Alban Jacquemart

Gender Quotas in the French Bureaucratic Elite The Soft Power of Restricted Coercion

Keywords:

Drawing on a collective study of gender and executive careers in the French Civil Service, this article provides an account of the genesis and implementation of the gender quota introduced by the 2012 "Sauvadet law" in senior executive positions of the State bureaucracy. Firstly, it shows the crucial, yet unobtrusive, role played by feminist actors in the process, in spite of the absence of any strong open politicization of the issue, from agenda setting to implementation. Secondly, the article points to the dual character of the reform. On the one hand, the gradual character and limited perimeter of the quota (to senior executive 'initial appointments') restricted its immediate quantitative impact. On the other hand, the constraining, technical dimension of the tool endowed it with a potential to foster incremental social change.

On March 12th, 2012 1 , with almost no media coverage and without prompting any parliamentary and public controversy, the "Sauvadet law" (named after François Sauvadet, minister for Civil Service at the time), introduced a series of mandatory gender quotas in decision-making positions of the French public administration, notably in appointments to senior executive positions 2 . This last provision (named here "the Sauvadet quota") imposed to all public administrations a gradual gender quota (up to an expected 40% in 2018) in the first appointments of senior executive positions in the three branches of the French Civil Service.

In the National-level Civil Service (Fonction publique d'Etat) 3 , which is at the center of our study, the gender quota provision applies to an average of 600 appointments a year, out of 3600 senior executive positions. At the time the provision was adopted (2012), women made up half of the National-level Civil Service workforce and accounted for less than 26% of senior executive positions.

The absence of visible controversies and struggles over the Sauvadet quota is not entirely surprising. The provision expanded to a new social field a gender policy tool that had gained increased legitimacy since the early 2000s [START_REF] Bender | Les quotas de femmes dans les conseils d'administration[END_REF]Bereni and Revillard, 2015;[START_REF] Bui-Xuan | L'égalité professionnelle entre hommes et femmes dans la fonction publique : une révolution manquée ?[END_REF][START_REF] Lépinard | From breaking the rule to making the rules: the adoption, entrenchment, and diffusion of gender quotas in France[END_REF]. A year earlier, the "Copé-Zimmermann" statute imposed incremental gender quotas in private sector corporate boards. Advocates of the Sauvadet quota presented the provision as a means to align the public sector on the private sector, against the backdrop of an ongoing bureaucratic reform under the auspices of new public management [START_REF] Bezes | New public management and professionals in the public sector. What new patterns beyond opposition?[END_REF].

Yet, on the other hand, the Sauvadet quota appeared as "a small revolution" ("une petite révolution"), as Sauvadet himself presented it at the Assemblée nationale at the time 4 . Indeed, the degree of constraint imposed by the quota represented a "turning point" in gender-equality policies in the public sector [START_REF] Bui-Xuan | L'égalité professionnelle entre hommes et femmes dans la fonction publique : une révolution manquée ?[END_REF][START_REF] Edel | Les instruments juridiques de l'égal accès des femmes et des hommes aux emplois publics : depuis le droit à l'égalité jusqu'aux politiques d'égalité[END_REF]. It introduced a breach into the ideology of meritocracy and equal treatment particularly associated with the French Civil Service, whose recruitment and promotion principles are deemed strictly neutral and bounded by statutory rules 5 . Moreover, whereas the quota put in place by the 2011 Copé-Zimmerman law only targeted corporate boards, leaving the more strategic executive ("Comex") and directory boards ("Codir") untouched, the Sauvadet quota targeted the highest positions of power (3 600 senior executive positions) in the national-level Civil Service. Because of this unprecedented constraining dimension, it faced strong -yet covered -resistances among most administrative elites and government members at the time, behind the public display of consensus. These resistances could only be overcome by the personal arbitration of President Sarkozy, according to Sauvadet's recent public account of the reform.

