Roula Ntefeh
email: roula.ntefeh@cerma.archi.fr

Daniel Siret

Christian Marenne

The internal courtyard of mixed use buildings, a device of thermal and luminous comfort

Keywords: Courtyard-Patio, Mixed use buildings, Natural Light, Sunlight

The historical patio-courtyard is an outdoor area largely or entirely surrounded by buildings or walls. Generally, the courtyards are useful to sunlighting because their open spaces preserve the solar access of the adjoining buildings. They allow sunlight to reach the facades so that side-lighting strategies can be used. In urban fabric of the Mediterranean countries, we observe a new generation of big patios with mixed-use buildings (housing, trade, offices). This may be a return to the traditional form of the patio, which allows a passive regulation of built environment, multi-use activities, and security of the inhabitants. This new interpretation of a traditional passive device suggests more problems of illumination on lower levels. It becomes necessary to assess the performances of different courtyard shapes that behave as shafts. This paper presents the influence of the courtyard shape and orientation, on sunlighting duration and illumination levels of ground and facades. The results enable to propose changes of the distribution of the uses through the facades, vertically or horizontally, according to the orientation preferred and the illumination for each use.

INTRODUCTION

The Mediterranean region is found between 30 and 40 degrees north of the equator, in south Europe, Middle East and North Africa. This region is characterized mainly by high summer daytime temperature, large diurnal temperature range and high solar radiation. For such difficult conditions, the urban design should achieve the following objectives: providing shade in summer in open spaces and courtyards, providing solar access in winter for the interior building, enabling good ventilation potential in evening, minimizing the dust level [START_REF] Givoni | Urban design for hot humid and hot dry regions in Architecture and urban space[END_REF].

In traditional urban fabric, the great majority of houses incorporate courtyards, which are in two high storeys, but the functions and use of courtyard is also an important factor for determining its size [START_REF] Al-Azzawi | Not every courtyard is necessarily a good one climatically, physical characteristics design of domestic courtyards in the hot-dry climates and the subtropics, Architecture and urban space[END_REF].

Currently, with new urban fabric of mediterranean countries, buildings are separated. In this case, it is essential to ensure greater ratio between the built volume and the envelope design. This implies vast residences surfaces and a great number of storeys. That also supposes many families or communities within one building [START_REF] Pinon | Maison en pays islamiques[END_REF].

In fact, in some Mediterranean countries, like Spain, Italy, and some Arab regions, we find a new generation of residential buildings whith big courtyard. They are mixed-use buildings: habitat, trade and office. The flexibility of this new type is due to the introduction of several uses into the building (see Figure1). This new type of mixed-use building with a large interior courtyard might achieve the objectives of sustainable development; use the renewable energy, reduce the electricity consumption, combine much function in one building, and so on.

However, this generation of building with interior courtyard is introduced into a new context. The space of courtyard has undergone some changes: the physical changes and the heat exchange between the envelope design and the exterior environment. These modifications are due to the absence of dense fabric of traditional cities characterized by narrow streets. Additionally, in a big courtyard, the direct daylight never reaches the lower parts of walls.

In this paper we discuss the solar and luminous interior environment in courtyard, by investigating the effects of five different courtyard configurations. The used configurations present a simplification of existing models with big courtyard of mid-rise building in mediterranean climate.

We achieve solar simulations in order to study the effect of its height and shape on the interior courtyard environment from luminous and thermal point of view. For that, we evaluate the sunlighting and the illumination on façades for each level, and in courtyard center. We consider the arriving of light through the courtyard as a principal daylight source for adjacent spaces and courtyards ground. Figure1 Fourssan Housing, Architect: Architectural and urban design studies, El-Mously Group& Hossam Eldien, Cairo, 1998. Typical housing floor plan and view of the building mass.

METHOD

The methodology adopted is based on simulation produced by SOLENE model developed by CERMA laboratory. This model uses the geometrical procedures for the calculation of sunshine duration, and the radiosity method for the daylight reflected calculation [START_REF] Groleau | A daylight simulation tool for urban and architectural spaces: application to transmitted direct and diffuse light trough glazing[END_REF].

The choice of shapes studied is based on a group of existing real examples found in mediterranean countries. The regular forms, square, rectangle, triangle and circle are mainly used, according to several orientations.

In fact, large patios are heated more quickly than traditional small ones, because of the higher exposure to the sky in the day and the overheating radiation diffused by the upper parts of walls at night. The degree of openness to the sky is given by the following relation, proposed by J. S. Reynolds, between floor area and square of average height of surrounding walls (aspect ratio used in daylighting design is found in Baker, Franchiotti and Steemers 1993 ) [START_REF] Reynolds | COURTYARDS, aesthetic, social, and thermal delight[END_REF]: The greater aspect ratio, the more exposure is the courtyard to the sky. This exposure allows heating by the sun by day, cooling by radiation to the cold sky at night, some entry to the wind and vice versa. For that, with big courtyards in mediterranean climate, the choice of courtyard scales is made in order to decrease the ratio between area and square height. In this study we use the ratio 1:3 which is detailed later.

In this case, we suggest the nocturnal ventilation, which appears the best with climatic condition such as the hot-dry climate, and that supports the chimney effect with the higher depth of courtyard. This has been studied in case of the residential buildings [START_REF] Piras | La cour intérieure des bâtiments en tant qu'espace de ventilation naturelle[END_REF].

Geometric parameters

We classify the outdoor area courtyard in terms of configuration and orientation, scale, and finally proportion.

Configuration and orientation: The forms suggested are: square, rectangle ratio1:2, rectangle ratio1:3, isosceles triangle and circle, the ratio is considered here between width and length (see figure .2). Each configuration has a ground plane and many adjoining façades. Solar access and luminous energy are evaluated for several configurations Scale: The building's type is the typical mid-rise building; number of the storeys is 8. With a height of 24m, the patio surface is approximately 200 m 2 , and finally, the position of courtyard is central.

Proportion: the geometrical shape is a vertical shaft, which provides the solar protection. In order to facilitate the comparison between different patios, we will fix the ratio (between surface and square of average height), which is approximately about 1:3, and the plan surface is a little over 200 m 2 which corresponds to a large courtyard of a residential multifamily building.

Simulations

Our analysis addressed the following parameters: the effects of geometry and orientation may be observed by using the internal shading of surfaces to indicate the degree of shading and sunlighting. We will consider the percentage of shade on vertical surface area and the percentage of ground area in shade. The examined dates are June 21 (summer solstice) and December 21 (winter solstice). The interval of time between two calculations is one hour for the latitude 35°N which corresponds to the average latitude of the Mediterranean region.

For the heat gain recommended during winter months, we considered that the threshold of sunlighting is limited to two hours minimum.

On the other hand on summer, if the facades receive more than four hours of sun, the overheating is probable; this is due to the inertia of the built masses, the solar energy received and absorbed by the walls of the patio, which will be diffused later towards the interior by heat convection [START_REF]Guide de l'architecture bioclimatique[END_REF].

As a measured of daylight distribution we have chosen the daylight factor DF%. Notice that the factor is obtained after reflexion. Values were computed on the centre of courtyard ground and at the middle level of each story (height 3 m). The model of sky used for simulation is the standard CIE overcast sky. The choice of this condition is to investigate the illumination distribution with the more undesirable climatic conditions. Values have been normalised in a similar manner to the sky component of the daylight factor % (ratio of the local illuminance to the simultaneous outdoor horizontal illuminance due to an unobstructed sky) [START_REF] Fontoynont | Daylight performance of buildings[END_REF]. The simulation was conducted on December 21, it was considered a ground and building façade reflectance of 0.8. The models were studied at different hours of the day; here we only show the results for 12:00h, where direct sunlight access is vertical and doesn't enter the interiors surrounding buildings.

