Lucie Percevault

Emmanuelle Limanton

Pauline Nicolas

Ludovic Paquin

Corinne Lagrost

Electrochemical Determination and Antioxidant Capacity Modulation of Polyphenols in Deep Eutectic Solvents

Keywords: Deep eutectics, polyphenols, electrochemistry, antioxidant capacity, long-term stability

Deep eutectic solvents (DES) are currently developed as ecofriendly and efficient solvents for polyphenols extraction. However, a challenge remains in these processes concerning the polyphenols determination that is routinely performed through colorimetric Folin-Ciocalteu assays. Besides these assays use toxic reagents, they may have serious limitations in some DES media due to possible side reaction or interferences with the DES components as found herein with ethaline (choline chloride / ethylene glycol (1:2)) or betaine/citric acid (2:3). An alternative method based on simple electrochemical analyses directly performed in DES-based media is proposed.

Since the direct electrochemistry of polyphenols in DES has been hardly explored, 9 polyphenolic standards including 7 flavonoids are examined and their electrochemical behaviour are found closely-related to those classically obtained in traditional solvents, notably in aqueous solutions.

A linear relationship is obtained between concentration and peak current intensity for quercetin in the range 10 -5 -10 -4 mol.L -1 , demonstrating the potential of the method for polyphenols contents quantitation. More importantly, their antioxidant capacity is evaluated by differential pulse voltammetry (DPV) through the measurements of the redox potentials. An interesting variation is observed according to the nature of the DES components, suggesting that antioxidant activity of polyphenols can be modulated through the choice of the DES. Moreover, a good stability of the polyphenols samples in the DES-water mixture under study is obtained after storage for 10 months at 30 ° C. These results open appealing perspectives in polyphenols extraction processes using eutectic solvents as extracting media that can be part of formulation for cosmetic or pharmaceutical applications, resulting in high-added values extracts.

INTRODUCTION

There is currently much interest in the development of green and efficient processes for the extraction of natural products from biomass. In this context, the solvents used in the processes either as extracting medium or for the recovery of target compounds are of paramount importance since they represent the largest part of the waste mass and contribute to environmental pollution while potentially hazardous to human health. To overcome these issues, deep eutectic solvents (DES) have been gaining increasing attention as sustainable and safe solvents. [START_REF] Zhang | Deep eutectic solvents: syntheses, properties and applications[END_REF][START_REF] Liu | Natural Deep Eutectic Solvents: Properties, Applications, and Perspectives[END_REF][START_REF] Murador | Bioavailability and biological effects of bioactive compounds extracted with natural deep eutectic solvents and ionic liquids: advantages over conventional organic solvents[END_REF] DES are generally viewed as mixture of two or three components whose melting temperature is lower than that of each individual component because the components are strongly interacting with each other, often through hydrogen bonding. [START_REF] Zhang | Deep eutectic solvents: syntheses, properties and applications[END_REF][START_REF] Percevault | Extraction of Plant and Algal Polyphenols Using Eutectic Solvents in Deep Eutectic Solvents for Medicine, Gas Solubilization and Extraction of Natural Substances[END_REF] A stricter definition of DES has been recently proposed as a mixture of two (or more) compounds for which the melting temperature corresponding to an eutectic point temperature is below that of an ideal liquid mixture with a significant negative deviation from ideality. [START_REF] Martins | Insights into the Nature of Eutectic and Deep Eutectic Mixtures[END_REF] DES are generally considered as greener and cheaper alternative to ionic liquids and share many physico-chemical properties with them, notably a great solubilizing power. [START_REF] Zhang | Deep eutectic solvents: syntheses, properties and applications[END_REF] However, they are much easier to synthesize, involving cheap biosourced and/or biocompatible components.

They are then regarded as biocompatible, biodegradable and non-toxic materials, even if this last point probably requires further dedicated investigations. [START_REF] De Morais | Ecotoxicity of Cholinium-Based Deep Eutectic Solvents[END_REF][START_REF] Nguyen | Deep Eutectic Solvents for Innovative Pharmaceutical Formulations in Deep Eutectic Solvents for Medicine, Gas Solubilization and Extraction of Natural Substances[END_REF] Thus DES have been successfully employed to substitute conventional volatile organic solvents in the extraction of various bioactive natural compounds including polyphenols. [START_REF] Percevault | Extraction of Plant and Algal Polyphenols Using Eutectic Solvents in Deep Eutectic Solvents for Medicine, Gas Solubilization and Extraction of Natural Substances[END_REF] Polyphenols are an important class of phytochemicals which are widely distributed as secondary metabolites in edible and non-edible plants. [START_REF] Quideau | Plant Polyphenols: Chemical Properties, Biological Activities, and Synthesis[END_REF] They have a wide range of biochemical activities, especially their antioxidant activity. [START_REF] Quideau | Plant Polyphenols: Chemical Properties, Biological Activities, and Synthesis[END_REF] The beneficial effects of the antioxidant properties for human health are nowadays known to a large public, [START_REF] Manach | Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies[END_REF] and attract growing interest in the fields of cosmetics, pharmaceutics and nutraceutics whose indutries are in demand of natural, greener and safer ingredients. This has led to the development of sustainable extraction methods where DES may improve the greenness and cost-effectiveness of the process.

Even more important than increasing extraction yields, the preservation of the bioactivity of the generated extracts and their non-toxicity are highly desirable, along with the option of keeping the DES itself as an active ingredient. This would result not only in an environmental friendly and safe approach but also in high added-value extracts. In this context, some works has reported improvement of stability or bioactivity thanks to DES. [START_REF] Dai | Natural deep eutectic solvents providing enhanced stability of natural colorants from safflower (Carthamus tinctorius)[END_REF][START_REF] Durand | Evaluation of the ROS Inhibiting Activity and Mitochondrial Targeting of Phenolic Compounds in Fibroblast Cells Model System and Enhancement of Efficiency by Natural Deep Eutectic Solvent (NADES) Formulation[END_REF][START_REF] De Gonzalo | Positive Impact of Natural Deep Eutectic Solvents on the Biocatalytic Performance of 5-Hydroxymethyl-Furfural Oxidase[END_REF][START_REF] Xu | Improving β-glucosidase biocatalysis with deep eutectic solvents based on choline chloride[END_REF][START_REF] Zeng | Purification of anthocyanins from saskatoon berries and their microencapsulation in deep eutectic solvents[END_REF] In addition, DES are nowadays considered in pharmaceutical science as part of the therapeutic formulation not only because they could advantageously increase the solubility of molecules and even macromolecules (proteins, nucleic acids, polysaccharides) of pharmaceutical interest compared to the aqueous media but also bring interesting properties such as antimicrobial activity, enhancement of drug bioavailability. [START_REF] Nguyen | Deep Eutectic Solvents for Innovative Pharmaceutical Formulations in Deep Eutectic Solvents for Medicine, Gas Solubilization and Extraction of Natural Substances[END_REF][START_REF] Santos | Therapeutic Deep Eutectic Systems for the Enhancement of Drug Bioavailability in Deep Eutectic Solvents for Medicine, Gas Solubilization and Extraction of Natural Substances[END_REF] However, a challenge concerns the quantification and identification of polyphenols in DES extracts which could still be regarded as the weak link in the whole extracting process. Usually, the quantification and/or identification are carried out with analytical chromatography techniques (HPLC, gas chromatography, with different detection systems), [START_REF] Fernández | Natural deep eutectic solvents-mediated extractions: The way forward for sustainable analytical developments[END_REF] or with colorimetric assays as classically employed in conventional media (Folin-Ciocalteu, FRAP, etc). [START_REF] Singleton | Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent[END_REF] Chromatography analyses are the most reliable tool to both identify and quantify the phenolics present in the extracts by establishing the phenolic profile with a great precision. But they require heavy equipment and trained operators. Consequently, for a quick determination of the total amount of phenolics contained in the extracts, colorimetric assays especially Folin-Ciocalteu assay are preferred as a routine method because they are quick and easy to handle. [START_REF] Singleton | Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent[END_REF][START_REF] Huang | The Chemistry behind Antioxidant Capacity Assays[END_REF] A huge volume of data is based on this assay including those concerning DES extracts. Although that Folin-Ciocalteu assay is widely accepted as a method to determine the total phenolic content, it overestimates the phenolic content because of the low selectivity of Folin-Ciocalteu reagents and possible interference of any reducing substrates. [START_REF] Singleton | Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent[END_REF][START_REF] Huang | The Chemistry behind Antioxidant Capacity Assays[END_REF] Especially, many components of DES could potentially interfere with reagents and make the colorimetric assay inaccurate.

Electrochemical techniques represent an alternative and powerful strategy for determining the polyphenols contents because they allow a quick, simple, inexpensive and sensitive detection. [START_REF] Hoyos-Arbeláez | Electrochemical methods as a tool for determining the antioxidant capacity of food and beverages: A review[END_REF][START_REF] Pisoschi | Electrochemical Methods for Total Antioxidant Capacity and its Main Contributors Determination: A review[END_REF] They do not require toxic reagents or complex sample preparation, even turbid solutions can be easily analysed. Moreover, another benefit of electrochemical approaches is the possibility to probe the overall reducing power of the polyphenols through the measurements of their redox potentials. [START_REF] Blasco | Direct Electrochemical Sensing and Detection of Natural Antioxidants and Antioxidant Capacity in Vitro Systems[END_REF] Low redox potential, i.e. a facile electron transfer, indicates a high antioxidant activity. Worth is to mention that electron transfer is the conceptual base of several antioxidant capacity assays like FRAP but also Folin-Ciocalteu which could be formally seen as a measurement of the antioxidant capacity. [START_REF] Huang | The Chemistry behind Antioxidant Capacity Assays[END_REF] Compared to colorimetric assays involving various reagents, voltammetric analyses can be considered as a direct evaluation of the antioxidant activity that depends on the physico-chemical properties of the active compounds. In addition, if the analyses are performed in the extracting media itself, possible interactions with the media could be probed since the electrochemical signal may be sensitive to specific solvation effects. [START_REF] Lagrost | Specific Effects of Room Temperature Ionic Liquids on Cleavage Reactivity: Example of the Carbon-Halogen Bond Breaking in Aromatic Radical Anions[END_REF][START_REF] Lagrost | The influence of room-temperature ionic liquids on the stereoselectivity and kinetics of the electrochemical pinacol coupling of acetophenone[END_REF] Thus, electrochemical determination of polyphenols contents or antioxidant capacity have proved to be efficient, even in complex matrices, including honey, [START_REF] Ismail | Evaluation of Radical Scavenging Capacity of Polyphenols Found in Natural Malaysian Honeys by Voltammetric Techniques[END_REF] human urine, [START_REF] Adam | Utilizing of Square Wave Voltammetry to Detect Flavonoids in the Presence of Human Urine[END_REF] , blood plasma, [START_REF] Chevion | Evaluation of Plasma Low Molecular Weight Antioxidant Capacity by Cyclic Voltammetry[END_REF] or wine. [START_REF] Kilmartin | Electrochemistry applied to the analysis of wine: A mini-review[END_REF] Surprisingly, there is no reported example of electrochemical analyses of polyphenols directly performed in DES. Only works based on the dilution in conventional aqueous electrolyte or the electrode drop-casting of DES containing polyphenols have been described to date. [START_REF] Gomez | Enhanced electrochemical detection of quercetin by Natural Deep Eutectic Solvents[END_REF][START_REF] Shabani | Deep eutectic solvents (DES) as green extraction media for antioxidants electrochemical quantification in extravirgin olive oils[END_REF] Herein, we examine the electrochemistry of phenolics in 3 DES media based on betaine and choline chloride. The phenolic compounds were chosen as standard and include 2 monophenols and 7 representative flavonoids, both aglycone or glycoside (Figure 1). The voltammetric signals are directly recorded in the DES-based media in order to show that electrochemical analyses could be a valuable alternative to Folin-Ciocalteu assay while the relevance of this colorimetric assay is carefully evaluated in the DES used in this work. In addition, the antioxidant capacity of the phenolics in the DES-based media is also discussed as a function of the DES nature, showing a potential synergy between DES and polyphenols. Finally, long-term storage of some flavonoids is also considered.