How was such a constraining policy tool adopted? To what extent have feminist actors and framings influenced its adoption and shaped its implementation? Did it favor gender transformation, in terms of both the access of women to the highest positions, and gender representations [START_REF] Engeli | Taking implementation seriously in assessing success: the politics of gender equality policy[END_REF]? Is it a coercive policy tool producing substantive effects, or a symbolic measure with only limited influence on state organizations' daily functioning [START_REF] Edelman | Working Law: Courts, Corporations, and Symbolic Civil Rights[END_REF][START_REF] Mazur | Gender Bias and the State: Symbolic Reform at Work in Fifth Republic France[END_REF]?

This article provides an account of the genesis and implementation of the Sauvadet quota, drawing on a collective research on gender and executive careers in the French public bureaucracy [START_REF] Marry | Le plafond de verre et l'État -La construction des inégalités de genre dans la fonction publique[END_REF][START_REF] Revillard | Gender, Class and Bureaucratic Power: the Production of Inequalities in the French Civil Service[END_REF]. We conducted a series of 100 in-depth interviews (one third with men and two third with women) with executives and senior executives working in four administrative departments pertaining to two ministries (ministry of Economic and Financial Affairs, ministry of Social Affairs), between 2011 and 2013.

While our research focus was mainly the mechanisms behind the production of the "glassceiling", we also granted attention to the reception of the Sauvadet quota by high civil servants and to its influence on their career perspectives (Bereni and Revillard, 2015). These data were complemented by a set of materials collected since the introduction of the provision (2012)(2013)(2014)(2015)(2016)(2017)(2018)(2019), documenting its genesis and its implementation: general and professional press articles, administrative reports, parliamentary debates, documentation issued by female high civil servants organizations. This research reveals the crucial, yet unobtrusive role played by feminist actors in the process, in spite of the absence of any strong open politicization of the issue, from agenda setting to implementation. In the terms of the Gender Equality Policy in Practice (GEPP) project (see Engeli and Mazur, this issue), this case reveals high substantive and moderate descriptive women's empowerment: (upper-class) women's interests have been taken into account, although women openly acting as women's rights advocates have become less visible. We draw on an analytical framework that envisions feminist mobilization in a broad perspective, including non-governmental as well as governmental actors [START_REF] Banaszak | The Women's Movement Inside and Outside the State[END_REF][START_REF] Revillard | Gender, Class and Bureaucratic Power: the Production of Inequalities in the French Civil Service[END_REF][START_REF] Katzenstein | Faithul and Fearless. Moving Feminist Protest Inside the Church and Military[END_REF]. Both the legitimization of the quota and its rigorous implementation can largely be explained by structural transformations of the 'women's cause field', defined as the relational structure of groups mostly devoted to the advancement of women in a variety of social settings, cutting across the line between civil society and political institutions (Bereni, 2019). The transformations at stake followed the adoption of the parity law in 2000, and were threefold: 1) a diffusion of the "parity grammar" [START_REF] Bereni | Des quotas à la parité : "féminisme d'État" et représentation politique (1974-2007)[END_REF] in dominant institutions; 2) a strengthening of the institutional pole of the women's cause field (women's policy agencies), and 3) a burgeoning of women's mobilizations among the business, professional, and bureaucratic elite. We argue that these structural transformations combined with the opening of political and discursive opportunities produced the conditions of a possible consensus of legislators around this constraining measure, and the overcoming of resistances stemming from administrative elites. Two opportunities were particularly key: the rise of a managerial rhetoric praising "gender diversity" ("mixité") in the private sector, and the implementation of new public management reforms and "state modernization" in the public sector.

The paper is divided in six sections, tracing the reform from its genesis to its outcomes.

The first two sections deal with the pre-adoption context, examining the slow emergence of equal employment policy in the public administration and the rise of the quota as a policy tool throughout the 2000s. The third section focuses on the adoption of the reform in 2011-2012.

As demonstrated in section 4, this genesis helps explain the tool's dual character, both restricted and relatively constraining. We then turn to the implementation and evaluation of the quota in practice: even though the actors in charge of implementation and evaluation were mainstream bureaucratic actors, they pressed in favor of a reinforcement of the measure, in a broader context of increased feminist mobilization in France. Finally, the last section assesses the outcomes of the reform, showing that in spite of its limited immediate effect, the tool is likely to result in significant gender transformation within the bureaucratic elite, over the years, due to its incremental effects.