Rotations are considered in order to present the effect of orientation on the courtyard geometry. We have carried out simulation every 15°ccw. In the interest of brevity, only simulations for eight shapes are presented in this paper. The simulation on the square shows, that square form supports solar protection in summer, the percentage of protected surfaces is equal to 53,1% in initial position (see Figure 3).

Simulation carried out on the rectangle (ratio1:2 elongated east-west) shows, that it seems to be not powerful in term of solar protection on summer. A greater part of the north, east, and west courtyard façades is exposed to direct solar access more than four hours in the day. The increased insolation minimizes the thermal comfort in adjacent spaces. On the contrary, the rectangle (ratio 2:1) elongated northsouth appears more efficient than the horizontal one; the percentage of shading on interiors façades is 60%. Moreover, during the day, the upper part of north façade reaches at minimum four hours of sun.

The rectangular courtyard (ratio1:3) elongated vertically is most efficient compared to the horizontal one. The vertical position ensures more protection on its long edges. Nevertheless, the two higher levels reach more than four hours, on north, east, and west courtyard façades.

On the other hand, it is found that isosceles triangle in initial position has less effect compared with second rotation. We noted that the results of the triangular form are similar to the results of the rectangle ratio 1:2. Also the rotation angle 60°ccw improves the solar protection.

Finally, for the circle, the percentage of the protected facades is approximately 30% and on the courtyard ground is 50% First of all, we search the surfaces, that receive a heat gain more than two hours by day.

The simulation results carried out on the square form, show that the percentage of vertical surfaces, which receive sun less than two hours on winter, is 18,75% max (see figure 4).

The vertical position of rectangular form (ratio1: 2) is better than the horizontal one. The percentage of the surfaces shown upon on winter is approximately 23% at vertical position, and fall down to 13% in horizontal position.

Similarly, rectangle elongated East-West (ratio 1:3) presents a lack of sunlighting. This is remarkable for both cases studied; the percentage of the internal facades, which reach the minimal hours recommended in winter, doesn't exceed 10% in initial position. Contrary to the vertical position the values reach to 18% on the interior façades. The triangular shape doesn't optimise the heat gain on winter without rotation; the percentage of the facades, which reached the threshold, is 16% and decrease to 11% with 60° rotation angle.

For the cylindrical shape, the percentage of interiors façades in sun is about 25% of total vertical faces.

For all studied cases, the ground plane never has any direct sunlight on winter. The improvement of sunshine duration is remarkable with rotation angle 90° for the rectangular forms (see figure.

Daylight in courtyard

Study of the various forms shows that the circle, the rectangle (1:2), the square, and the triangle rotated 60°ccw optimise illumination on the patio ground. The daylight factor reaches to 16.5%, 9.87%, 9.5%9.45% respectively. (See table I).

However, the minor values are monitored in the rectangle ratio 1:3 (DF: 9.2%), the same result for rectangle ratio 3:1, rectangle ratio 2:1 (DF: 9,32%), and triangular form (DF: 9.41%)

On the other hand, analysis of illumination on the façade shows that the light decreases gradually from top to bottom. By using the average daylight factor to indicate the best form, we found the following results. The rotation angle 90°ccw doesn't improves the average daylight factor in both rectangle ratio (2:1) and rectangle (3:1).

Besides, the rotation angle 60°ccw applied on the triangle, gives a value more than the initial orientation one. Also for all rotations angle 90° and 60°ccw, the difference between results is slight (see figure 6).

Also, the square shape provides good illumination on the patio ground. The average value reaches to 12.81% while the maximum value obtained is in the circular form with 18.82%.

We noted that in all cases studied the upper levels receive sufficient amount of light, the values rise to 35.35% at least in rectangle 1:2 and reaches to 33.18% in the triangle rotation 60°ccw.

The daylight factor measured on the façades can clarify the influence of the patio shapes on the illumination of surrounding walls, while these calculations are not sufficient in order to evaluate the interior illumination quality and the visual comfort. By comparing the various studied forms, the results of solar simulations show that the rectangle ratio (2:1) presents the highest values in terms of solar protection on summer and heat gain on winter. As the square form and the rectangle ratio (3:1), it appears more adequate compared to the others. Additionally, the square shows a good illumination in both façades and courtyard itself. The rotation angle 90°ccw gives an improvement in terms of solar protection and heat gain on winter, as well as the rotation angle 60° applied to the triangle. The results obtained in this case are more powerful compared to triangular one.

Height on façade (m)

On the contrary, the results of the circle, are contradictory. This form presents a highest level of illumination and heat gain in winter, and the lowest level of solar protection in summer. We noted that its presence in urban fabric of Mediterranean cities is seldom excluding the court of the small urban islands.

On the other hand, the different studied forms show an insufficiency of the solar gain in winter; the patio ground doesn't receive enough sun. Moreover, on the summer, the percentage of surfaces, which receive less than four hours, is to 50% at minimum, while the ground is approximately protected.

Nevertheless, in this Shaft form of patio, the uppers level are almost exposed to the sun, and the four lower floors can't receive sufficient light and heat gain in winter. It seems that the obstruction of the sky, due to the higher number of level, has not provided the best solution for the thermal comfort and human use in habitat, specially in terms of heat gain on winter. The degree of the openness to the sky is more important for illumination and improving heat gain on winter.

By considering the requirements of the sunlighting recommended in winter, it seems that the apartments should be placed in the sunny part of building, in upper levels or on the western, eastern, and the southern façades. This implies a solar protection in summer especially on the last proposition On the other hand, the offices can be sited on the north façade which receive a minimum of sun compared to the other ones.

Also, the problem of luminosity on the first floors has to be solved. It seems that the combination between trim materials and the colors could ensure the improvement of lighting on winter and minimizes the risk of discomfort glare on the interior on summer time.

The trades which are normally placed on the lower floors in order to facilitate their access. They are well protected from the direct solar access, however, they cannot benefit from natural daylight. A bilateral lighting can improve the illumination in this part.

This study aims at clarifying the influence of geometry and orientation on the courtyards environment. We will carry out, later, an analysis of luminous and thermal aspects inside the interior parts of buildings. This future step is in order to distribute the functions according to obtained results. The objective of studies examines the new shapes of big patios, which exists in an urban Mediterranean context. This new type of mixed-use building with the large interior courtyard might achieve the objectives of sustainable development.

Figure 2 :

 2 Figure 2: The configurations of enclosed courtyards

3 .

 3 DISCUSSION OF RESULTS 3.1. Summer monitoring (June 21)

Figure 3 : 2 .

 32 Figure 3: simulation results for sunshine duration in June 21.

5 )Figure 4 :

 54 Figure 4: solar simulations; in grey the surfaces receive less than two hours on winter from axonometric view

Figure 5 :

 5 Figure 5: comparison between different shapes of the patio

N

Figure 6 :

 6 Figure 6: average daylight factor% on façades from ground floor to upper level


Mark Bastourous
email: mark.bastourous@univ-lehavre.fr

François Guérin
email: francois.guerin@univ-lehavre.fr

Frédéric Guinand
email: frederic.guinand@univ-lehavre.fr

Eric Lemains
email: eric.lemains@univ-lehavre.fr

Decentralized High Level Controller for Formation Flight Control of UAVs

Keywords: Distributed control, Formation control, Robotics, Autonomous vehicles, UAVs I

The main contribution of this paper is the design of a decentralized and tuning-less high level controller able to maintain without tracking errors a Leader-Follower (LF) configuration in case of lack or degraded communications (latencies, loss…) between the leader and followers UAVs. The high level controller only requires simple tunings and rests on a predictive filtering algorithm and a first order dynamic model to recover an estimation of the leader UAV velocities and avoid the tracking errors.