EXPERIMENTAL SECTION

Chemicals. Pyrogallol, quercetin, (+)-catechin were purchased from Sigma-Aldrich. Gallic acid (98 %), myricetin, citric acid (99.6 %), choline chloride (99 %) and ethylene glycol (99.5 %) were from Acros. Betaine anhydre was supplied by TCI. Luteolin, isoquercetin and rutin were from Merck, and epigallocatechin gallate from Alfa Aesar. All chemicals were used without further purification. DES with 30 % of water (w/w) were mixed with ultrapure water (18.2 MΩ).

Synthesis of DES.

The choline chloride/ethylene glycol DES mixture was prepared with a molar ratio 1:2. The constituents were mixed under stirring at 80°C for 2 h until a stable clear homogeneous liquid was obtained. The betaine/citric acid DES mixture was prepared with a molar ratio 2:3, the constituents and water were mixed under stirring at 60°C for 4 h until a stable clear homogeneous liquid was formed. The DES were allowed to cool. The water content was determined by Karl Fischer titration (831 KF Coulometer with generator electrode with diaphragm) through an average of three measurements. Finally, the water content was adjusted to 30% of water (w/w).

Folin-Ciocalteu assay. Folin-Ciocalteu assay for total phenolic content was prepared and calibrated with phloroglucinol as a standard. 80 µL of sample, 520 µL of water and 40 µL of Folin-Ciocalteu's reagent were mixed with a vortex. After 1 min, 160 µL of 20% sodium bicarbonate solution was added to the mixture, then vortexed. The mixture is left at room temperature in darkness for 1h30. The absorbance was monitored at 760 nm with a spectrophotometer Fluostar Omega (BMG labtech).

Preparation of the phenolics sample for electrochemistry measurements. Except for myricetin for which 70 µmol.L -1 solutions were prepared, the different phenol compounds were added in the 3 DES-based media for preparing aliquot solutions of concentration equal to 10 -2 mol.L -1 . After vortexing, a ten-fold dilution of the aliquot is performed by adding DES media. A variation of the solubility of the compounds as a function of DES is observed (Table S1). Turbid but homogeneous solutions are generally obtained with BCA-water 30 % and ethaline-water 30

RESULTS AND DISCUSSION

In this work, we consider 3 DES media based on 2 different DES: betaine/citric acid 2:3 (BCA)

and ethaline (choline chloride/ ethylene glycol 1:2). While ethaline represents an archetypal DES, based on the classical choline chloride salt and used in a wide range of applications including extraction, BCA is more rarely employed despite its great potential. Interestingly, these DES, both biosourced, were chosen because of distinct structures, and different pH. BCA and ethaline are diluted with water and ethaline is also employed as "pure" material as explained in the following.

BCA and ethaline are used as they contain a considerable amount of water i.e. 30 % (w/w). This amount is specifically chosen for lowering the viscosity of the solvents in the event of applications in extractions processes for which this parameter affecting transport phenomenon is of primary importance. [START_REF] Percevault | Extraction of Plant and Algal Polyphenols Using Eutectic Solvents in Deep Eutectic Solvents for Medicine, Gas Solubilization and Extraction of Natural Substances[END_REF][START_REF] Martins | Insights into the Nature of Eutectic and Deep Eutectic Mixtures[END_REF] In a recent work, the effect of different concentration of water (10%, 30%, 75% w/w) in natural DES was evaluated in the extraction of flavonoids. [START_REF] Bajkacz | Development of a Method Based on Natural Deep Eutectic Solvents for Extraction of Flavonoids from Food Samples[END_REF] The best extraction yield is found for a water concentration of 30 % while extraction efficiency markedly decreases for further dilution. Similarly, 20 % of water (w/w) was found optimal for the extraction of rutin in choline chloride-based DES. [START_REF] Huang | Green and efficient extraction of rutin from tartary buckwheat hull by using natural deep eutectic solvents[END_REF] The presence of such amount of water may drastically change the structure of DES, notably through hydrogen bonds disruption, leading to a loss of the supramolecular structure. [START_REF] Dai | Tailoring properties of natural deep eutectic solvents with water to facilitate their applications[END_REF][START_REF] Passos | Are Aqueous Biphasic Systems Composed of Deep Eutectic Solvents Ternary or Quaternary Systems? ACS Sustain[END_REF] Interestingly, an NMR study of reline demonstrates that the hydrogen bonding structure subsists with addition of 14 % water (w/w) , with a full loss for concentration above 67 % (w/w). [START_REF] Gutiérrez | Freeze-Drying of Aqueous Solutions of Deep Eutectic Solvents: A Suitable Approach to Deep Eutectic Suspensions of Self-Assembled Structures[END_REF] The molar ratio of water which is compatible with the stability of DES actually depends on the nature of the DES composition. [START_REF] Dai | Natural deep eutectic solvents as new potential media for green technology[END_REF] However, many works have pointed out that dilution of water using a moderate concentration of water does improve the extraction efficacy, [START_REF] Percevault | Extraction of Plant and Algal Polyphenols Using Eutectic Solvents in Deep Eutectic Solvents for Medicine, Gas Solubilization and Extraction of Natural Substances[END_REF] and 30 % w/w of water is generally viewed as a maximum limit able to preserve the specific properties of DES towards extraction. [START_REF] Liu | Natural Deep Eutectic Solvents: Properties, Applications, and Perspectives[END_REF][START_REF] Bajkacz | Development of a Method Based on Natural Deep Eutectic Solvents for Extraction of Flavonoids from Food Samples[END_REF][START_REF] Huang | Green and efficient extraction of rutin from tartary buckwheat hull by using natural deep eutectic solvents[END_REF][START_REF] Dai | Tailoring properties of natural deep eutectic solvents with water to facilitate their applications[END_REF][START_REF] Benvenutti | Which is the best food emerging solvent: IL, DES or NADES?[END_REF] Ethaline with 0.86 % water, being an archetypal DES, is also used for comparison purposes. BCA-water 30 % exhibits a pH equal to 2.5 while ethaline and ethaline-water 30 % has pH equal to 7 and 7.3, respectively. Obviously, considering their structure, polyphenols properties are expected to be sensitive to pH. It is noteworthy, for instance, that their electrochemical behaviour are pH-dependent. [START_REF] Pisoschi | Electrochemical Methods for Total Antioxidant Capacity and its Main Contributors Determination: A review[END_REF][START_REF] Gil | Flavonoid electrochemistry: a review on the electroanalytical applications[END_REF][START_REF] Martinez | Catechin antioxidant action at various pH studied by cyclic voltammetry and PM3 semi-empirical calculations[END_REF][START_REF] Sokolová | The oxidation of natural flavonoid quercetin[END_REF][START_REF] Ramešová | The oxidation of luteolin, the natural flavonoid dye[END_REF][START_REF] Huang | High sensitivity simultaneous determination of myricetin and rutin using a polyfurfural film modified glassy carbon electrode[END_REF] Folin-Ciocalteu assay. As explained in the introduction, Folin-Ciocalteu assay is routinely employed for determining the total phenolics content in extracts. As in conventional media, the method is equally widely used in DES extracts but there is a lack of critical assessment of the relevance of the method in DES. The Folin-Ciocalteu reagent (FCR) is prepared from a mixture of phosphomolybdic and phototungstic acids, with lithium sulfate that gives a yellow color to the solution. The exact chemical nature of the reagent is not known but probably based on heteropolyanions of phosphotungstates-molybdates. [START_REF] Singleton | Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent[END_REF][START_REF] Huang | The Chemistry behind Antioxidant Capacity Assays[END_REF] Phenolic compounds can react with FCR under strong basic conditions, and pH is usually fixed at pH~10 using sodium carbonate. The reaction between phenols and FCR leads to an intense blue coloration which is spectrophotometrically detected and, the corresponding absorbance is supposed to be proportional to the phenolics concentration. The blue color is believed to originate from species like (PMoW11O40) [START_REF] Percevault | Extraction of Plant and Algal Polyphenols Using Eutectic Solvents in Deep Eutectic Solvents for Medicine, Gas Solubilization and Extraction of Natural Substances[END_REF]-obtained from the reduction of FCR by phenolate compounds possibly through electron-transfer mechanisms. [START_REF] Huang | The Chemistry behind Antioxidant Capacity Assays[END_REF] The two kind of DES used in this work are incompatible with FCR, precluding any reliable determination of phenolics content with Folin-Ciocalteu assay. Figure 2 clearly shows a precipitation of the FCR in the presence of ethaline. On the other hand, a side reaction occurs between FCR and BCA-water 30 %, notably due to the acidic character of the DES (Figure 2).

These observations fully support the need for alternative methods dedicated to polyphenols quantitation or antioxidant activity evaluation because Folin-Ciocalteu assay may be particularly difficult if impossible to achieve with some DES. based media composed of an organic salt as hydrogen bond acceptor associated to an organic hydrogen bond donor (so-called Type III DES, [START_REF] Percevault | Extraction of Plant and Algal Polyphenols Using Eutectic Solvents in Deep Eutectic Solvents for Medicine, Gas Solubilization and Extraction of Natural Substances[END_REF][START_REF] Smith | Deep Eutectic Solvents (DESs) and Their Applications[END_REF] ).