The slow construction of equal employment policy in the public administration Prior to 2000

The issue of women's access to senior executive positions in the Civil Service was not put on the political agenda before the early 2000s. During the 1970s and 1980s, gender equality policies in the public sector focused on equal employment principles, following a series of reforms in the private sector initiated by women's policy structures. The first equal employment public policies mainly aimed at ensuring formal equal rights for men and women, be it in the private sector or in the public administration. A combination of external pressure from the UN and the European commission, and internal lobbying on the part of women's policy structures, favored the gradual legal assertion of a principle of non-discrimination [START_REF] Mazur | Gender Bias and the State: Symbolic Reform at Work in Fifth Republic France[END_REF][START_REF] Edel | Les instruments juridiques de l'égal accès des femmes et des hommes aux emplois publics : depuis le droit à l'égalité jusqu'aux politiques d'égalité[END_REF][START_REF] Revillard | La cause des femmes dans l'Etat[END_REF]. As of the end of the 1960s, the Comité du Travail Féminin (a women's rights advisory board) raised the issue of gender discrimination in access to administrative exams ("concours de la Fonction publique"), and issued reports on gender inequalities in the public administration in 1974 and 1978 [START_REF] Revillard | L'expertise Critique, Force d'une Institution Faible ? Le Comité Du Travail Féminin et La Genèse d'une Politique d'égalité Professionnelle En France (1965-1983)[END_REF]. A 1975 law eliminated the possibility of gender distinctions in exams, except in limited cases listed by a Conseil d'Etat decree.

Governmental action in favor of equal employment gained more visibility with Yvette Roudy's appointment as minister for women's rights in 1981. At the beginning of the 1980s, women already accounted for half of public employees, but they were almost absent from the highest positions: in 1983 there weren't a single female prefect, and there were only 3% female ambassadors and 5% female heads of national level ministerial departments 6 . In 1983, general Civil Service regulations were modified to prohibit all distinction based on sex, following a law adopted on the same day as the more famous "Loi Roudy" on equal employment in the private sector. Beyond formal non-discrimination, attempts to promote more actively the advancement of women in public administration were very limited, and took the form of rather vague incentives, lagging behind the more structured equal employment policy put in place in the private sector by Roudy's equal employment law. For example, a 1983 executive memorandum 7 prompted ministerial departments to act to "tackle disparities" between women and men through various measures in terms of recruitment and promotion. Up until the 2000s, equal employment policy, at the heart of French state feminism [START_REF] Revillard | La cause des femmes dans l'Etat[END_REF], mainly targeted the private sector. Because of a dominant framing in terms of (formal) equal employment, the issue of women's access to senior executive positions in the public administration remained marginal, leading to no significant policy outcomes (Mazur, 2002). Only when it started being framed in terms of access to power, in line with the diffusion of the parity grammar throughout the 1990s, did a coercive measure start being envisioned.

From the gender parity reform to the routinization of gender quotas in decision-making sites (2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010) French political and intellectual elites opposed for a long-time any form of affirmative action, conceived as an infringement of the Republican ideals of meritocracy and of "indifference to differences" between citizens [START_REF] Scott | Parité: Sexual Difference and the Crisis of French Universalism[END_REF]). Yet the campaign for gender parity shattered this consensus at the end of the 1990s, progressively making gender quotas compatible with republican universalism [START_REF] Bereni | French Feminists Renegotiate Republican Universalism. The Gender Parity Campaign[END_REF]Lépinard, 2007). The controversy around gender quotas, which was initially strong even among feminist advocates, suddenly faded after the passing of the gender parity law in 2000 (see Mazur et al. in this issue). Increasingly justified by "pragmatic" reasons, quotas became the default solution to the "underrepresentation" of women in power positions (Bereni and Revillard, 2015;[START_REF] Lépinard | From breaking the rule to making the rules: the adoption, entrenchment, and diffusion of gender quotas in France[END_REF], while the term "quota" itself, still contentious, was replaced by more consensual phrasings, such as "balanced representation" (représentation équilibrée), "parity" (parité) or "gender diversity" (mixité). In 2008, a new provision was added into the first article of the Constitution, stating that "the law favors the equal access of women and men to elected offices and positions, as well as professional and social responsibilities". The new paragraph expanded beyond the political field a principle that had been initially introduced in 1999 into article 3 of the Constitution as part of the parity reform, and thus paved the way for the use of gender quotas in non-political decision-making sites. Those were first introduced in the private sector: the Copé-Zimmermann law (named after the two deputies who introduced the bill), passed in 2011, mandated a 40% quota of persons of each sex in corporate boards of the largest firms [START_REF] Bender | Les quotas de femmes dans les conseils d'administration[END_REF], before being extended to the first appointments of the senior executive positions in the public sector by the Sauvadet law (2012). The gender parity campaign contributed in three respects to the routinization of gender quotas in decisionmaking sites throughout the 2000s, through framing, institutional, and mobilization effects.