INTRODUCTION

Multi-Robot Systems (MRS) have been widely studied since the 90s [START_REF] Arai | Editorial: Advances in multi-robot systems[END_REF], [START_REF] Cao | Cooperative mobile robotics: Antecedents and directions[END_REF]. Many advantages can be expected from using a group of robots: increase and share of the payload [START_REF] Mellinger | Cooperative Grasping and Transport Using Multiple Quadrotors[END_REF], reduction of the time needed for the achievement of a task [START_REF] Khamis | Multi-robot Task Allocation: A Review of the State-of-the-Art[END_REF], fault tolerance and resilience of the system [START_REF] Zeng | Wireless communications with unmanned aerial vehicles: opportunities and challenges[END_REF], [START_REF] Tuna | Unmanned aerial vehicle-aided communications system for disaster recovery[END_REF], or use of simpler and cheaper robots for adaptability to the environment [START_REF] Tosun | Perception-informed autonomous environment augmentation with modular robots[END_REF]. However, making a group of robots evolving together in the same environment entails the resolution of some problems. Coordination is one of the most critical ones, especially considering decentralized settings. Coordination between the members of the group can be achieved through various ways, behavior or force-based methods, Virtual Rigid Body (VRB) structure building, or Leader-Follower (LF) schemes. In the present work, we focus on the last one and investigate the problem of maintaining such a configuration in case of degraded communications between the leader and the followers. The question of building and maintaining such a formation without communication has been addressed many times during the last two decades and most of the time the proposed solution relies on a sensor-based mechanism for compensating the absence of communication. Very often, works are considering cameras; pan controlled camera [START_REF]Adaptive leader-follower formation control of nonholonomic mobile robots using active vision[END_REF] or omnidirectional camera like in [START_REF] Morbidi | Observer design via immersion and invariance for vision based leader follower formation control[END_REF], [START_REF] Guo | A unified leader-follower scheme for mobile robots with uncalibrated on-board camera[END_REF], or other types of sensors like Kinect [START_REF] Liu | Vision-based Leader/Follower formation control of multiagents with visibility constraints[END_REF]. From the data obtained by the sensors, each follower of the formation attempts to estimate the pose of the global or of its local leader. In [START_REF]Adaptive leader-follower formation control of nonholonomic mobile robots using active vision[END_REF], the authors consider this problem with a pan-controlled camera. Since no communication occurs, the leader's velocities are unknown. The goal of their work is the design of both an adaptive formation controller and an adaptive camera controller. The authors in [START_REF] Guo | A unified leader-follower scheme for mobile robots with uncalibrated on-board camera[END_REF] proposed a method for LF formation control considering a robotic system in which each follower is equipped with an uncalibrated omnidirectional or perspective camera. No communications are allowed and the velocity of the leader is considered unknown as well. In that work the authors present an adaptive estimator based on several feature points. In [START_REF] Liu | Vision-based Leader/Follower formation control of multiagents with visibility constraints[END_REF], the goal is to build and maintain a LF formation without communication between the robots. Each robot is equipped with only one type of sensor, a Kinect, for estimating the pose (relative orientation and position of the leader). The choice of the Kinect is justified by the fact that it provides color and depth information from which can be directly deduced angles and distances measurements but it suffers from a limited field of view. The controller depends on both the visibility maintenance and the minimization of position errors. With respect to these previous works, our main contribution is the design of a decentralized and tuning-less high level controller able to maintain a LF configuration. The proposed controller combines an omnidirectional camera with filtered and predicted measurements and a first order dynamic model of the UAV to get the leader velocities (even in case of degraded communications (latencies, loss) between the leader and followers UAVs) and avoid the tracking errors.

II. PROBLEM STATEMENT

The goal is to construct a geometrical shape sometimes known as Virtual Rigid Body (VRB) in order to establish the desired formation. The virtual structure between several robots aims at facilitating the agile control of several multirotor formations. The goal of each follower UAV is to follow the leader by keeping predefined separation distances XSP1, YSP1, ZSP1 as illustrated on Fig. 1. The leader UAV is assumed to track perfectly a virtual target which moves along a defined trajectory (straight line, circle …). The controller of the leader UAV is not described in this work. The description of the high level controller will be done for the follower UAV number 1. The vision-based system provides the distance (d1) and the bearing angle (θ1) of the leader UAV.

III. DESIGN OF THE HIGH LEVEL CONTROLLER

A. Double Exponential Smoothing (DES) algorithm

The proposed method assumes the availability of an estimation of both the distance (d1) and of the bearing angle (θ1) whose measurements are naturally degraded by noise and other uncertainties. A filtering stage to overcome these problems is mandatory. Such algorithm has to be fast, accurate, robust to different motions, and easy to implement. We assume that the leader UAV moves regularly over time with a trend. In [START_REF] Joseph | Double Exponential Smoothing: An Alternative to Kalman Filter-Based Predictive Tracking[END_REF], [START_REF] Joseph | An Experiment Comparing Double Exponential Smoothing and Kalman Filter -Based Predictive Tracking Algorithms[END_REF], a comparison is drawn between Double Exponential Smoothing (DES) algorithm and Kalman filter. The authors show that DES algorithm runs faster than Kalman filter with equivalent prediction performances and simpler implementations. That is why DES algorithm can be a good choice to generate smoothed estimates and forecast of distance (d1) and bearing angle (θ1). The DES algorithm at time instant n.Te, where Te is the sampling period and n is the discrete-time index, implemented for both the distance (d1) and bearing angle (θ1) measurements is given as follows:

= . + (1 - ). ( + ) (1) = . - + (1 - ). ( 2) = . + (1 - ). ( + ) ( 3) = . - + (1 - ). (4)
where: d1n, θ1n are the values of d1 and θ1 at nth sample. , are the smoothed values of d1n and θ1n. , are the trend values of d1n and θ1n.