Actually, the electrochemistry of the different phenolics under study, especially the flavonoids has been widely studied in conventional media, [START_REF] Gil | Flavonoid electrochemistry: a review on the electroanalytical applications[END_REF][START_REF] Sokolová | The oxidation mechanism of the antioxidant quercetin in nonaqueous media[END_REF][START_REF] Masek | Electrooxidation of flavonoids at platinum electrode studied by cyclic voltammetry[END_REF] mainly in aqueous buffered solutions. [START_REF] Sokolová | The oxidation of natural flavonoid quercetin[END_REF][START_REF] Ramešová | The oxidation of luteolin, the natural flavonoid dye[END_REF][START_REF] Huang | High sensitivity simultaneous determination of myricetin and rutin using a polyfurfural film modified glassy carbon electrode[END_REF][START_REF] Hendrickson | Electrochemistry of catechol-containing flavonoids[END_REF][START_REF] Kummer | Comparison of Electrochemical Oxidation of Flavonols and Calculated Proton Affinity and Electron Transfer Enthalpy in Water[END_REF][START_REF] Zielińska | Determination of quercetin and its glucosides in onion by electrochemical methods[END_REF][START_REF] Novak | Electrochemical Characterization of Epigallocatechin Gallate Using Square-Wave Voltammetry[END_REF][START_REF] Abdel-Hamid | Electrochemical behavior of antioxidants: I. Mechanistic study on electrochemical oxidation of gallic acid in aqueous solutions at glassy-carbon electrode[END_REF][START_REF] Liu | Electrochemical synthesis of a nanocomposite consisting of carboxy-modified multi-walled carbon nanotubes, polythionine and platinum nanoparticles for simultaneous voltammetric determination of myricetin and rutin[END_REF][START_REF] Komorsky-Lovrić | Abrasive stripping voltammetry of myricetin and dihydromyricetin[END_REF][START_REF] Medvidović-Kosanović | Electrochemical and Antioxidant Properties of (+)-Catechin, Quercetin and Rutin[END_REF] The electrochemical behaviour of flavonoids is related to the electroactivity of the A and B phenol units while ring C generally has much less influence (see Figure 1). In aqueous media, the redox behaviour of these units is characterized by proton-electron transfer mechanisms which depend on the pH. The first oxidation peak is generally due to B ring and corresponds to the oxidation of catechol or pyrogallol moiety. Due to electron delocalisation, the phenoxy radicals in B ring are relatively stabilized, therefore the oxidation process is quasi-reversible (Figure S1). An oxidation peak at more positive potentials is related to the oxidation of 5,7 substituent on the A ring (Figure 1). An additional peak due to the hydroxyl group at C3 position in the C ring may occur and is generally located at potentials between the two former. Typically, this peak is not observed for luteolin, rutin or isoquercetin.

In contrast to conventional media, less is known about the flavonoid electrochemistry in DES because they have been not yet explored in these media. We can only mention the electrochemical analysis of vanillic and caffeic acids dissolved in a DES (lactic acid: glucose: water) and dropcasted onto an electrode, immersed in a conventional electrolyte, [START_REF] Shabani | Deep eutectic solvents (DES) as green extraction media for antioxidants electrochemical quantification in extravirgin olive oils[END_REF] or quercetin studied in phosphate buffer containing 10 % of DES. [START_REF] Gomez | Enhanced electrochemical detection of quercetin by Natural Deep Eutectic Solvents[END_REF] Although that some DES, notably type III DES, are suitable media for performing electrochemical measurements, [START_REF] Fryars | Diffusion of redox active molecules in deep eutectic solvents[END_REF][START_REF] Zhen | Electron Transfer Kinetics in a Deep Eutectic Solvent[END_REF] , no study of flavonoids or related compounds has been carried out directly using DES as electrolyte. The electrochemical behaviour of quercetin in the 3 DES-based media is comparable to those reported in aqueous media (Figure 3). [START_REF] Sokolová | The oxidation of natural flavonoid quercetin[END_REF][START_REF] Hendrickson | Electrochemistry of catechol-containing flavonoids[END_REF][START_REF] Medvidović-Kosanović | Electrochemical and Antioxidant Properties of (+)-Catechin, Quercetin and Rutin[END_REF][START_REF] Brett | Electrochemical Oxidation of Quercetin[END_REF] . Two oxidation peaks are observed and could be attributed to the oxidation of 3',4'-dihydroxy substituent in B ring (first peak) and hydroxyl group at C3 position in C ring (second peak). This assignation is further supported by the electrochemical behaviour of luteolin which displays only one peak (Figure 4). The oxidation associated to the A ring at higher potential is probably hidden by the oxidation of the DES. The oxidation potentials in ethaline or ethaline water 30 %, on the one hand and in BCA-water 30% on the other hand, are different, indicating that the redox behaviour of the flavonoid is probably sensitive to the DES composition and properties such as pH. This point will be further analysed below. However, it is interesting to compare the values of oxidation potentials of quercetin obtained in ethaline and ethaline -water 30 % with those reported in aqueous solutions of same pH, i.e. pH=7.

Peak potentials values of 0.15 V and 0.5 V vs Ag/AgCl were found in phosphate buffer solution, [START_REF] Hendrickson | Electrochemistry of catechol-containing flavonoids[END_REF] while values equal to 0.53 and 0.48 V for the first peak and to 0.73 and 0.76 V for the second peak were obtained in "pure ethaline" and ethaline-water 30 %, respectively. These results show that oxidation of quercetin is more difficult to achieve in ethaline than in aqueous conventional medium, suggesting strong interaction between the DES components and the polyphenol.

Differential pulse voltammetry (DPV) is an electrochemical technique able to minimize

the capacitive contribution in the current measurement. Thus DPV has a good sensitivity even at low concentration of analytes (typically 10 -7 -10 -8 mol.L -1 ). The technique has been largely employed in conventional media for polyphenols quantitation in extracts. [START_REF] Hoyos-Arbeláez | Electrochemical methods as a tool for determining the antioxidant capacity of food and beverages: A review[END_REF] The characteristic response signal of DPV is a voltammogram with a peak whose intensity is directly proportional to the concentration of the analyte. As observed in Figure 5 with quercetin in BCA -water 30 %, a good linear correlation (r 2 = 0.98) is obtained between the peak current intensity and the concentration of quercetin in the 10-100 µmol.L -1 range. In this concentration range, quercetin is well soluble in the DES-based media (no apparent turbidity). Although that the concentration range might be overestimated compared to the expected amount of polyphenols in extracts, this result is very promising and validates the use of DPV as practical alternative to Folin-Ciocalteu assay for the quantitation of polyphenols in DES-based media. As an illustration, we have performed extraction of thyme and green tea in BCA-Water 30 % and the extracts were analyzed using DPV (Fig. S1). Oxidation peaks with intensity in the range 15-10 µA are typically observed, suggesting that the amount of phenolics contained in the extracts could be evaluated by electrochemistry and expressed for instance as quercetin equivalents from calibration exemplified in Figure 5. Further work combining electrochemistry and chromatography of real extracts is currently in progress to fully evaluate the potential of the technique and will be published elsewhere. Antioxidant capacity in DES-based media. Importantly, electrochemical techniques have been also proved to be very efficient for evaluating and classifying the antioxidant capacity (antioxidant power) of phenolics compounds on the basis of the measurement of their redox potentials. [START_REF] Hoyos-Arbeláez | Electrochemical methods as a tool for determining the antioxidant capacity of food and beverages: A review[END_REF][START_REF] Blasco | Electrochemical approach for discriminating and measuring predominant flavonoids and phenolic acids using differential pulse voltammetry: towards an electrochemical index of natural antioxidants[END_REF] It has been demonstrated that the lowest oxidation potential rules the antioxidant power. 58-59 47, 60 In other words, the ring leading to the radical with the lower redox potential corresponds to the active antioxidant moiety in any flavonoid. Indeed, the conjugation between the rings, especially B and A, in the flavonoid radicals is very inefficient. When B ring is a catechol or pyrogallol unit as for the flavonoids under study, it is responsible of the antioxidant action. [START_REF] Jovanovic | Reduction potentials of flavonoid and model phenoxyl radicals. Which ring in flavonoids is responsible for antioxidant activity?[END_REF] The lower the redox potential, the higher is the antioxidant capacity.

Therefore, we especially focus on the first oxidation peak in the following. DPV measurements in the 3 DES-based media were recorded for all the 9 standard flavonoids used in the work (Figure 6). Table 2 gathers the values of the redox peak potentials obtained from DPV analyses. For each of the DES-based media, the redox potentials of the different flavonoids remain in the same range, in good agreement with their close chemical structure. Pyrogallol unit is known to oxidize at slightly lower potentials than catechol moiety, probably because of a better stabilization of the formed radicals thanks to additional hydroxyl group in B ring. [START_REF] Kummer | Comparison of Electrochemical Oxidation of Flavonols and Calculated Proton Affinity and Electron Transfer Enthalpy in Water[END_REF] . Thus considering the series of flavonoids in BCA-water 30 %, it is not surprising that myricetin and epigallocatechin gallate exhibit slightly lower redox potentials than quercetin, rutin, isoquercetin or catechin. Note that the redox potentials of gallic acid and pyrogallol are in line with those of myricetin and epigallocatechin gallate. The most interesting result is the variation of the redox potential according to the nature of the DES. There are striking differences when comparing values in ethaline or ethaline -water 30 % with those in BCA-water 30 %. The values in ethaline and ethaline -water 30 % are relatively close, with slightly lower values in ethaline-water 30 %, probably because of slightly higher pH in the water diluted DES. [START_REF] Pisoschi | Electrochemical Methods for Total Antioxidant Capacity and its Main Contributors Determination: A review[END_REF] In contrast, the redox potentials are negatively-shifted in BCA -water 30 %, indicating that the phenolics are much easier oxidized in BCA than in ethaline. This result is especially remarkable because the redox potentials of flavonoids are known to shift to more positive values as the pH is decreased. For instance, in aqueous electrolyte, the peak potentials of myricetin and rutin at pH = 7 were found at 0.05 and 0.25 V vs SCE, respectively while the values were 0.25 and 0.4, respectively at pH = 4. [START_REF] Huang | High sensitivity simultaneous determination of myricetin and rutin using a polyfurfural film modified glassy carbon electrode[END_REF] A theoretical paper demonstrates that the antioxidant capacity of catechin increases as the pH increases. [START_REF] Martinez | Catechin antioxidant action at various pH studied by cyclic voltammetry and PM3 semi-empirical calculations[END_REF] In sharp contrast, the antioxidant activity of the 9 phenolics under study is clearly higher in the acidic BCA DES-based medium than in the two neutral ethaline media.

These observations indicate that the antioxidant activity of polyphenols can be modulated through the choice of the DES. The interactions between the DES and the polyphenols, depending on the nature of the DES might explain these observations. Notably, hydrogen bonding that plays a key role in the formation of DES, 5 is expected to influence the interaction between DES and polyphenols. Polyphenols groups are good hydrogen bond donors due to their hydroxyl groups and form bonds with good hydrogen bond acceptors. [START_REF] Ruesgas-Ramón | Application of Deep Eutectic Solvents (DES) for Phenolic Compounds Extraction: Overview, Challenges, and Opportunities[END_REF] A recent paper using a combination of methyl--cyclodextrin and glycerol-based DES illustrates the importance of interactions between polyphenols and extracting media regarding their antioxidant activity. [START_REF] Athanasiadis | Methyl β-cyclodextrin as a booster for the extraction for Olea europaea leaf polyphenols with a bio-based deep eutectic solvent[END_REF] The authors show that the cyclodextrin boosts the extraction yield of polyphenols while the antioxidant activity of the extracts is not significantly higher due to specific interactions between cyclodextrin and polyphenols. [START_REF] Athanasiadis | Methyl β-cyclodextrin as a booster for the extraction for Olea europaea leaf polyphenols with a bio-based deep eutectic solvent[END_REF] .