A "balanced representation" at the top: the diffusion of the parity grammar

The gender parity reform participated to the diffusion of a parity grammar [START_REF] Bereni | Des quotas à la parité : "féminisme d'État" et représentation politique (1974-2007)[END_REF], i.e. a new standard of gender equality based on the numerical co-presence of men and women in historically male-dominated sites. Against this discursive backdrop, gender equality policies that gained momentum in the bureaucracy during the 2000s particularly turned attention to women's access to the highest positions [START_REF] Edel | Les instruments juridiques de l'égal accès des femmes et des hommes aux emplois publics : depuis le droit à l'égalité jusqu'aux politiques d'égalité[END_REF][START_REF] Jacquemart | Femmes hautes fonctionnaires en France[END_REF].

In 1999, high civil servant Anne-Marie Colmou delivered a report to the Civil Service minister documenting the "glass-ceiling" (at the turn of the 2000s, women accounted for only 12% of executive level positions in public administration 8 ), and making the case for a "balanced representation of men and women" [START_REF] Colmou | L'Encadrement supérieur de la fonction publique : vers l'égalité entre les hommes et les femmes[END_REF]. Importing the "parity grammar" from the political to the administrative field, the report paved the way, with subsequent reports (e.g., [START_REF] Versini | La diversité dans la fonction publique[END_REF], for legitimizing the idea of "representative bureaucracy" [START_REF] Peters | Politics of Representative Bureaucracy: Power, Legitimacy and Performance[END_REF], historically unfamiliar in the French Civil Service (Bui-Xuan, 2015;[START_REF] Calvès | Refléter la diversité de la population française" : naissance et développement d'un objectif flou[END_REF][START_REF] Meier | Ethnic Conflict in France: A Case for Representative Bureaucracy?[END_REF]. The report issued a series of recommendations, including gender monitoring and ministerial plans of actions with quantified targets. A 2000 memorandum issued by the Prime Minister 9 prompted ministerial departments to adopt "multi-annual plans to improve women's access to executive positions", setting goals in terms of "feminization rates". The same year, a Steering committee for the equal access of women and men to senior management positions in the Civil Service was set up. The committee issued three reports developing a quantitative assessment of the glass ceiling as well as recommendations [START_REF] Le Pors | Piloter l'accès des femmes aux emplois supérieurs : premier rapport du comité de pilotage pour l'égal accès des femmes et des hommes aux emplois supérieurs des fonctions publiques[END_REF][START_REF] Le Pors | Promouvoir la logique paritaire: deuxième rapport du Comité de pilotage pour l'égal accès des femmes et des hommes aux emplois supérieurs des fonctions publiques[END_REF], 2005). A 2001 statute (the "Genisson" law) 10 provided that the government must deliver every two years to Parliament a report on men's and women's employment in the different branches of the Civil Service and at different hierarchical levels -this provision, however, was suppressed in 2007.

Therefore, throughout the 2000s, efforts to promote women in the higher ranks of the public bureaucracy rested mainly on soft, incentive tools, such as gender monitoring. In the report issued at the end of its mandate, in 2005, the Le Pors committee stressed the inefficiency of the legal provisions, the lack of political will, and the weakness of the ministerial plans that were adopted [START_REF] Le Pors | Vouloir l'égalité: troisième rapport du Comité de pilotage pour l'égal accès des femmes et des hommes aux emplois supérieurs des fonctions publiques[END_REF]. Throughout the decade, the presence of women among senior executives of the National-level Civil Service (Fonction publique d'Etat) increased, from 12% in 1999 to 24% in 2010, although women still accounted for less than 10% of prefects and 15% of ambassadors in 2010 11 .