Equations ( 1) and ( 3) smooth the values of the sequence of measurements by taking into account the trend, whilst ( 2) and ( 4) smooth and update the trend. The m (m > 0) steps ahead forecast of the next distance ( ) and bearing angle ( ) estimations are:

= + . = + .
(

) 5
The initial values given to , , and are:

= , = , = - , = - (6)
Usually, α (0 ≤ ≤ 1) is called the data smoothing factor and λ (0 ≤ ≤ 1) is called the trend smoothing factor. A compromise has to be found for the values of α and λ. High values make the DES algorithm follow the trend more accurately whilst small values make it generate smoother results. For the follower UAV 1, the filtered (7) and predicted (8) distance errors (x, y axes) are the following ones (Fig. 1):

̂ = * cos - ̂ = * sin - (7) ̃ = * cos - ̃ = * sin - (8)

B. Design of the tuning-less high level controller

We assume that the low level controllers (inner loop) implemented in the UAVs allow them to reach the desired references velocities (according to x and y axes) and the desired references positions (according to z axis (altitude, yaw angle)) with a short response time and without static errors, damping or oscillations. That means that: ⃗ = ⃗ with ⃗ = [ ] the velocity vector of the follower UAV 1 and ⃗ = [ ] its control inputs. All the UAVs are assumed to fly at the same altitude with the same yaw angle (altitude and yaw control can be controlled separately and are not detailed in this work). The control objective is to regulate the distance errors as follows:

lim → ̂ ( ) = 0 lim → ̂ ( ) = 0 (9)
To fulfill this control objective, we propose the following error dynamics equation:

̂̇ ̂̇ = - ̂ . . ∑ , - , . ∑ , - , = . ⃗ -⃗ (10)
= cos( -) -sin( -) sin( -) cos( -) ̂ =

. ̂ ⃗ = [ ] is the leader velocity vector. Ay is an attenuation parameter which makes decrease the effect of the linear control (x axis) when the lateral error (y axis) is not null. with:

= ̂ ̇ = ̃ ̂ . = ̃ -̂ = ( , 1,1) = , 1,1 = ( . . , 1 ,1)
with:

= ̂ ̇ = ̃ ̂ . = ̃ -̂
The saturation function (f) is defined below. It allows to take into account the UAV limitations [START_REF] Guo | A unified leader-follower scheme for mobile robots with uncalibrated on-board camera[END_REF]. , are the maximum allowed velocities according to x and y axes. The maximum allowed velocity of the follower is reached as soon as the components of its distance, to the leader, are greater or equal to , . The saturation function (f) is also used to normalize the values of and between -1 and +1. From [START_REF] Guo | A unified leader-follower scheme for mobile robots with uncalibrated on-board camera[END_REF], we get the following control law:

⃗ = . ⃗ + ̂ . . ∑ , - , . ∑ , - , (11)
In steady state [START_REF] Guo | A unified leader-follower scheme for mobile robots with uncalibrated on-board camera[END_REF], the follower will fly at the speed of the leader without tracking errors ( ⃗ = . ⃗ ) only if it receives, through a communication network, the leader velocity ( ⃗ ) without disturbances, latencies or losses.

C. Leader UAV velocities estimation

To compensate the tracking errors due to disturbances, latencies or loss of communication, the leader UAV velocity vector ( ⃗ ) is estimated by using a first order dynamic model of the UAV [START_REF] Sanabria | ARDrone Simulink Development Kit Version 1.0[END_REF]:

⃗ ̇ = - 0 0 - . ⃗ + 0 0 . ⃗ (14)

⃗ = [ ] and ⃗ = [

] are the velocity and control input vectors of the model respectively. In the earth frame (Fig. 1), its position ⃗ = [ ] is:

⃗ ̇ = . ⃗ with = cos( ) -sin( ) sin( ) cos( ) (15)
Let us consider the following filtered distance errors (Fig. 1):

̂ ⃗ = * cos * sin + . ⃗ -⃗ - (16)
with:

̂ ⃗ = [ ̂ ̂ ]
The control objective is to regulate the distance errors as follows:

lim → ̂ ( ) = 0 lim → ̂ ( ) = 0 (17)
To fulfill this control objective, we propose the following error dynamics equation:

̂ ⃗ ̇ = - 0 0 . ̂ ⃗ = . ⃗ -⃗ (18)
with: = =

In steady state [START_REF] Sanabria | ARDrone Simulink Development Kit Version 1.0[END_REF]18), ⃗ = ⃗ and ⃗ = . ⃗ . The tracking errors (18) will be null only if the dynamic model get the leader UAV velocities without disturbances, latencies or losses. Otherwise, the tracking errors will depend on the values of the proportional gains Kx, Ky. Indeed, let us consider the following candidate Lyapunov function:

= ̂ ⃗ . ̂ ⃗ = . ( ̂ + ̂ ) (19)
Its time derivative can be written as:

̇= . ̇ + . ̇ Thus, ̇< 0 if | ̂ | ≥
and ̂ ≥ so that the size of the tracking errors | ̂ |and ̂ are uniformly and ultimately bounded by and respectively, where and represent bounds on the leader UAV velocity components satisfying | | < and < . These tracking errors don't have any effects since they concern the dynamic model. This interesting result allows to get the leader UAV velocities even in case of degraded (latencies, loss,…) communications. From (18), we get for the dynamic model the following control law:

⃗ = . ⃗ + 0 0 . 1 ⃗ (20)
Finally, by combining ( 11) and (20), we get:

⃗ = ⃗ + ̂ . . ∑ , - , . ∑ , - , (21)
IV. SIMULATION RESULTS

The simulations have been carried out on Matlab-Simulink software. They are based upon a dynamic model of the UAV [START_REF] Sanabria | ARDrone Simulink Development Kit Version 1.0[END_REF] combined with usual low level controllers (PID control for horizontal velocities and Phase Lead control for altitude and angular (yaw) positions). The simulation parameters are the following ones:

-Simulation time: T=800s -Sampling period: Te=0.01s On Fig. 4,5,6,7 we can see that the tracking errors according to x and y axes converge toward zero when the leader UAV flies at different speeds and even when the communication between the leader and the followers is not available. As long as the communication is available, the model errors are null and the follower UAV 1 uses the speed information sent by the UAV leader. When the communication is lost, the model errors are not null and allows to recover the speed information.

V. CONCLUSION AND PERSPECTIVES

In conclusion, the formation control can work in a fully decentralized way. Indeed, unlike other VRB methods, which rely on the group barycenter to steer the formation [START_REF] Brandao | On the guidance of multiple uav using a centralized formation control scheme and delaunay triangulation[END_REF], our approach requires neither positioning nor tracking from a centralized point. The high level controller will be implemented in C++ language in a companion computer (Odroid XU4) embedded in DJI F450 quadrotors. The static and dynamic performances of the formation control will be evaluated for different shapes (lines, traingle, diamond,...) and trajectories (straight lines, circles,...), and the impact of communication losses will be analyzed.

Figure 1 .

 1 Figure 1. Problem statement

(

  ∈ [-1. . +1]) and ( ∈ [-1. . +1]) are defined as follows:

-

  Altitude (after the take-off phase): 10m -Omnidirectional camera position: Loc = 0.15m -Attenuation parameter : Ay=0.3 -Maximum velocities: , = 3 / -Maximum distances: , = 15 DES algorithm: αd,θ = 0.5, λd,θ = 0.5, m = 10 -Time constants (first order model): τx = 0.2s, τy = 0.2s -Take-off conditions (m, deg) / linear-circle trajectories : -Leader (XL, YL, δL) : [-82,-82,0°] -[-95,0,90°] -Follower 1 (X1, Y1, δ1) : [-90,-82,0°] / [-95,-25,90°] -Follower 2 (X2, Y2, δ2) : [-90,-74,0°] / [-110,-15,90°] -Follower 3 (X3, Y3, δ3) : [-90,-90,0°] / [-80,-15,90°] In order to study the LF formation and the global aspect of the VRB, linear and circle trajectories (radius of the circle: 90m -leader velocity according to x and y axes: 1m/s) have been chosen. The VRB requirements are the following ones: -Circle trajectory (diamond): -XSP1 = +20m -YSP1 = 0m -ZSP1 = 0m -XSP2 = +10m -YSP2 = -10m -ZSP2 = 0m -XSP3 = +10m -YSP3 = +10m -ZSP3 = 0m -UAVs yaw angle: δ1,2,3 = 90° -Linear trajectory (line): -XSP1 = +10m -YSP1 = 0m -ZSP1 = 0m -XSP2 = +10m -YSP2 = -10m -ZSP2 = 0m -XSP3 = +10m -YSP3 = +10m -ZSP3 = 0m -UAVs yaw angle: δ1,2,3 = 0°

Figure 2 .