On the whole, the possibility to vary the antioxidant activity of polyphenols with the DES composition could be of great interest to produce extracts with beneficial properties in which the extracting medium could serve as an active component. The extract could be directly employed in cosmetic or pharmaceutical uses by being part of the formulation. [START_REF] Jeong | Multi-functioning deep eutectic solvents as extraction and storage media for bioactive natural products that are readily applicable to cosmetic products[END_REF] Stability in BCA-water 30 %. Solutions of catechin, epigallocatechin gallate, luteolin and rutin were stored for 10 months in a heat chamber at 30°C. DPV analyses were carried out in order to probe the stability of the polyphenols in BCA-water 30%. The DPV signals are fairly similar to those recorded originally with no significant variation of their redox potentials (< 30 mV, see Table S2), showing no degradation of the polyphenols after 10 months (Figure 7), with the exception of rutin (Figure 7D). The DPV signal for rutin displays a shoulder peak at about 0.17-0.19 V. This shoulder peak probably corresponds to the signal of quercetin formed upon the partial degradation of rutin into its aglycone counterpart in the acidic DES-based medium.

However, the DPV analyses demonstrate a remarkable stability of the polyphenols in the BCAwater 30 % DES, even under rather stringent conditions. This result is very promising for cosmetic or pharmaceutical formulation. Since the DES components are cosmetic-compatible, DES could be envisioned as functioning medium for the extraction and storage of bioactive phenolics, without the need of polyphenols isolation and recovery.

CONCLUSIONS

The direct electrochemistry of 9 phenolics, including 7 flavonoids, was investigated for the first time in 3 DES-based media using cylic voltammetry and DPV. Well-defined signals were readily obtained at glassy carbon electrodes or using commercial SPE carbon system. Electrochemical analyses of phenolics in DES extracts can then constitute a potent alternative for the quantification of polyphenols contents, especially when considering that the routine Folin-Ciocalteu assay could be difficult to handle or even inaccurate in some DES media. Electrochemical techniques allow quick, simple and almost inexpensive analyses without requiring a complex sample preparation or the use of non-environmentally friendly reagents. This makes it possible the development of a fully green process for polyphenols extractions that includes their quantification. A study with real extracts of herbs and medicinal plants using the DES-based media is currently in progress.

The antioxidant capacity of the polyphenols in the 3 DES-based media has been evaluated and compared through the values of their first redox peak potentials. The antioxidant capacity varies as a function of the DES composition, probably due to interactions between polyphenols and DES components. Thus, lower redox potentials are obtained in the acidic betaine-citric acid-water 30 % DES-based media than in the two neutral ethaline media, suggesting higher antioxidant activity of the flavonoids in betaine-citric acid-water 30 %. In addition, the polyphenols redox signal show a good to very good stability in betaine-citric acid-water 30 % after 10 months storage at 30 °C.

These results open interesting perspectives in polyphenols extraction using DES where the extracting medium could serve as an active material in the formulation for cosmetic or pharmaceutical uses with beneficial effects for long-term storage and tuning of the antioxidant activity.

ASSOCIATED CONTENT

Supporting Information. DPV of real thyme and green tea extracts and table of redox potentials of polyphenols.

Figure 1 .

 1 Figure 1. Chemical structure of monophenolics and polyphenols : a) pyrogallol, b) gallic acid, c)

Figure 2 .

 2 Figure 2. Folin-Ciocalteu assay with DES-based media containing 0, 25, 50, 75 and 100 µg/mL

Figure 3 .

 3 Figure 3. Cyclic voltammetry of quercetin at 0.2 V/s on a glassy carbon electrode (Ø 1mm) in

Figure 4 .

 4 Figure 4. Cyclic voltammetries of luteolin in A) ethaline, B) ethaline-water 30 % and C) BCAwater 30 %. A and B are recorded using home-made three-electrode cell while C is obtained at SPE electrode. * are impurities present in the DES batch.

Figure 5 .

 5 Figure 5. Linear variation of oxidation peak current intensity (first peak) as a function of concentration of quercetin in BCA -water 30 % water. Concentration range [10-100 µmol. L -1 ]

Figure 6 .

 6 Figure 6. DPV signals at the first oxidation peak for 9 phenolics in BCA -30 % water recorded

Figure 7 .

 7 Figure 7. DPV signals of catechin (A), luteolin (B), epigallocatechin gallate (C) and rutin (D) in

Table 1 .

 1 Solubility appearance of the polyphenols solution in the 3 DES media. while 200 µL of solution was employed with the home-made cell. In this latter case, the glassy carbon electrode was carefully polished with a SiC paper and diamond paste (Struers), then extensively rinsed with ultrapure water and finally dried with an Argon flow prior to experiments. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were carried out with an Autolab PGSTAT 30 potentiostat/galvanostat (Metrohm Autolab B.V.). For CV, the potential was scanned between -0.5 to 0.8 V at 0.1 V.s -1 . DPV measurements were performed with a step potential of 2.55 mV, a pulse amplitude of 50 mV, a pulse width of 30 ms and scan rate of 4 mV s -1 . Voltammetric scans were recorded between -0.5 to 1.2 V.

	Solubility	QCT	LUT	ISOQCT RUT	CAT	EGCG	AG	Pyrogallol
	appearance
					BCA
	10 -2 M	turbid	turbid	turbid	turbid	soluble	turbid	soluble	soluble
	« 10 -3 M »	partially	partially	turbid	turbid	soluble	Partially	soluble	soluble
		soluble	soluble				soluble
				Ethaline-water 30 %
	10 -2 M	partially	partially	nd	turbid	partially	nd	nd	soluble
		soluble	soluble			soluble
	« 10 -3 M »	soluble	soluble	nd	partially	soluble	nd	nd	soluble
					soluble
					Ethaline
	10 -2 M	soluble	soluble	nd	soluble	soluble	nd	soluble	soluble
	« 10 -3 M »	soluble	soluble	nd	soluble	soluble	nd	soluble	soluble
	Electrochemical measurements. The measurements were performed either with carbon screen
	printed electrodes (carbon SPE, DRP 110 -Dropsens) purchased from Metrohm or with an home-
	made three-electrode cell, 30 using a glassy carbon working electrode (disk of 1 mm of diameter),

QCT : quercetin ; LUT : luteolin ; ISOQCT : isoquercetin ; RUT :rutin ; CAT :catechin ; ECGC :epigallocatechin gallate ; AG :gallic acid. a platinum wire as a counter-electrode and a silver wire as a quasi-reference. A drop of 50 µL of solution of phenolics in DES-based media was used with SPE allowing to cover the three electrodes system

Table 2 .

 2 Peak potentials values obtained from DPV measurements corresponding to the first

	oxidation peak (V vs Ag)
	Phenolics Pure	Ethaline-	BCA	-
		ethaline	water	water
	GA	nd	nd	0.16
	pyrogallol nd	0.50	0.15
	QCT	0.55	0.46	0.18
	ISOQCT 0.66	0.59	0.31
	LUT	0.71	0.48	0.25
	RUT	0.68	0.44	0.24
	myricetin nd	nd	0.13
	CAT	0.69	0.54	0.18
	EGCG	nd	nd	0.11-0.20 a
	GA, gallic acid; QCT, quercetin; ISOQCT, isoquercetin; LUT, luteolin; RUT, rutin; CAT, catechin, EGCG,
	epigallocatechin gallate.

a the signal for EGCG is characterized by a "double" peak

mol.L -1 aliquot solutions correspond to saturated conditions except for "pure" ethaline.

ACKNOWLEDGMENT

The authors thank ministry of research (FUI project OCEACTIF) and university of Rennes 1 (Défis Scientifiques) for financial support.

AUTHOR INFORMATION

Corresponding Author

Corinne Lagrost, Email: corinne.lagrost@univ-rennes1.fr Ludovic Paquin, Email: ludovic.paquin@univ-rennes1.fr

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


Yu-Wai-Man Patrick

Nancy J Newman

Valerio Carelli

Mark L Moster

Valerie Biousse

Alfredo A Sadun

Thomas Klopstock

Catherine Vignal-Clermont

Robert C Sergott

Günther Rudolph

Patrick Yu-Wai-Man

La Chiara

Morgia

Rustum Karanjia

Magali Taiel

Laure Blouin

Pierre Burguière

Gerard Smits

Caroline Chevalier

Harvey Masonson

Yordak Salermo

Barrett Katz

Serge Picaud

David J Calkins

José-Alain Sahel
email: sahelja@upmc.edu

Bilateral visual improvement with unilateral gene therapy injection for Leber hereditary optic neuropathy

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Leber hereditary optic neuropathy (LHON) is a maternally-inherited blinding bilateral optic neuropathy [START_REF] Carelli | International consensus statement on the clinical and therapeutic management of Leber hereditary optic neuropathy[END_REF]. It is the most common primary mitochondrial DNA (mtDNA) disorder, affecting approximately 1 in 30,000 to 1 in 50,000 people, particularly young adult males [START_REF] Yu-Wai-Man | The epidemiology of Leber hereditary optic neuropathy in the north east of England[END_REF]. The pathophysiology of LHON is characterized by selective loss of retinal ganglion cells (RGCs) and their axons, which leads to rapidly progressive bilateral vision loss. The visual prognosis is poor and most patients progress to vision worse than 20/200 within the first year following disease onset [START_REF] Yu-Wai-Man | A neurodegenerative perspective on mitochondrial optic neuropathies[END_REF]. Three mtDNA point mutations account for approximately 90% of all LHON cases, namely m.3460G>A (MT-ND1), m.11778G>A (MT-ND4), and m.14484T>C (MT-ND6), with m.11778G>A being the most common mutation worldwide [START_REF] Carelli | International consensus statement on the clinical and therapeutic management of Leber hereditary optic neuropathy[END_REF][START_REF] Yu-Wai-Man | A neurodegenerative perspective on mitochondrial optic neuropathies[END_REF][START_REF] Newman | The clinical characteristics of pedigrees of Leber's hereditary optic neuropathy with the 11778 mutation[END_REF]. These mutations affect complex I subunits of the mitochondrial respiratory chain, impairing mitochondrial function and increasing the production of reactive oxygen species. RGCs appear to be selectively vulnerable to mitochondrial dysfunction resulting in apoptotic cell death, optic nerve degeneration and the development of optic atrophy [START_REF] Yu-Wai-Man | A neurodegenerative perspective on mitochondrial optic neuropathies[END_REF].

The current treatment for LHON remains limited [START_REF] Jurkute | Treatment strategies for Leber hereditary optic neuropathy[END_REF]. Idebenone (Raxone, Santhera) was granted market authorization in the European Union for treatment of LHON under exceptional circumstances [START_REF]2015 Committee for Medicinal Products for Human Use (CHMP) -Assessment Report Raxone (Idebenone)[END_REF]. LHON is especially amenable to gene therapy as it has a well-defined onset with an expected natural history. Furthermore, the biological targets, the RGCs, are easily accessible for gene delivery through standard intravitreal injection [START_REF] Yin | Intravitreal Injection of AAV2 Transduces Macaque Inner Retina[END_REF].