Reinforced women's policy agencies mobilizing for gender quotas

Another significant effect of the campaign for gender parity was to broaden the range of women's policy agencies within the government: The Observatoire de la parité was created in 1995, and Parliamentary Delegations for women's rights were set up at the Assemblée nationale and the Sénat in 1999 [START_REF] Savinel | Les paradoxes d'une institution militante : la Délégation aux droits des femmes, « affaire de bonnes femmes » ou nouvelle « carte de visite[END_REF]. These women's policy agencies, although merely advisory, played a major role in the process of legitimation of the gender quota beyond the political field, through their "critical expertise" [START_REF] Revillard | L'expertise Critique, Force d'une Institution Faible ? Le Comité Du Travail Féminin et La Genèse d'une Politique d'égalité Professionnelle En France (1965-1983)[END_REF] and through their discreet, unobtrusive activism in the hallways of republican institutions. They played the role of "support structures" for the "entrenchment and diffusion of gender quotas" [START_REF] Lépinard | From breaking the rule to making the rules: the adoption, entrenchment, and diffusion of gender quotas in France[END_REF].

Marie-Jo Zimmermann, a Gaullist (UMP) deputy who had been a key advocate of the parity reform against most of her own political party, and who had been appointed at the same time the head of both the Delegation for Women's rights at the National Assembly and the Parity Observatory, orchestrated this feminist campaign within the Parliament and the Government. She seized president Sarkozy's project of constitutional reform, launched in the name of « state modernization » in the first year of his tenure, as an opportunity to extend the constitutional basis for gender quotas beyond the electoral sphere. Indeed, a provision of the 2005 equal pay law setting quotas in corporate board had been ruled out by the Conseil constitutionnel in 2006 based on the argument that the 1999 constitutional reform only authorized the enactment of gender quotas in political offices and functions. A constitutional reform was thus needed in order to extend this tool to other domains. To this effect, Zimmermann mobilized her nongovernmental, governmental and parliamentary networks across party lines (notably through the parliamentary Delegations for women's rights). As a result of this successful mobilization, the paragraph opening the possibility for gender quotas was extended and moved from article 3 to article 1 of the Constitution in 2008, without any major parliamentary opposition.

Made possible by this constitutional reform, the 2011 Copé-Zimmermann law imposing gender quotas in corporate boards also resulted from the parliamentary and bureaucratic activism of a coalition of state feminist actors. Marie-Jo Zimmerman notably drew on a 2009 report on equal employment delivered by Brigitte Grésy, former head of the women's rights bureau (Service des droits des femmes et de l'égalité), arguing in favor of a law imposing an obligation of quasi-parity (40%) in a "reasonable timeframe" to feminize the executive boards of major firms.

As the Copé-Zimmermann law was enacted in January, 2011, right-wing deputy and member of the Parliamentary Delegation for women's rights Françoise Guégot delivered to President Sarkozy a report entitled "Equal employment between men and women in public administration". One of her recommendations was to adopt quotas in order to produce a "balanced representation" of men and women at the highest levels of the state. She made the case for "an obligation to succeed" in the attempt to increase the share of women, given the "strong initiative" developed in parallel in the private sector through the Copé-Zimmermann law. Alignment with the private sector thus became a strong rationale for the promotion of quotas at the highest levels of the public administration.