 2 Figure 2. Circle trajectory

Figure 4 .Figure 5 .Figure 6 .Figure 7 .

 4567 Figure 4. Follower 1 (linear trajectory) x uav tracking error (e1x) -model control input (U1mx) -x model error (e1mx)

ACKNOWLEDGMENT

The authors would like to thank Le Havre Town Council (LHSM) for their support under research grants.


Laura Muraine

Mona Bensalah

Jamila Dhiab

Gonzalo Cordova

Ludovic Arandel

Alix Marhic

Maud Chapart

Stéphane Vasseur

Sofia Benkhelifa-Ziyyat

Anne Bigot

Gillian Butler-Browne

Vincent Mouly

Elisa Negroni

Capucine Trollet
email: capucine.trollet@upmc.fr

Transduction efficiency of AAV serotypes after local injection in mouse and human skeletal muscle

Adeno-associated viral vector (AAV) is an efficient tool for gene delivery in skeletal muscle.

AAV-based therapies show promising results for the treatment of various genetic disorders, including muscular dystrophies. These dystrophies represent an heterogeneous group of diseases affecting muscles and typically characterized by progressive skeletal muscle wasting and weakness and the development of fibrosis. The tropism of each AAV serotype has been extensively studied using systemic delivery routes, but very few studies have compared their transduction efficiency via direct intramuscular injection. Yet, in some muscular dystrophies, where only a few muscles are primarily affected, a local intramuscular injection to target these muscles would be the most appropriate route. A comprehensive comparison between different rAAV serotypes is therefore needed. Here, we investigated the transduction efficiency of rAAV serotypes 1 to 10 by local injection in skeletal muscle of control C57BL/6 mice. We used a CMV-nls-LacZ reporter cassette allowing nuclear expression of LacZ to easily localize targeted cells. Detection of beta-galactosidase activity on muscle cryo-sections demonstrated that rAAV1, 7, 8, 9 and 10 were more efficient than the others with rAAV9 being the most efficient in mouse. Furthermore, using a model of human muscle xenograft in immunodeficient mice, we observed that in human muscle rAAV8 and rAAV9 had similar transduction efficiency. These findings demonstrate for the first time that human muscle xenograft can be used to evaluate AAV-based therapeutical approaches in a human context.

Introduction

Adeno-associated virus (AAV) recombinant vectors (rAAV) are tools of choice for a wide range of gene therapy applications in humans, due to their good safety profile in clinical trials including an ability to transduce both dividing and non-dividing cells, to elicit a limited cytotoxicity [START_REF] Colella | Emerging Issues in AAV-Mediated In Vivo Gene Therapy[END_REF] and to allow long-term transgene expression. [START_REF] Wang | Adeno-associated virus vector as a platform for gene therapy delivery[END_REF] . Many serotypes of AAV from human and primate source have been identified, including AAV1 3 , AAV2 [START_REF] Srivastava | Nucleotide sequence and organization of the adeno-associated virus 2 genome[END_REF] , AAV3 [START_REF] Muramatsu | Nucleotide Sequencing and Generation of an Infectious Clone of Adeno-Associated Virus 3[END_REF] , AAV4 [START_REF] Chiorini | Cloning of adeno-associated virus type 4 (AAV4) and generation of recombinant AAV4 particles[END_REF] , AAV5 [START_REF] Chiorini | Cloning and characterization of adeno-associated virus type 5[END_REF] , AAV6 [START_REF] Rutledge | Infectious clones and vectors derived from adeno-associated virus (AAV) serotypes other than AAV type 2[END_REF] , AAV7 [START_REF] Gao | Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy[END_REF] , AAV8 [START_REF] Gao | Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy[END_REF] , AAV9 [START_REF] Gao | Clades of Adeno-associated viruses are widely disseminated in human tissues[END_REF] , AAV10 [START_REF] Mori | Two novel adeno-associated viruses from cynomolgus monkey: pseudotyping characterization of capsid protein[END_REF] , each of them with a specific transduction and tropism profile. Recombinant AAV (rAAV) have been shown to efficiently infect many tissues including liver [START_REF] Grimm | Preclinical in vivo evaluation of pseudotyped adeno-associated virus vectors for liver gene therapy[END_REF][START_REF] Paulk | Bioengineered AAV Capsids with Combined High Human Liver Transduction In Vivo and Unique Humoral Seroreactivity[END_REF] ; lung [START_REF] Halbert | Adeno-associated virus type 6 (AAV6) vectors mediate efficient transduction of airway epithelial cells in mouse lungs compared to that of AAV2 vectors[END_REF] , eye [START_REF] Auricchio | The Future Looks Brighter After 25 Years of Retinal Gene Therapy[END_REF] and skeletal muscle [START_REF] Harper | Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy[END_REF][START_REF] Greelish | Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adenoassociated viral vector[END_REF] , their tissue transduction targeting being dependent both on the capsid properties [START_REF] Ellis | A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype[END_REF] and the route of administration [START_REF] Wang | Adeno-associated virus vector as a platform for gene therapy delivery[END_REF] .

Skeletal muscles constitute 40-50% of the body mass in adult humans, and encompasses a broad range of different muscles. Each of these muscles is composed of contractile muscle fibres, post-mitotic multinucleated cells, surrounded by a specialized plasma membrane -the sarcolemma -and by a layer of extracellular matrix known as the basement membrane.

Tropism evaluation of rAAV serotypes in muscle has mainly been studied by systemic administration: rAAV1, 6, 7, 8 and 9 showed efficient transduction of skeletal muscle in most animal models [START_REF] Gregorevic | Systemic delivery of genes to striated muscles using adeno-associated viral vectors[END_REF][START_REF] Wang | Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart[END_REF][START_REF] Zincarelli | Analysis of AAV Serotypes 1-9 Mediated Gene Expression and Tropism in Mice After Systemic Injection[END_REF] . But to our knowledge, no study has provided a comprehensive side-byside comparison of the transduction efficiency and pattern of serotypes 1 to 10 after a direct in vivo local intramuscular injection, a route of delivery needed in some cases of muscular dystrophies where discrete muscles need to be targeted.

Muscular dystrophies are a heterogeneous group of genetic disorders characterized by progressive muscle wasting and weakness. These diseases are often associated with an excessive accumulation of extracellular matrix leading to muscle fibrosis. This is for example the case in Duchenne muscular dystrophy (DMD), where mutations in the dystrophin gene lead to cycles of degeneration/regeneration and the eventual development of fatty infiltration and fibrosis. This is also the case for a much rarer disease called oculopharyngeal muscular dystrophy (OPMD) where an abnormal triplet expansion in the PABPN1 gene [START_REF] Brais | Short GCG expansions in the PABP2 gene cause oculopharyngeal muscular dystrophy[END_REF] leads primarily to pharyngeal and eyelid muscle weakness and exacerbated fibrosis in these affected muscles [START_REF] Gidaro | Atrophy, Fibrosis, and Increased PAX7-Positive Cells in Pharyngeal Muscles of Oculopharyngeal Muscular Dystrophy Patients[END_REF] . Regarding OPMD, promising preclinical AAV-based gene replacement therapy data have been obtained via intramuscular injection in an OPMD mouse model [START_REF] Malerba | PABPN1 gene therapy for oculopharyngeal muscular dystrophy[END_REF] and the AAV gene-replacement product has now received orphan drug designation from EAM and FDA. Whereas a whole-body transduction efficiency has to be reached for DMD -three AAV trials for DMD are on going in the US based on the delivery of various engineered microdystrophins via systemic delivery using rAAV9 and rAAVrh74 25 (NCT03368742 Solid Biosciences; NCT03375164 Nationwide Children's Hospital; NCT03362502 Pfizer) 2 in OPMD patients, a local intramuscular injection would probably be preferred since a restricted number of muscles are primarily affected. Fibrotic muscular substitution is a complex process characterized by an excessive accumulation of collagens and other components of ECM [START_REF] Serrano | Regulation and dysregulation of fibrosis in skeletal muscle[END_REF] surrounding muscle fibers and fibroblasts that have an increasingly appreciated role as an autocrine source of profibrotic stimuli [START_REF] Wynn | Cellular and molecular mechanisms of fibrosis[END_REF] . The capacity to target fibrotic interstitial cells with rAAV has never been evaluated.