Over the past decade, substantial progress has been made in the application of gene therapy to monogenic blinding diseases, with the first treatment approved by both American and European regulatory agencies for an inherited retinal degenerative disorder, namely Leber congenital amaurosis caused by recessive RPE65 mutations [START_REF] Russell | Efficacy and safety of voretigene neparvovec (AAV2-HRPE65V2) in patients with RPE65-mediated inherited retinal dystrophy: a randomized, controlled, open-label phase 3 trial[END_REF]. Gene therapy in mitochondrial disorders is challenging as the wild-type protein needs to reach the mitochondrial matrix compartment by crossing the mitochondrial outer and inner membranes. The allotopic expression strategy involves the nuclear expression of the wild-type recoded replacement mitochondrial gene, which having been engineered to carry a mitochondrial targeting sequence (MTS), results in mRNA translation and co-translocation of the protein into mitochondria [START_REF] Gray | Allotopic expression of mitochondrial ATP synthase genes in nucleus of Saccharomyces cerevisiae[END_REF]. This strategy has been successfully applied in cell models and safely translated in induced rodent LHON models with preservation of RGCs and visual function [START_REF] Guy | Rescue of a mitochondrial deficiency causing Leber hereditary optic neuropathy[END_REF][START_REF] Koilkonda | LHON gene therapy vector prevents visual loss and optic neuropathy induced by G11778A mutant mitochondrial DNA: biodistribution and toxicology profile[END_REF][START_REF] Koilkonda | Induction of rapid and highly efficient expression of the human ND4 complex I subunit in the mouse visual system by self-complementary adeno-associated virus[END_REF][START_REF] Cwerman-Thibault | Nuclear expression of mitochondrial ND4 leads to the protein assembling in complex I and prevents optic atrophy and visual loss[END_REF][START_REF] Bonnet | The optimized allotopic expression of ND1 or ND4 genes restores respiratory chain complex I activity in fibroblasts harboring mutations in these genes[END_REF]. rAAV2/2-ND4 (GS010) is a recombinant replication-defective adeno-associated virus, serotype 2, which contains a modified cDNA encoding the human wild-type mitochondrial ND4 protein and a specific MTS for translocation of the protein into the mitochondrial matrix. rAAV2/2-ND4 has been shown to efficiently rescue an induced defect in mitochondrial respiratory chain complex I in a rat model of the disease [START_REF] Cwerman-Thibault | Nuclear expression of mitochondrial ND4 leads to the protein assembling in complex I and prevents optic atrophy and visual loss[END_REF], and it was able to restore complex I activity and ATP synthesis in cultured LHON fibroblasts carrying the m.11778G>A mutation [START_REF] Bonnet | The optimized allotopic expression of ND1 or ND4 genes restores respiratory chain complex I activity in fibroblasts harboring mutations in these genes[END_REF]. Based on these preclinical studies, a first-in-human trial (GS-LHON-CLIN-01)

showed that a single intravitreal administration of increasing doses of rAAV2/2-ND4 was safe and well-tolerated in LHON subjects [START_REF] Vignal | Safety of rAAV2/2-ND4 gene therapy for Leber hereditary optic neuropathy[END_REF][START_REF] Bouquet | Immune response and intraocular inflammation in patients with Leber hereditary optic neuropathy treated with intravitreal injection of recombinant adeno-associated virus 2 carrying the ND4 gene: a secondary analysis of a phase 1/2 clinical trial[END_REF]. Three ongoing clinical trials are examining the efficacy of intravitreal injection of rAAV2/2-ND4 (GS010) in LHON subjects with the m.11778G>A mutation and with vision loss within one year (RESCUE NCT02652767, REVERSE NCT02652780 and REFLECT NCT03293524). Here, we report the 96-week results of the phase 3 REVERSE trial in 37 LHON subjects carrying the m.11778G>A mutation and with duration of vision loss between 6 and 12 months upon inclusion. REVERSE was designed with unilateral intravitreal injection of rAAV2/2-ND4 and sham-treated fellow eye, under the assumption that the therapeutic effects of treated versus untreated eyes would be compared. The unexpected observation of a bilateral improvement of visual function prompted us to further investigate the possible transfer of rAAV2/2-ND4 viral vector to the contralateral sham-treated eye by conducting a non-human primate (NHP) study equivalent to the human REVERSE study. We confirmed the presence of viral vector DNA in the contralateral visual pathway, providing a plausible explanation for the clinical results observed in REVERSE.

Results

Demographics and Baseline Characteristics of the Study Population

Thirty-seven subjects with LHON due to the m.11778G>A mutation and duration of vision loss between 6 to 12 months were enrolled in the multicenter REVERSE trial between February 2016 and February 2017. Subjects were predominantly males (78.4%) with a mean age of 34.2 years at enrollment (Table S1). Mean duration of vision loss for rAAV2/2-ND4-treated and sham-treated eyes was 8.8 months and 9.3 months, respectively. At baseline, the mean (standard deviation [SD]) best-corrected visual acuity (BCVA) for rAAV2/2-ND4-treated and sham-treated eyes was 1.67 (0.50) and 1.55 (0.42) logarithm of the minimal angle of resolution (LogMAR), respectively, and this difference was not significant (p = 0.152 by paired t-test) (Table 1). At enrollment, 54% of all eyes scored 0 logarithm contrast sensitivity (LogCS) on the Pelli-Robson chart, meaning that no more than one out of 3 letters was read correctly at highest contrast (100%). The mean (SD) baseline score for contrast sensitivity for rAAV2/2-ND4-treated and sham-treated eyes was 0.25 (0.40) and 0.35 (0.46) LogCS, respectively, with sham-treated eyes reporting significantly better contrast sensitivity (p = 0.032 by paired t-test) (Table S2). Fourteen subjects had prior use of idebenone and they all had discontinued this medication at least 7 days prior to enrollment.

Efficacy Data at Week 96

The mean (SD) change in BCVA from baseline to Week 48 was -0.218 (0.055) and -0.211 (0.055) LogMAR in rAAV2/2-ND4-treated and sham-treated eyes, respectively. The difference of the change in BCVA between these two groups at Week 48 was -0.007 LogMAR (p = 0.894, analysis of covariance, ANCOVA). The primary endpoint, which was defined as a clinically significant difference of 0.3 LogMAR between treated and sham-treated eyes, was, therefore, not met. At Week 96, rAAV2/2-ND4-treated eyes showed a mean (SD) improvement in BCVA of -0.308 (0.068) LogMAR, equivalent to a gain of 15 Early-Treatment Diabetic Retinopathy Study (ETDRS) letters (p < 0.0001 for change from baseline) (Table 1). An average improvement of -0.259 (0.068) LogMAR was observed in the sham-treated eyes, equivalent to a gain of 13 ETDRS letters (p = 0.0001 for change from baseline). The mean change from baseline in BCVA increased continuously and bilaterally over the 96-week period following treatment (Fig. 1A).

The proportion of eyes with a clinically relevant response (CRR) in the rAAV2/2-ND4treated eye group (62%) was significantly higher than in the sham-treated eye group (43%) (p = 0.0348). Twenty-five subjects (68%) showed a CRR in at least one eye at Week 96 and 23 of these 25 subjects (92%) had a CRR in the rAAV2/2-ND4-treated eye (Table S3). Fourteen (38%) subjects showed a CRR in both eyes and 9 (24%) subjects in the rAAV2/2-ND4-treated eye only (Fig. 1B). Two subjects (5%) showed a CRR in the sham-treated eye only. In a second responder analysis, an eye responder at Week 96 was defined as an on-chart improvement of at least -0.3 LogMAR (3 ETDRS lines) or an improvement from off-chart to on-chart vision with a final BCVA of 1.4 LogMAR or better (equating to at least the first 3 ETDRS lines of the chart being read at a distance of 1 meter). Using these criteria, the eye responder rate was 35% for rAAV2/2-ND4-treated eyes and 27% for sham-treated eyes (Table S3). Fifteen subjects (41%) had this response in at least one eye. Based on a generalized estimating equation model to assess treatment effect, rAAV2/2-ND4-treated eyes were 3.6 times more likely to achieve a BCVA better than 20/200 (p = 0.0032). Based on the identical statistical model, rAAV2/2-ND4-treated eyes were 2.8 times more likely to achieve a BCVA better than or equal to 20/200 (p = 0.0094).

Most subjects showed similar BCVA changes in both eyes, as indicated by the distribution of data points close to the bisector, and a bilateral improvement in BCVA was observed in 29 (78%) subjects (Fig. 1B). Amongst subjects with a bilateral improvement in BCVA from baseline, 44% showed an earlier improvement in the rAAV2/2-ND4-treated eye than in the sham eye, 15% showed an earlier improvement in the sham eye, and 41% had a simultaneous improvement in both eyes. On average, rAAV2/2-ND4-treated eyes improved 66 days earlier than the sham eyes.

The mean improvement from the nadir (worst BCVA for each eye of each subject) for rAAV2/2-ND4-treated eyes and sham-treated eyes was -0.570 LogMAR (28.5 ETDRS letters) and -0.490 LogMAR (24.5 ETDRS letters), respectively (Table 2). In total, 78% of REVERSE subjects presented a CRR from the nadir in at least one eye.

Contrast sensitivity improved in rAAV2/2-ND4-treated and sham-treated eyes with a mean (SD) increase from baseline to Week 96 of 0.22 (0.06) and 0.12 (0.06) LogCS, respectively (Fig. S1 and Table S2). The mean deviation on automated perimetry showed a mean (SD) improvement of 2.70 (0.90) and 2.57 (0.90) dB in rAAV2/2-ND4-treated and sham-treated eyes, respectively (Table S2). There were no differences in the change of spectral-domain optical coherence tomography (SD-OCT) parameters from baseline to Week 96 in rAAV2/2-ND4treated and sham-treated eyes (Table S2).

Patient-reported outcome measures were evaluated using the National Eye Institute Visual Function Questionnaire-25 (NEI VFQ-25). The change in quality of life was assessed and compared with the baseline values before treatment. The composite score, which is an average of the vision-targeted subscales scores excluding the general health rating question, showed a mean improvement of 9.5 points. Improvements were reported for the following subscales [START_REF] Suñer | Responsiveness of NEI VFQ-25 to changes in visual acuity in neovascular AMD: validation studies from two phase 3 clinical trials[END_REF]: dependency (+18.5, +130.2%), mental health (+16.1, +108.2%), role difficulties (+15.9, +78.9%), near activities (+13.3, +78.1%), peripheral vision (+10.8, +41.0%), distance activities (+10.7, +47.4%), color vision (+6.9, +21.6%), general vision (+6.5, +32.4%), and social functioning (+4.7, +32.8%) (Table S4).