Women elite networks promoting the women's elite cause Mobilizations of MPs and bureaucrats acting within women's policy offices unfolded hand in hand with (equally unobtrusive) mobilizations from members of female upper civil servant organizations. Throughout the 2000s, in a wider context of feminist revival, elite professional women's associations mushroomed. The most visible "women's networks" put together female alumni from French elite universities ("Grandes écoles") and corporate executives, lobbying to improve women's access to strategic business positions. Fueled by the parity grammar, they also drew on the "diversity" rhetoric that gained momentum in the French business world by the mid-2000s [START_REF] Bereni | Faire de la diversité une richesse pour l'entreprise": la transformation d'une contrainte juridique en catégorie managériale[END_REF]. On the one hand, these networks promoted incentive tools pertaining to "market feminism" (coaching, mentoring, benchmarking, good practices, rankings, etc.) [START_REF] Blanchard | Une cause de riches ? L'accès des femmes au pouvoir économique[END_REF][START_REF] Kantola | From State Feminism to Market Feminism?[END_REF][START_REF] Pochic | L'égalité élitiste ou le féminisme de marché. Genre et pouvoir dans une multinationale[END_REF]. But on the other hand, they also more discreetly mobilized in favor of "constraining measures", including gender quotas. In parallel, similar networks also burgeoned in the public sector. By 1998, the professional women's organization "Administration moderne" (Admod) brought together women upper civil servants, mostly énarques, working in various administrations, to promote an increased representation of women among the bureaucratic elite. These mobilizations at the top of the public administration unfolded against the backdrop of the diffusion of new public management (NPM) in the bureaucracy, presenting the adoption of management practices coming from the private sector as a way to improve government functioning [START_REF] Bezes | New public management and professionals in the public sector. What new patterns beyond opposition?[END_REF]. Endorsing this dominant discourse, they argued that the feminization of the Civil Service was a key driver for modernizing the bureaucracy [START_REF] Revillard | Gender, Class and Bureaucratic Power: the Production of Inequalities in the French Civil Service[END_REF]. At the end of the 2000s, while the Copé-Zimmermann bill was to be adopted, the leadership of Admod began lobbying in favor of the adoption of a quota. Two of them were called to give testimony by Guégot in preparation for her report, notably Nathalie Tournyol Duclos, who was to be appointed head of the women's rights bureau a few month later. Admod members also helped minister Sauvadet rally members of parliament and higher civil servants to the idea of quotas behind closed doors.

The adoption of the Sauvadet law: an opportunity under constraints (2011)(2012) The bill François Sauvadet introduced in the Senate on September 7 th , 2011, was mainly devoted to favoring access to tenure in the Civil Service. Only one -largely symbolicprovision dealt with gender equality: the obligation for ministerial departments to deliver an annual report on gender equality policy in the public service. Even though the Government had officially taken a stand in favor of gender quotas in "professional responsibilities" as of 2011, the introduction of a gender quota provision into the Sauvadet law was the outcome of a new intra-institutional mobilization of women's rights advocates. It was also made possible by avoiding the term "quota", which was replaced by the more consensual idea of "balanced representation" (Bui-Xuan, 2012).

In 2010, when President Sarkozy commissioned deputy Françoise Guégot to prepare a report on women's access to executive positions in the Civil Service, what was at stake for him was to promote measures that would publicize his presidencies action on gender equality and bureaucratic modernization when he would run for reelection in 2012. A measure such as the quota had the advantage to signal a commitment to gender equality, without entailing any major budgetary cost; this was particularly crucial in the aftermath of a financial crisis. One month before the report was officially delivered in March 2011, the State Secretary for Civil Service, Georges Tron, announced that he endorsed the idea of quotas, and hinted at the possibility of financial sanctions in case of non-compliance 12 . On March 8 th , after a bargaining meeting with public employees unions, he presented the Assemblée nationale with a series of measures, including the gender quota, which would be included into a future bill on "the fight against insecurity" in the Civil Service. Public employee unions strongly called for such a bill in the context of accelerating New Public Management reforms, and their representatives were formally involved in the preparation of the bill. However, the gender quota provision disappeared from the bill that François Sauvadet, Tron's successor (the latter resigned because of charges of sexual assault) 13 , introduced in Parliament in September 2011.

One reason for the omission was that public employees unions, including gender equality advocates within them, were ambivalent about introducing a gender quota for senior executive positions: even though this policy tool broke with the incentive, soft approach that had prevailed so far, most union representatives saw it as a minimal provision, focused on a narrow administrative elite, and which would turn the attention away from the broader structural reforms needed to favor gender equality in the bureaucracy (equal pay, work-family reconciliation, career assessment…) 14 . More importantly, the absence of a gender quota provision in the first draft of the bill, while Sauvadet declared himself in favor of the measure 15 , was due to resistances from administrative elites in the upper echelons of the bureaucracy, and even more crucially, to the ambivalence of Prime minister François Fillon 16 .