In this study, we evaluated the transduction efficiency of rAAV serotypes following intramuscular injection. Furthermore, we investigated the transduction translatability into human skeletal muscle, using a model of human muscle xenograft into immunodeficient mice allowing a side-by-side comparison in mouse and human context, an essential step for preclinical therapeutic studies.

Material and Methods

AAV production

AAV pseudotyped vectors containing a CMV-nlsLacZ expression cassette were produced using a three plasmid transfection in HEK-293 cells as described previously [START_REF] Grieger | Production and characterization of adenoassociated viral vectors[END_REF] using the pAAV-CMV-LacZ plasmids with the pXX6 plasmid coding for the Ad helper genes essential for AAV production and the pRepCap plasmid coding for AAV capsids. Vector particles were purified on an iodixanol gradient and concentrated on Amicon Ultra-15 100 K columns (Merck-Millipore). The titer corresponding to the number of viral genomes per milliliter (vg/ml) was determined by quantitative real-time PCR on a StepOnePlus (Applied Biosystems), by using the following primers and probe:

CTCCATCACTAGGGGTTCCTTG (forward), GTAGATAAGTAGCATGGC (reverse)

and TAGTTAATGATTAACCC (Taqman MGB probe, Life Technologies) amplifying a region between the ITR and the promoter. The pAAV plasmid was used as a control to establish the standard curve for absolute quantification.

In vivo injection

3 month old male C57BL/6J (Janvier Labs) mice were injected in the tibialis interior (TA)

with the different AAV serotypes (containing 3.5x10 10 vg per injection) diluted in 25μl PBS using a Hamilton syringe. Injections were carried out under general anesthesia using xylasine (80 mg/kg) /ketamine (10 mg/kg). Animal studies conform to the French laws and regulations concerning the use of animals for research and were approved by an external Ethical committee (approval no.16650-2018090514008520 delivered by the French Ministry of Higher Education and Scientific Research).

Muscle analysis

Four weeks post-injection mice were sacrificed by cervical dislocation; TA muscles were collected and snap-frozen in liquid nitrogen-cooled isopentane. Transverse 5μm sections were made with a cryostat (Leica CM1850) throughout the whole muscle with 500 μm between each section (supp figure 1A). Additional tissue between the sections was collected for DNA and/or protein extraction, ensuring representation of the whole muscle during data collection.

Xenografts

Three to four month old male or female immunodeficient Rag2-/-Il2rb-/-mice were used ) after all subjects had signed informed consent. The strip of human muscle was placed in the empty anterior compartment and tied, at both ends, with non-absorbable suture (Ethicon) to the tendons of the peroneus longus. Skin was sutured with absorbable suture (Ethicon). Buprenorphine (0.1 mg/kg) was given subcutaneously at the beginning of the surgery and each 12 hours (h) for the first 48h after surgery, for pain control. 4 months after surgery, when the human muscle was fully regenerated, the grafts were injected with the different AAV serotypes as described previously.

β-galactosidase histochemistry and histological analysis

Muscle sections were fixed with 0,25% glutaraldehyde for 5 minutes and stained for several hours with a mixture of 500 µg/mL X-Gal (5-bromo-4-chloro-3-indolyl-β-Dgalactopyranoside), 5 mM potassium hexacyanoferrate(III), 5mM potassium hexacyanoferrate(II) trihydrate, and 2 mM magnesium chloride in PBS at 37°C. Laminin staining was performed by incubating the sections in H2O2 3% for 5 min, then blocking in FBS 2% for 30 min and incubating with anti-laminin antibody (1:400 diluted in PBS, Z0097, Dako, Agilent Technologies France, Les Ulis, France) and the biotinylated secondary antibody (1:200 diluted in PBS, E0432, Dako). Laminin staining was revealed with Diaminobenzidine with 0,01% H2O2. For the assessment of tissue morphology and visualization of fibrosis and connective tissue, muscle sections were stained with hematoxylin and eosin and sirius red for light microscopic examination. Pictures were taken with a slide scanner (Hamamatsu). For each muscle, we selected the largest cross section area among all the muscles sections of a TA muscle and on this section we counted the total number of β-gal-positive nuclei. The relative transduction efficiency was calculated as βgal positive nuclei/mm2/gc, with the genome copy (gc) number calculated as per μg DNA (see below).

Western-blot analysis

Immunofluorescence staining

Muscle sections were blocked with 2% FBS in 1xPBS for 30 min. The sections were incubated with primary antibodies against human-specific spectrin

Vector genome quantification

Transduction efficacy was evaluated by quantification of viral genomes in injected muscles.

Genomic DNA was extracted from mouse muscles using Puregene Blood kit (Qiagen, To specifically amplify the viral genome sequence, we used the same primers as the one for AAV titration (above). A control pAAV plasmid was 10-fold serially diluted (from 10 7 to 10 1 copies) and used as a control to establish the standard curve for absolute quantification.

All genomic DNA samples were analyzed in duplicates.

Statistical analysis

All data are mean values ± standard error of the mean. Statistical analyses were performed using the Student t-test or the one-way ANOVA with the Bonferroni post-hoc analysis as indicated. GraphPad Prism (version 4.0b; GraphPad Software, San Diego California, USA)

was used for the analyses. A difference was considered to be significant at *P < 0.05, **P< 0.01 or ***P < 0.001.

Results

Transduction efficiency in mice

We first investigated the transduction efficiency of single stranded rAAV serotypes by local injection in skeletal muscle of control C57BL/6 mice. We used a CMV-nls-LacZ reporter cassette to precisely localize transduced nuclei. 10 different serotypes of rAAV-CMV-nls-LacZ (1, 2, 3, 4, 5, 6, 7, 8, 9 and 10) each at 3.5x10 [START_REF] Gao | Clades of Adeno-associated viruses are widely disseminated in human tissues[END_REF] vector genomes (vg) were injected in the tibialis anterior (TA) of control C57BL/6 mice. Four weeks after injection, muscles were harvested and frozen for histological and biochemical analysis (figure 1A). For each transduced TA, reporter nls-LacZ expression was quantified on the largest cross section area of the muscle by manual counting of all lacZ-positive nuclei (figure 1B and supp figure 1A-B) and by Western Blot (supp figure 2A). The number of vector genomes was quantified by quantitative PCR (qPCR) (supp figure 2B). Four weeks post-injection, serotype 9 showed the most robust transduction (figure 1C). All serotypes, except rAAV6, showed a limited inflammatory response. It should be noted that all of the rAAV6-injected TA's presented an intense inflammatory response with large areas of regenerating myofibers (supp figure 2C), thus maybe impairing the quantification of transduction efficiency. This was observed in independent experiments.