Safety Data at Week 96

Treatment with viral vector was tolerated well, without any occurrences of discontinuation over the 96 weeks of follow-up. One serious adverse event (a retinal tear requiring hospitalization) was reported in the sham-treated eye of one subject, which was deemed clinically unrelated to the study drug or procedure. No prophylactic oral or topical steroids were provided before or immediately after the intravitreal injection. In rAAV2/2-ND4-treated eyes, the most frequent ocular adverse event was intraocular inflammation, which was documented in 34 (92%) eyes and assessed as mild for 28 (76%) eyes. Anterior and intermediate uveitis was reported in 27 (73%) and 25 (68%) eyes, respectively, with no posterior uveitis affecting the retina and optic nerve reported. The anterior uveitis was graded as mild in 23 eyes and moderate in 4 eyes. There was no incidence of severe anterior uveitis. The intermediate uveitis was graded as mild in 21 eyes and moderate in 3 eyes. One eye developed severe intermediate uveitis. Intraocular inflammation resolved without sequelae following standard therapy in all patients. For the 34 eyes that developed intraocular inflammation, 29 (85%) eyes received topical steroids for a mean duration of 211 days. A course of oral steroids was prescribed for 9 (26%) patients based on the clinician's judgement. An increase in intraocular pressure was reported in 27% of eyes and was mostly mild elevation, resolving with standard therapy. No subject developed chronic ocular hypertension or glaucoma during follow-up. Viral vector biodissemination was assessed for all patients at 2 weeks post-treatment. All the tested samples showed negative quantitative PCR (qPCR) results, demonstrating no systemic diffusion of GS010.

Biodistribution of rAAV2/2-ND4 (GS010) DNA Following a Single Eye Intravitreal Injection in Cynomolgus Monkeys

We conducted a study using non-human primates to probe a possible mechanism underlying the unexpected improvement in visual function in the contralateral untreated eye following unilateral intravitreal administration of the viral vector in the REVERSE clinical study. Viral DNA was detected and quantified using ND4 transgene-specific qPCR three months following unilateral intravitreal injection of rAAV2/2-ND4 in three animals and vehicle formulation in one control animal. The qPCR protocol used specifically targeted the ND4 transgene of rAAV2/2-ND4.

Viral DNA was neither detected (below the limit of detection, [BLD]) nor quantified (below the limit of quantification, [BLQ]) in the 13 sentinel control samples that were concomitantly extracted, indicating no cross-contamination during the extraction and the qPCR analysis. All the tissue samples from both eyes of the control animal (Group 1) showed no detection (BLD) of viral DNA three months after unilateral intravitreal injection of vehicle formulation in the right eye (Table 3).

Three months after unilateral intravitreal injection of rAAV2/2-ND4 in the right eye, rAAV2/2-ND4 DNA was detected in all the tissue and fluid samples tested for the 3 animals (Table 3 and Fig. 2). When quantified, the mean quantity of rAAV2/2-ND4 ranged from 2.84x10 2 to 3.21x10 6 vg/µg of DNA in tissue samples, and 1.65x10 3 and 8.70x10 4 copy/µL in the aqueous and vitreous humors, respectively. rAAV2/2-ND4 DNA was detected in the contralateral non-injected eyes and visual pathways in the following tissues: anterior segment of the eye (all 3 animals; quantifiable for the 3 animals), retina (all 3 animals; quantifiable for 2 animals, BLQ for 1 animal), optic nerve (all 3 animals; quantifiable for the 3 animals), optic tract (2 animals; BLQ for the 2 animals), and lateral geniculate nucleus (2 animals; BLQ for the 2 animals). Viral DNA was detected in the optic chiasm of all 3 animals (Table 3 and Fig. 2). No viral DNA was detected in the contralateral visual cortex. When quantified, the mean quantity of rAAV2/2-ND4 in the contralateral non-injected eyes ranged from 3.39x10 3 to 1.00x10 4 vg/µg of DNA. No PCR inhibition was detected in any sample.

Discussion

REVERSE is the largest phase III clinical trial of gene therapy in LHON for which results are now reported. The subjects recruited were representative of the LHON population in terms of demographics and baseline visual characteristics [START_REF] Carelli | International consensus statement on the clinical and therapeutic management of Leber hereditary optic neuropathy[END_REF][START_REF] Yu-Wai-Man | The epidemiology of Leber hereditary optic neuropathy in the north east of England[END_REF][START_REF] Yu-Wai-Man | A neurodegenerative perspective on mitochondrial optic neuropathies[END_REF][START_REF] Newman | The clinical characteristics of pedigrees of Leber's hereditary optic neuropathy with the 11778 mutation[END_REF]. Both the rAAV2/2-ND4-treated and sham-treated eyes showed an improvement in BCVA when compared with baseline and nadir measurements. The sustained improvement observed in the contralateral sham-injected eyes was unexpected and resulted in the primary endpoint at Week 48 not being met. At Week 96, the change from baseline of -0.308 LogMAR (+15 ETDRS letters) in rAAV2/2-ND4-treated eyes and -0.259 LogMAR (+13 ETDRS letters) in sham-treated eyes represents a clinically meaningful bilateral improvement of vision. As some subjects were still in the dynamic phase of the disease process upon enrollment, the visual gain from the nadir was of an even larger magnitude, reaching 28.5 ETDRS letters for rAAV2/2-ND4-treated eyes and 24.5 ETDRS letters for sham-treated eyes. At the time of study design, it was assumed that the BCVA in sham-treated eyes would act as the placebo arm, remaining stable over time or worsening if the nadir of vision had not been reached at baseline. As a result, REVERSE lacks a true internal control group, resulting in our dependence on comparisons with previously published natural history figures. In the RHODOS study, which was a randomized controlled trial comparing idebenone with placebo in LHON patients treated within 5 years of the onset of visual loss, an improvement from baseline of -0.139 LogMAR (+6 ETDRS letters) was noted at Week 24 in the cohort of 35 patients with the m.11778G>A mutation treated with placebo [START_REF] Klopstock | A randomized placebo-controlled trial of idebenone in Leber's hereditary optic neuropathy[END_REF]. Similar natural history results were reported in a larger retrospective study of a LHON cohort of idebenone-treated patients compared with those untreated [START_REF] Carelli | Idebenone treatment in Leber's hereditary optic neuropathy[END_REF]. In a retrospective study that included all three common mtDNA LHON mutations, 82% of patients had a BCVA of 20/200 or worse at the last available data point, which was on average 14.9 months after the onset of vision loss (range from 2.3 to 56.7 months) [START_REF] Silva | Natural history of Leber's hereditary optic neuropathy (LHON): findings from a large patient cohort[END_REF][START_REF] Silva | Natural history findings from a large cohort of patients with Leber's hereditary optic neuropathy (LHON): new insights into the natural disease course[END_REF]. In that study, spontaneous CRR was defined as an improvement from baseline either from off-chart BCVA to reading at least 5 letters on the ETDRS chart at 1 meter, or as an on-chart improvement of at least 10 letters on the ETDRS chart. The analysis was run on a cohort of 86 subjects, including childhood-onset cases. For the subgroup of 61 subjects that carried the m.11778G>A mutation, 15% (9/61) experienced a spontaneous CRR from baseline in at least one eye at the last follow-up visit. In comparison, 68% of subjects (25/37) in REVERSE showed a similar improvement 2 years after treatment.

In a prospective natural history study of LHON, 12 subjects with LHON due to the m.11778G>A mutation and with vision loss of up to 12 months had no improvement of their ETDRS score after 12 months of follow up, and a slight decrease in ETDRS score at 24 months in 9 subjects [START_REF] Lam | Trial end points and natural history in patients with G11778A Leber hereditary optic neuropathy[END_REF]. Including patients with vision loss of more than 12 months, 13 out of 88 eyes (15%) showed a spontaneous improvement of at least 15 ETDRS letters during follow-up, accounting for 18% of enrolled subjects. Childhood-onset LHON carries a better prognosis for spontaneous clinical recovery of vision when compared with adult-onset cases [START_REF] Majander | Childhood-onset Leber hereditary optic neuropathy[END_REF]. The prospective natural history study included 8 subjects who were below the age of 15 years at the time of onset and 3 (38%) of those subjects were reported as being responders in at least one eye The NEI VFQ25 questionnaire has been validated as a responsive and sensitive measure of vision-related quality of life [START_REF] Suñer | Responsiveness of NEI VFQ-25 to changes in visual acuity in neovascular AMD: validation studies from two phase 3 clinical trials[END_REF]. In REVERSE, the composite NEI VFQ25 score showed a mean improvement of 9.5 points, which is more than the clinically relevant threshold estimated at between +3.9 and +4.3 points [START_REF] Suñer | Responsiveness of NEI VFQ-25 to changes in visual acuity in neovascular AMD: validation studies from two phase 3 clinical trials[END_REF]. LHON has a major detrimental impact on activities of daily living and the substantial improvement in composite score suggests a clinically meaningful improvement in patient-reported outcome measures [START_REF] Kirkman | Quality of life in patients with Leber hereditary optic neuropathy[END_REF].

The unexpected bilateral improvement in vision observed in REVERSE is in contrast to the results obtained in other gene therapies targeting photoreceptors or the retinal pigment epithelium with a surgical subretinal injection of the viral vector [START_REF] Yin | Intravitreal Injection of AAV2 Transduces Macaque Inner Retina[END_REF][START_REF] Cideciyan | Leber congenital amaurosis due to RPE65 mutations and its treatment with gene therapy[END_REF]. However, an improvement in visual function in the untreated eye following unilateral intravitreal administration of the viral vector has been demonstrated in other gene therapy trials for LHON [START_REF] Guy | Gene therapy for Leber hereditary optic neuropathy: lowand medium-dose visual results[END_REF][START_REF] Yang | Long-term outcomes of gene therapy for the treatment of Leber's hereditary optic neuropathy[END_REF][START_REF] Liu | Factors associated with rapid improvement in visual acuity in patients with Leber's hereditary optic neuropathy after gene therapy[END_REF][START_REF] Yuan | Seven-Year Follow-up of Gene Therapy for Leber's Hereditary Optic Neuropathy[END_REF].

Regarding potential mechanisms for the observed contralateral improvement, the most intriguing explanation would be the inter-eye transfer of the rAAV2/2-ND4 viral vector. In one rodent study, fluorogold nerve tracer dye injected intravitreally into rat eyes was detected in the optic nerves of the contralateral non-injected eyes, implying axonal or glial transfer through the anterior visual pathways [START_REF] Yang | Chemical and material communication between the optic nerves in rats[END_REF]. There is also evidence supporting the transneuronal spread of AAV, possibly through synaptic transfer mechanisms [START_REF] Zingg | AAV-Mediated Anterograde Transsynaptic Tagging: Mapping Corticocollicular Input-Defined Neural Pathways for Defense Behaviors[END_REF]. Mitochondria have been shown to migrate long distances in axons to distribute energy and allow for distal neuronal activity [START_REF] Mandal | Axonal transport and mitochondrial function in neurons[END_REF].

Mitochondria within RGC axons can also be engulfed into vesicles and exported to astrocytes that are found at a high density in the optic nerve head and then stored in endosomes [START_REF] Burdett | Neuroscience. Astrocytes eyeball axonal mitochondria[END_REF][START_REF] Falchi | Astrocytes shed large membrane vesicles that contain mitochondria, lipid droplets and ATP[END_REF].

