGGATGAGCTTCGAGA G TCCATTGAAATTATTAACAAGACCATCTTGTCTTTCTGAA [SEQ ID N.20]

WPRE

Aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgtt gctccttttacgctatgtggatacgctgctttaatgcctttgtat catgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctg tctctttatgaggagttgtggcccgttgtcaggcaacgtggc gtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgt cagctcctttccgggactttcgctttccccctccctat tgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgtt gggcactgacaattccgtggtgttgtcggggaa gctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtc cttctgctacgtcccttcggccctcaatccagcggac cttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcg [SEQ ID N.21]

BGHpA

Gcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgcct tccttgaccctggaaggtgccactcccactgtcctttccta ataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtgg ggtggggcaggacagcaagggggaggattggga agacaatagcaggcatgctgggga [SEQ ID N.22]

References

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