Transduction efficiency in human muscle using xenograft model

In order to evaluate intramuscular AAV transduction in a human context, we used a human muscle xenograft model: we transplanted fresh human skeletal muscle into the empty tibial compartment of Rag2-/-Il2rb-/-immunodeficient mice. Four months post-transplant, the engrafted human muscle regenerated as previously described [START_REF] Zhang | Human skeletal muscle xenograft as a new preclinical model for muscle disorders[END_REF] . Xenografts were composed entirely of human myofibers (expressing spectrin) (figure 2B) with comparable fiber type composition compared to the original donor biopsy (Supp figure 3A-B). rAAV 8 and 9

vectors expressing CMV-nls-LacZ were injected into these xenografts, and mice were analyzed 4 weeks later (figure 2A). These AAVs were selected as they showed a good efficiency in mouse and are currently being tested in several clinical trials for muscular dystrophy in patients. The position of the human xenografts was confirmed by immunofluorescence staining using anti-human spectrin and anti-human lamin A/C antibodies to stain specifically human muscle fiber membranes and human nuclei, respectively (figure 2B and Supp figure 3C). In human muscle xenografts, rAAV8 and rAAV9 gave comparable transduction efficiency (figure 2C-D) and transduced both slow and fast fibers (figure 2E).

When compared to mouse, we observed a similar level of transduction for both rAAV serotypes (figure 2F) even though there is significant muscle fibrosis in the human xenograft (figure 2G). Interestingly, we never observed LacZ expression in the human interstitial cells surrounding the muscle fibers (figure 2H).

Discussion

Achieving efficient transduction of rAAV in diseased skeletal muscle, notably in the presence of fibrosis, is a major challenge for the treatment of muscular dystrophies. For many dystrophies a systemic administration of the vector is required, however for some more localized diseases a local intramuscular rAAV delivery might be sufficient. Whereas the comparison of serotypes has been largely described via systemic delivery, to our knowledge very few studies have compared their efficiency after a local intramuscular injection. In addition, while it is known that the potency of rAAV gene delivery can be impacted by several host factors, such as the molecular interactions between the capsid and target cell surface receptors [START_REF] Huang | Parvovirus glycan interactions[END_REF] or the interaction with serum proteins in the circulation [START_REF] Denard | AAV-8 and AAV-9 Vectors Cooperate with Serum Proteins Differently Than AAV-1 and AAV-6[END_REF] , the impact of fibrosis by extracellular matrix deposition around muscle fibers on rAAV transduction efficiency and the transduction of interstitial fibrosis cells has not been evaluated.

In this study we therefore tested the efficiency of rAAV1 to 10 after direct intramuscular injection into the TA of mice and human xenografted muscle.

When comparing the rAAVs in mouse we found that rAAV9 was the most efficient vector.

Serotypes 1, 7 and 8 were also efficient although less so following intramuscular injection.

rAAV1 gave high number of vector copy genome not correlated with high level of expression suggesting impaired disassembly in the nucleus as previously shown in Hela cells [START_REF] Keiser | Unique Characteristics of AAV1, 2, and 5 Viral Entry, Intracellular Trafficking, and Nuclear Import Define Transduction Efficiency in HeLa Cells[END_REF] . In the human xenograft model, rAAV8 and rAAV9 were found to be equally efficient at transducing both slow and fast human muscle fibers. It should be noted that rAAV9 and rAAV8 are already used in several ongoing clinical trials using systemic delivery in different myopathies:

a Phase I/II trial with Solid Biosciences (NCT03368742) and one with Pfizer (NCT03362502)

for DMD as well as a trial for Spinal muscular atrophy type 1 (NCT02122952) and a trial for X-linked Myotubular Myopathy (NCT03199469).

In our study, rAAV6-injected muscles systematically presented an acute inflammatory response with the degeneration and regeneration of the majority of the muscle fibers. Such a phenotype has been described previously for other transgenes [START_REF] Riaz | Differential myofiber-type transduction preference of adeno-associated virus serotypes 6 and 9[END_REF][START_REF] Wang | Immunity and AAV-Mediated Gene Therapy for Muscular Dystrophies in Large Animal Models and Human Trials[END_REF][START_REF] Arnett | Immune Responses to rAAV6: The Influence of Canine Parvovirus Vaccination and Neonatal Administration of Viral Vector[END_REF] even though rAAV6 has also been used to transduce skeletal muscle [START_REF] Arnett | Adeno-associated viral vectors do not efficiently target muscle satellite cells[END_REF][START_REF] Qiao | Adeno-Associated Virus Serotype 6 Capsid Tyrosine-to-Phenylalanine Mutations Improve Gene Transfer to Skeletal Muscle[END_REF][START_REF] Winbanks | Transduction of Skeletal Muscles with Common Reporter Genes Can Promote Muscle Fiber Degeneration and Inflammation[END_REF] . As suggested by Winbanks et al, discrepancies in rAAV6 transduction and related inflammatory response could be due either to the nature of the transgene or to the dose used [START_REF] Winbanks | Transduction of Skeletal Muscles with Common Reporter Genes Can Promote Muscle Fiber Degeneration and Inflammation[END_REF] . Regarding the nature of the transgene, the absence of an inflammatory response with all of the other rAAV serotypes used in our study suggests that the lacZ reporter gene is not itself deleterious. Regarding the dose, the question remains as to whether a lower dose of AAV6 would have allowed a better level of expression without any related inflammatory response. We can only conclude here that at this same dose rAAV6 induces an inflammatory response whereas other serotypes do not, and this is not related to the quality of the rAAV6 production (data not shown).

AAV use glycan receptors and co-receptors, which play a role in cell attachment, and these interactions have been well characterized for a number of serotypes 30 : α2-3 and α2-6 Nlinked sialic acid for AAV1 ; heparan sulfate proteoglycan for AAV 2 and 3 ; α2-3 O-linked and α2-3 N-linked sialic acids for AAV 4 and 5 ; heparan sulfate and α2-3 and α2-6 N-linked sialic acid for AAV6 ; and N-linked galactose for AAV9. A "new" cellular AAV receptor (AAVR, also named KIAA0319L) has also recently been described as essential for AAV cell entry following attachment [START_REF] Pillay | Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor[END_REF][START_REF] Pillay | An essential receptor for adeno-associated virus infection[END_REF] . In our study, none of the vectors were able to induce the expression of LacZ in interstitial cells (fibroblasts and inflammatory cells) (figure 2H) or satellite cells (supp figure 4), as previously described for rAAV6 [START_REF] Arnett | Adeno-associated viral vectors do not efficiently target muscle satellite cells[END_REF] and rAAV9 [START_REF] Stitelman | Developmental stage determines efficiency of gene transfer to muscle satellite cells by in utero delivery of adenoassociated virus vector serotype 2/9[END_REF][START_REF] Nance | AAV9 edits muscle stem cells in normal and dystrophic adult mice[END_REF] . We cannot exclude that these cells are transduced but unable to express the transgene, maybe due to an uncoating issue. Otherwise, this could be due to differences in cell surface receptors or mitotic status of myogenic and non-myogenic cells respectively. Further studies should include the analysis of the expression of these receptors on non myogenic/fibrotic cells compared to myogenic cells. Although we do not see any satellite cells targeted in our study, other have shown an efficient AAV transduction after systemic delivery with a CMV promoter [START_REF] Goldstein | In Situ Modification of Tissue Stem and Progenitor Cell Genomes[END_REF] . An important feature of our study is the observation that the fibrosis present in the muscle xenograft does not impair rAAV8 and rAAV9 transduction of muscle fibers which is promising for future gene therapy strategies. Interestingly, rAAV transduction does not allow expression in interstitial muscle cells and further studies should look at other vectors which could target these non myogenic cells with a potential anti-fibrotic gene therapy purpose. Elucidating tropism and transduction efficiency is critical to implement targeted and specific applications in a dynamic environment such as pathological muscle.