The dense network of interconnected astrocytic processes could, therefore, allow for the longdistance cell-to-cell transfer of cytoplasmic elements via membrane junctions [START_REF] Ariazi | Tunneling nanotubes and gap junctions-their role in long-range intercellular communication during development, health, and disease conditions[END_REF]. Another hypothetic underlying mechanism for the contralateral improvement is brain plasticity with reorganization of the visual areas contributing to the visual improvement in the contralateral noninjected eyes [START_REF] Hrvatin | Single-cell analysis of experience-dependent transcriptomic states in the mouse visual cortex[END_REF][START_REF] Sabel | Residual vision activation and the brain-eyevascular triad: dysregulation, plasticity and restoration in low vision and blindness -a review[END_REF].

To explore potential inter-eye transfer, we conducted an equivalent biodistribution study in healthy cynomolgus monkeys. Viral vector DNA was detected in quantifiable amounts in the anterior segment, retina and optic nerve of the non-injected eye three months following unilateral injection of rAAV2/2 ND4. Although the presence of viral vector DNA in the optic chiasm provides a possible clue to the diffusion pathway, our study was not designed to address the exact mechanisms underlying this transfer. The detection of viral vector DNA in the anterior segment of the non-injected eye is also interesting. Additional work is needed to clarify these important observations that have broader relevance to the design of gene therapy trials for optic neuropathies.

The results of REVERSE raise a number of fundamental questions about the prospect of gene therapy for LHON and the best approach to correct the underlying pathogenic mtDNA mutation. The extent of visual recovery seen in patients treated with rAAV2/2-ND4 is more than what one would have expected based on the analysis of the published natural history data, which shows a much lower rate of recovery for this specific mutation [START_REF] Newman | Visual outcomes in Leber hereditary optic neuropathy patients with the m. 11778>A (MTND4) mitochondrial DNA mutation[END_REF]. However, we did not have a prospective control group of untreated patients carrying the m.11778G>A mtDNA mutation assessed using the same comprehensive protocol with whom to make a direct comparison. If we consider that the improvement of vision does not reflect the natural history of LHON, but a true biological effect, the mechanisms that underpin the rescue of RGCs warrant further mechanistic investigations. Although speculative, it is also possible that the enhanced survival of RGCs could arise, at least partly, from the secondary effects of the intravitreal injection with the resulting inflammatory response increasing the expression of coactivators and transcription factors that upregulate mitochondrial biogenesis, which has been shown to be neuroprotective in the presence of a LHON mtDNA mutation [START_REF] Giordano | Efficient mitochondrial biogenesis drives incomplete penetrance in Leber's hereditary optic neuropathy[END_REF][START_REF] Pisano | Targeting estrogen receptor β as preventive therapeutic strategy for Leber's hereditary optic neuropathy[END_REF][START_REF] Cherry | Regulation of mitochondrial biogenesis and its intersection with inflammatory responses[END_REF]. In turn, these mediators could find their way to the non-injected eye accounting for the unexpected bilateral improvement in visual function seen with a unilateral injection of rAAV2/2-ND4. Investigating this hypothesis would require a trial design that assigns one arm to receiving placebo intravitreal injection, which entails a number of safety and ethical considerations, in addition to patient acceptability. Furthermore, the intraocular inflammation seen with rAAV2/2-ND4 is generally mild and transient and it is unlikely to fully account for the sustained enhanced visual rescue. Another hypothesis, besides gene transfer, is the possible exchange of metabolic resources between the optic pathways [START_REF] Coopera | Redistribution of Metabolic Resources through Astrocyte Networks Mitigates Neurodegenerative Stress[END_REF].

In conclusion, the REVERSE study showed bilateral improvement of visual function in LHON subjects treated with a unilateral intravitreal injection of rAAV2/2-ND4. The unexpected visual improvement observed in the contralateral non-injected eyes may reflect transfer of viral vector DNA from one eye to the other as demonstrated in our complementary NHP study.

However, further investigations are needed to confirm these findings and their underlying mechanisms. This study, providing both clinical and preliminary experimental evidence for a bilateral effect of unilateral intravitreal injections targeting RGCs suggests that interocular diffusion of viral DNA vector could occur. These findings could have major implications for gene therapy clinical trial design and outcome measures.

Materials and Methods

Study Design

REVERSE (NCT026527080) was a randomized, double-masked, sham-controlled, phase III clinical trial to evaluate the efficacy of a single intravitreal injection of rAAV2/2-ND4 in LHON subjects with the m.11778G>A mutation and vision loss occurring in both eyes in the prior 6 to 12 months. A total of 37 subjects were enrolled in seven centers (France, Germany, Italy, United Kingdom and United States of America). The objective was to evaluate the efficacy of rAAV2/2-ND4 compared with a sham injection at Weeks 48 and 96, with the change from baseline in visual acuity expressed as the LogMAR as the primary efficacy endpoint. An interim analysis was planned at Week 72 to bring additional insights to Week 48 results.

The right eye of each subject was randomly allocated to receive either treatment with rAAV2/2-ND4 via intravitreal (9E 10 viral genomes in 90 μl per eye) or sham-treatment. The fellow (left) eye received the treatment not allocated to the right eye, in a 1:1 ratio. Treatment with prophylactic oral or topical steroids was not provided. Both eyes received standard antiseptic preparation with administered topical ocular anesthetic agent and underwent pupillary dilation. Before treatment, an intraocular pressure lowering agent was administered. The viral vector rAAV2/2-ND4 was administered in a single intravitreal injection. Following the same preparation steps preceding an intravitreal injection, a sham injection was performed on the sham-treated eye using the blunt end of a syringe and applying pressure to the eye at a typical injection site. Only the pharmacy team, the injecting physician and the medical team assisting in treatment were unmasked to treatment allocation. The unmasked study team performed the first assessment the day after treatment, whereas a separate medical team masked to treatment allocation performed all following ocular examinations.

The protocol was reviewed and approved by independent ethic committees at all sites.

The study was conducted in accordance with the principles and requirements of the International Conference on Harmonization Good Clinical Practice. An independent Data Safety Monitoring Board periodically reviewed study data to ensure the continued safe conduct of the trial and protection of subjects.

To be enrolled, LHON subjects had to be 15 years or older, with duration of vision loss in both eyes between 6 and 12 months and visual acuity of at least counting fingers in each eye. Documented genotyping was required to confirm the presence of the m.11778G>A mutation in the MTND4 gene and the absence of other primary LHON-associated mutations (m.3460G>A in MTND1 or m.14484T>C in MTND6). The main exclusion criteria were known mutations in other genes involved in pathological retinal or optic nerve conditions, previous treatment with an ocular gene therapy product, glaucoma, optic neuropathy other than LHON, history of amblyopia, previous vitrectomy in either eye or ocular surgery of clinical relevance within 90 days. Any prior use of idebenone was required to have ceased at least 7 days prior to enrollment.

Outcome Measures

Ophthalmic evaluations included assessment of BCVA using the ETDRS chart at 1 or 4 meters, assessment of contrast sensitivity using the Pelli-Robson chart, Humphrey Visual Field (HVF) 30-2 testing, Farnsworth-Munsell 100-Hue Color Vision testing, slit-lamp biomicroscopy, Goldmann applanation tonometry, fundoscopy SD-OCT, and color fundus photography.

When subjects could not read any letters on the ETDRS chart, they were asked to count the assessor's fingers or to detect hand motion. An off-chart Snellen equivalent was derived using both the distance at which the assessment was made and the size of the assessor's fingers, as described by Karanjia et al [START_REF] Karanjia | Correcting finger counting to Snellen acuity[END_REF]. The method was also adapted to hand motion visual acuity to provide conversion into a LogMAR value. Light perception and no light perception visual acuities were assigned a value of 4.0 and 4.5 LogMAR, respectively.

Contrast sensitivity (CS) -the reciprocal of contrast threshold -was measured using the Pelli-Robson chart at 1 meter, performed according to test instructions and expressed as a logarithm (LogCS). Subjects who could not read any letter on the Pelli-Robson chart were assigned the worst possible score (0 LogCS). Intraocular inflammation was assessed and graded according to the Standardization of Uveitis Nomenclature (SUN) [START_REF] Jabs | Standardization of Uveitis Nomenclature (SUN) Working Group. Standardization of uveitis nomenclature for reporting clinical data. Results of the First International Workshop[END_REF] and the National Institutes of Health Grading Scale for Vitreous Haze [START_REF] Nussenblatt | Standardization of vitreal inflammatory activity in intermediate and posterior uveitis[END_REF]. Spectral domain-OCT was performed with the Spectralis OCT (Heidelberg Engineering).

Parameters were measured for the optic nerve (Retinal Nerve Fiber Layer [RNFL], Ganglion

Cell Layer [GCL], RNFL of the Papillo Macular Bundle [PMB]) and posterior pole per standard protocols included in the Spectralis software. At pre-specified visits, certified technicians performed one "Optic Nerve Head -Radial Scan and Concentric Circle Scan" (ONH-RC) and one "Posterior Pole N Scan" (PPoleN) for each eye, after maximal dilation. A reading center masked to treatment allocation -the Optic Nerve Research Center (ONRC) -performed quality control, analysis, and interpretation of all SD-OCT data.

The standardized automated HVF 30-2, Central Threshold, SITA-FAST procedure was performed with the HVF Analyzer II. The reliability of the HVF test results were quality controlled by the ONRC reading center and the HVF test was repeated if considered unreliable by that center (defined as fixation losses ≥ 15%, false positive errors ≥ 20%, or false negative errors ≥ 33%).

Quality of life was assessed at enrolment and Week 96 using the NEI VFQ-25 [START_REF] Suñer | Responsiveness of NEI VFQ-25 to changes in visual acuity in neovascular AMD: validation studies from two phase 3 clinical trials[END_REF]. The VFQ-25 consists of 25 vision-targeted questions representing 11 vision-related constructs, and a General-Health rating question. All items were scored so that a high score on a 0 to 100 scale represents better functioning. An overall Composite Score was calculated as the average of the vision-targeted subscale scores, excluding the General-Health rating question. For each subscale, change from baseline was calculated in terms of average score increase/decrease, and as the average of percent changes in scores.

Statistical Analyses

The primary endpoint for the REVERSE study was the change from baseline to Week 48 of LogMAR BCVA following treatment. A difference of -0.3 LogMAR (15 ETDRS letters equivalent) between the change from baseline in the rAAV2/2-ND4-treated eyes and the sham-treated eyes was considered clinically significant based on the U.S. Food and Drug Administration (FDA) recommendations. The sample size calculation was based on the primary endpoint and on the paired comparison of rAAV2/2-ND4-treated and sham-treated eye. The sample size calculation through paired comparison required assumptions due to the absence of published data on the within-subject correlation between right and left eyes and on the withinand between-subject variance of the LogMAR acuity. Based on available data [START_REF] Bonnet | The optimized allotopic expression of ND1 or ND4 genes restores respiratory chain complex I activity in fibroblasts harboring mutations in these genes[END_REF], the sample size required to have a power of 85% was 36 subjects.

The baseline value of functional endpoints (BCVA, CS, HVF 30-2) was defined as the last reported value prior to treatment administration, considering the subacute progression of visual signs. For OCT parameters, baseline was defined as the average value of screening and baseline visits assessments. We defined the "nadir" during the course of the study as the worst BCVA for each eye of each subject from baseline to Week 96 (including baseline and Week 96 values). By definition, the change in BCVA from nadir for each eye results in no change or improvement. A statistical analysis plan was prepared after the study protocol was approved before the database lock.

The efficacy analyses were run using the intent-to-treat population including all subjects who were randomized and received the actual study treatment (rAAV2/2-ND4). The safety analyses were run using the safety population including all subjects who received study treatment (rAAV2/2-ND4).

The change of LogMAR BCVA from baseline to Week 96 in rAAV2/2-ND4-treated eyes was compared to that in sham-treated eyes (intra-subject comparison) using a mixed-effects analysis of covariance (ANCOVA) model with subject and eyes of the subject as random factors, treatment as a fixed effect, and baseline LogMAR BCVA as a covariate. The difference in the mean change from baseline between the two treatment groups and associated 95% confidence interval were reported. Statistical significance was assessed using an alpha of 0.05.

In order to compare our results with previously published data on the natural history of LHON, two responder analyses were performed using different responder definitions. In the first responder analysis, a CRR at Week 96 was defined as either an eye that was on-chart (able to see letters on the chart) at baseline and that showed an improvement of at least 10 ETDRS letters, or an eye that was off-chart (not able to see letters on the chart) at baseline and that became able to read 5 ETDRS letters on-chart at 1 meter [START_REF]2015 Committee for Medicinal Products for Human Use (CHMP) -Assessment Report Raxone (Idebenone)[END_REF][START_REF] Silva | Natural history of Leber's hereditary optic neuropathy (LHON): findings from a large patient cohort[END_REF][START_REF] Silva | Natural history findings from a large cohort of patients with Leber's hereditary optic neuropathy (LHON): new insights into the natural disease course[END_REF]. A subject responder was defined as having this response in at least one eye at Week 96. In the second responder analysis, an eye responder at Week 96 was defined as an on-chart improvement of at least -0.3 LogMAR (3 ETDRS lines), or an improvement from off-chart to on-chart vision with a final BCVA of 1.4 LogMAR or better (able to read at least the first 3 ETDRS lines of the chart at 1 meter) [START_REF] Lam | Trial end points and natural history in patients with G11778A Leber hereditary optic neuropathy[END_REF]. A subject responder was defined as having this response in at least one eye at Week 96. We opted for the first responder analysis, CRR, as this approach was considered by the European Medicines Agency (EMA) in its assessment of the efficacy of idebenone (Raxone, Santhera) in LHON [START_REF]2015 Committee for Medicinal Products for Human Use (CHMP) -Assessment Report Raxone (Idebenone)[END_REF].

A group of experts have also endorsed the use of CRR as a valid outcome measure when assessing the effect of treatment with idebenone in LHON (1).

Non-Human Primate Study

Methods

Four healthy male purpose-bred cynomolgus monkeys (Macaca fascicularis) were allocated to groups using a computerized stratification procedure so that the average body weight of each group was similar. At the beginning of the treatment period, the animals were at least 24 months old and with body weights between 3 and 5 kg. Formulations were administered by unilateral intravitreal injection in the right eye on Day 1 under a volume of 90 μL. Following injection, the animals were kept for an observation period of 3 months ± 2 days (with day 1 corresponding to the day of treatment). Group 1 included one control animal receiving unilateral intravitreal injection of vehicle, composed of formulation buffer (balanced sterile saline solution [BSSS buffer] supplemented with 0.001% Pluronic F68). Group 2 included 3 test animals receiving unilateral intravitreal injection of rAAV2/2-ND (S222/DP0014/FC003 batch, Novasep), formulated in BSSS buffer supplemented with 0.001% Pluronic F68 to reach a dose of 4.

Between-eye

Difference in

Change from

Baseline (1) n 35

LS Mean (95% CI) 0.2 (-2.2, 2.7) p-value 0.8465

  at their last follow-up visit. Direct comparisons between the Lam et al. study and our study are problematic because of the relatively small numbers in the subgroups in the Lam et al. study and the potential confounders of inclusion of subjects with young age at onset, enrollment of some patients with visual loss duration of less than 6 months, and possible patient concurrent use of idebenone (21). However, 38% of rAAV2/2-ND4-treated eyes and 32% of sham-treated eyes in REVERSE showed an improvement from baseline of at least 15 ETDRS letters at week 96, representing 46% of the entire study cohort, suggesting a possible better responder rate than in any subgroups of the Lam et al. study. The bilateral gain in BCVA in the REVERSE study was consistent with the trend observed for other visual parameters, including contrast sensitivity and visual field perimetry.

  3x1010 viral genomes (vg) in 90 μL injected in the right eye. The 4.3x1010 vg dose was determined based on animal toxicology studies previously conducted and it corresponds to the human injected rAAV2/2-ND4 dose, proportional to the vitreous volume of the cynomolgus monkeys.At the end of the 3-month observation period, the animals were necropsied, and the following tissues were sampled from each animal (with right and left sides being collected and preserved separately): vitreous humor, aqueous humor, anterior segment of the eye (including cornea, iris, lens and choroid), lacrimal gland, retina, optic nerve, optic chiasm, lateral geniculate nucleus, optic tract and visual cortex. Analyses consisted of qPCR (DNA) targeting the ND4 transgene of rAAV2/2-ND4. Special attention was given to sample collection to avoid crosscontamination. Tissues and fluids were collected first for Group 1 control animals. Amongst the treated animals, tissues and fluids related to the non-treated eye were collected first. A clean set of disposable sterile instruments was used for each animal and for each tissue collected. The precise times of sacrifice of the animals and of snap freezing of the tissues were recorded.DNA from tissues was extracted using the Nucleospin Tissue kit (Macherey-Nagel). Tissue samples, except the anterior segment of the eye samples, were homogenized in 1 mL of T1 buffer. The anterior segment of the eye was entirely sampled and weighed. All the samples were homogenized into 2 mL of T1 buffer. For each tissue sample, 400 μL of the homogenized tissue were used for the extraction. The remaining homogenized tissue was kept at -80°C until further notice. Each tissue was weighed before the DNA extraction step. To the extent possible, tissue samples of minimum 20-30 mg (around 3 x 3 x 3 mm for small organs) and tissue sample of minimum 80 mg (around 5 x 5 x 5 mm for larger organs) were used. The volume of each fluid was measured before the DNA extraction step. Sentinel controls are samples composed by the first reagent that are usually added onto the biological material to monitor the potential crosscontamination between samples during the extraction process. Sentinel controls were included within each DNA extraction batch. DNA concentration and purity were determined by UV spectrophotometry. DNA was then stored at -20°C until analysis.The extracted DNA was used as a template for qPCR amplification. The TaqMan qPCR protocol specifically targets the ND4 transgene of rAAV2/2-ND4. The qPCR protocol was validated in a previous dedicated study and the analytical parameters for sensitivity, specificity and accuracy were determined. Each qPCR contained 5 μL of DNA corresponding to 400 ng of DNA when feasible (or 400 ng of herring sperm for NTC sample, or 5 μL of ultrapure nucleasefree water for no DNA sample or 5 μL of eluate from sentinel controls), and 20 μL of the qPCR master mix was composed of PCR Master Mix 2X (12.5 μL), forward-primer and reverse-primer (300 nM each), TaqManProbe (150 nM), and ultrapure nuclease-free water (qsp 20 μL). PCR plates were run on an Applied Biosystems 7900HT.

Fig 1 .

 1 Fig 1. (A) Mean Change from Baseline in BCVA up to 96 Weeks Post-Administration of

Acknowledgments:

We are grateful to the study teams that have contributed to the conduct of REVERSE in the various recruitment centers (supplementary materials). We would also like to thank the patients who took part in this gene therapy study.

Funding: GenSight Biologics fully funded and sponsored the study. PYWM is supported by a Clinician Scientist Fellowship Award (G1002570) from the Medical Research Council (UK), and also receives funding from Fight for Sight (UK), the Isaac Newton Trust (UK), the National Eye Centre UK), the UK National Institute of Health Research (NIHR) as part of the Rare Diseases Translational Research Collaboration, and the NIHR Biomedical Research Centre based at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. VC is supported by grants from the Italian Ministry of Health (RF-2018-12366703), the Italian Ministry of Research (20172T2MHH), and Telethon-Italy (GUP15016). VC is also supported by patients' organizations MITOCON and IFOND, and patients' donations. TK is supported by the German Federal Ministry of Education and Research (BMBF, Bonn, Germany) through grants to the German Network for Mitochondrial Disorders (mitoNET, 01GM1906A) and to the E-Rare project GENOMIT (01GM1920B). JAS and SP are supported by the Agence Nationale de la Recherche within the Programme Investissements d'Avenir, Institut Hospitalo-Universitaire FOReSIGHT [ANR-18-IAHU-0001].

Data and Materials Availability:

All data associated with this study are available in the main text or the supplementary materials.

Between-eye

Difference in

Change from

Baseline (1) n 37 The mean improvement in BCVA from nadir to Week 96 was converted from LogMAR to "letters equivalent" by multiplying the LogMAR value by -50 [START_REF] Holladay | Proper method for calculating average visual acuity[END_REF]. Nadir is defined as the worst BCVA recorded in any of the visits in REVERSE, including the baseline visit immediately prior to the injection. BCVA = best-corrected visual acuity; LogMAR = logarithm of the minimal angle resolution.

LS

Nervous System

The bar graph indicates the number of animals (out of 3) in which rAAV2/2-ND4 DNA was present above the limit of detection (15.6 copies/μg of DNA).

SUPPLEMENTARY FIGURES AND TABLES

Between-eye

Difference in

Change from

Baseline (1) n 36

LS Mean (95% CI) 0.013 (-0.016, 0.042) p-value 0.3528

(1) A mixed-effects analysis of covariance (ANCOVA) model was used with change from baseline at as the response, and subject and eyes of the subject as random factors, treatment as a fixed effect, and the baseline LogMAR value as covariate. P-value is used to assess the significance of the difference between All-rAAV2/2-ND4 and All-Sham with respect to change from baseline.

(

(1) CRR was defined per treatment group as either an eye which is on-chart at baseline with an improvement at Week 96 of at least 10 ETDRS letters, or an eye which is off-chart at baseline that became on-chart with at least 5 letters read at Week 96. A subject responder was defined as having a CRR in at least one eye at Week 96.

(2) p-value from McNemar test compares the rates of eye responders between treatments.

(3) Responder was defined as an on-chart improvement by at least -0. (2) The mean percent change from baseline was calculated from individual percent changes from baseline.

(3) The Composite Score is the average of vision-targeted sub-scale scores, excluding the General Health rating question.