It is always a challenge to translate results obtained in mice into larger species. For example different profiles have been observed between transduction efficiency in mice and dogs [START_REF] Pan | AAV-8 is more efficient than AAV-9 in transducing neonatal dog heart[END_REF] . We used for the first time a muscle xenograft model to evaluate AAV transduction efficiency in a human context. We did not observe any difference between rAAV8 and rAAV9 confirming their efficient transduction profile in human muscle. Interestingly, while different transduction efficiency have been observed on different fiber types in mice [START_REF] Riaz | Differential myofiber-type transduction preference of adeno-associated virus serotypes 6 and 9[END_REF][START_REF] Bostick | Systemic AAV-9 transduction in mice is influenced by animal age but not by the route of administration[END_REF] we observed transduction of both slow and fast fiber type in the xenograft model. While bearing in mind that the human xenograft corresponds to a regenerated human muscle, we believe that this xenograft model is an ideal tool for pre-clinical studies to translate results found in mice into an in vivo human context and to further validate therapeutic strategies. The presence of a high degree of fibrosis in these xenografts could be a very useful model to evaluate therapeutic gene-based strategies in a fibrotic environment, similar to that observed in most muscular dystrophies.

  as recipients for human xenografts. Control donor human tensor Fascia Lata muscle was cut into strips (8x3x1mm). Mice were anaesthetized with an intraperitoneal injection of ketamine hydrochloride (80 mg/kg) and xylazine (10 mg/kg) (Sigma-Aldrich, St Louis, MO) in accordance with the French and European Community legislation (approval no. 4165-2016021715164682 delivered by the French Ministry of Higher Education and Scientific Research). The TA and extensor digitorum longus (EDL) were removed. Human muscle samples were obtained as surgical waste during routine surgical procedures from the MYOBANK, a tissue bank affiliated to EuroBiobank (authorization from the French Ministry ref 2013-1968

( 1 :

 1 50 mouse monoclonalIgG2b NCL-spec1, Novocastra, Leica Biosystems, Wetzlar Germany) human-specific lamin A/C (1:300 mouse monoclonal IgG1 Ab40567, Abcam, Cambridge, UK), or laminin (1:400 diluted, Z0097, Dako) for 1 hour and further incubated with appropriate secondary antibodies for 1 hour. When necessary, βgal detection was performed using a chicken antibody (Invitrogen AB9361; 1:1000; 1 hour). Pax7 staining was performed using a mouse monoclonal antibody (Developmental Studies Hybridoma Bank, Iowa City, IA; 1:20) after a 10 min fixation with 4% paraformaldehyde. Fiber type composition was obtained using an anti-MYHC-I antibody (A4.840, 1:50; DSHB) and an anti-MYHC-II (A4.74, 1:50; DSHB).Macrophages and neutrophils were stained with F4/80 antibody (MCA497BB, 1:50, Biorad) and Ly6g antibody (Ab25377, 1:100, Abcam) respectively, after a 10 min fixation with 4% paraformaldehyde. Finally, the sections were incubated 10 min with Hoechst (0.5μg/ml, Hoechst No. 33258; Sigma-Aldrich) and mounted with a mounting medium (Cytomation fluorescent mounting Medium, S3023, Dako, Agilent).

  Courtaboeuf, France) according to the manufacturer's protocol. Copy numbers of AAV genome and genomic DNA were measured on 100ng of genomic DNA by absolute quantitative real-time PCR on a StepOnePlus TM (Applied Biosystems) using Taqman probe and the Taqman R Universal Master Mix (Applied Biosystems)

Fig. 1 .Fig. 2 .Fig. 1 .

 121 Fig. 1. Intramuscular AAV transduction efficiency in control C57BL/6 mice. rAAV serotypes 1 to 10 were injected into the TA (3.5x10 10 vg/TA) . Mice were sacrificed 4 weeks post-injection and TA were analyzed (N=4 to 10 TA) A. Cryostat sections of TA muscles of C57BL/6 mice injected with rAAV1 to 10 carrying a nls-LacZ gene reporter and stained for β-galactosidase activity and laminin. (scale bar: 250μm) B. Quantification of cross sectional β-galactosidase positive nuclei by mm 2 (N=4 to 10 TA) C. Transduction efficiency is expressed as the β-gal positive nuclei/mm 2 relative to the AAV genome copy (gc) number quantified by absolute Taqman qPCR. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 ANOVA Tukey test)

  Proteins were extracted from muscle sections by sonicating the cells in RIPA buffer (0.15 M

	NaCl, 0.1% SDS, 50 mM Tris-HCl (pH 8), 2 mM EDTA and 10% Triton-X-100 with
	protease inhibitor cocktail (Complete, Roche Diagnostics) and phosphatase inhibitor cocktail
	(Santa Cruz; sc-45064). Protein concentration was determined by colorimetric detection
	method (Pierce BCA Protein Assay; Thermo Fisher). Proteins were separated on 4-12% Bis-
	Tris gels (Invitrogen) and transferred onto a PVDF membrane for 1 h at constant 250 mA at
	4°C. Membranes were blocked by incubation in 5% BSA in 1X TBS, 0.1% Tween-20 (TBST)
	for 1h at room temperature under agitation. Membranes were stained with anti-beta
	galactosidase antibody (Thermofisher A-11132 ; 1 :1000 1 hour) and tubulin antibody (Sigma
	T5168 1 :1000 1 hour). Membranes were washed in TBST and incubated with appropriate
	secondary antibodies. The G:Box system (Syngene) was used to detect the signals from the
	membranes.

Acknowledgments

We thank Françoise Balter, Véronique Blouin and the vector core CPV at UMR 1089 (Nantes, France) for AAV production. We also thank the Penn Vector Core (Gene Therapy Program, University of Pennsylvania, Philadelphia) for providing the pAAV-1 (p0001), pAAV2 (p5E18), pAAV-5 (p0050-R), pAAV-6 (P0004-R), pAAV-7 (p5E18-VD27), pAAV-8 (p0007-R), pAAV-9 (p5E18-VD29), pAAV-rh10 (P0009-8) plasmids and Laura Julien from the MyoVector platform of the Centre of Research in Myology-UMRS974 (Paris, France) for AAV productions. We thank Cécile Peccate and Alison Oliver for technical assistance. We thank the MYOBANK-AFM of the Institut de Myologie (BB-0033-00012) for the access to human muscle samples. The Pax7, MYHC-I (A4.840) and MYHC-II (A4.74) antibodies were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. We acknowledge funding from the Association Française contre les Myopathies (AFM Telethon), Inserm, Sorbonne Université, Fondation pour la Recherche Médicale (FRM) and the Fondation Maladie Rare (AP-RM-16-035).

Author disclosure statement

No competing financial interests.