Still, at the beginning of 2012, women's rights advocates in Parliament renewed their efforts to introduce a gender quota provision within the Sauvadet bill. Appointed as Rapporteur for the bill, Socialist Catherine Tasca, who had been one of the most prominent advocates of the parity law in Parliament at the end of the 1990s, criticized in her report the weakness of the measures regarding gender equality 17 . In the senatorial debate over the bill, communist Senator Brigitte Gonthier-Maurin, president of the Delegation for women's rights, pressed for the introduction of quotas, quoting the Guégot report 18 . While the Senate did not introduce any quota provision when it first adopted the bill on January 26 th , 2012, Sauvadet announced a few days later that the Government would introduce a gender quota amendment during the parliamentary debate. The quota provision would set a target of 40% by 2018, and Sauvadet alluded to possible sanctions. This announcement was made possible by president Sarkozy's arbitration against Fillon's reluctance towards quotas 19 .

Women's rights advocates from the right-wing majority, notably deputies Marie-Jo Zimmermann and Françoise Guégot, backed by minister Sauvadet, introduced new gender equality provisions into the bill during its examination in parliamentary commission at the Assemblée nationale. François Sauvadet finally introduced a gender quota in senior executive appointments during the public debates at the Assemblée. As he argued, "even though quotas are no panacea, to this day they remain the only way to really make things change; in the government we are convinced of this" 20 . He also stressed the impact of women's rights advocates (mentioning Françoise Guégot and Marie-Jo Zimmermann) in putting the reform on the agenda, and changing his own opinion on the issue 21 . Meanwhile, the union bargaining process (which included demands in favor of broader gender equality measures) was put to a halt with the 2012 presidential election looming.

Cheered by women's rights advocates across the political spectrum, backed by promoters of neo-managerial reforms keen on "modernizing" the public administration to align it with private sector standards, the quota was only publicly opposed by conservative deputy Hervé Mariton 22 , and was adopted by a large majority. While the consensus over the Sauvadet quota can be accounted for by the diffusion of the parity grammar within institutional circles, it could also be explained by the technical restrictions of the legislation.

Firstly, the statute provided for a gradual implementation of the quota, going from 20% in 2013 from 40% in 2018. This contributed to convince skeptics about the "feasibility" of the provision, and dismissed the argument that "there are not enough female candidates" to these positions. This incremental framework was also in line with the mandated quota introduced a year earlier in private-sector corporate boards; this policy precedent helped mainstream the idea of gender quotas as a relevant policy tool. Secondly, the choice to target only the flux of "first appointments" (unlike the Copé-Zimmermann law on corporate boards), rather than the whole stock of senior executive positions, reassured already appointed men regarding the absence of threat on their personal situation. As clearly stated at the time by François Sauvadet.

We need to make sure that men who have already been appointed will be renewed within the limit defined by Civil Service regulations, or that they will be appointed to a similar position. For example, a prefect moving to another department must escape the quota system. 23 This consensus, however, was no more than a public façade; Sauvadet recently reported how higher civil servants and members of parliament discreetly but vehemently opposed the quota: "Nobody can imagine what I had to face, as minister for Civil Service: the resistance was as numerous as it was fierce, coming from all administrative bodies, with relays on the benches of the National assembly and the Senate." 24 In this respect, what the public consensus reflected was also the increased illegitimacy of an open criticism of the gender quota, in the context of its diffusion as a tool of gender equality policy.

A coercive and monitored policy tool

Following the parliamentary debate, the Sauvadet legislation of March, 12 th , introduced a series of mandatory gender quotas in several decision-making positions in the civil service: in executive boards of public institutions, in entrance examination committees, in joint administrative committees, in Civil Service advisory boards, and, last but not least, in appointments to senior executive positions. The statute mandated a minimal representation of "each sex category" of 20% in 2013, 30% in 2015 and 40% in 2018 in appointments to senior executive positions. The law provided that the quota was to be reached on a yearly basis and at the level of each ministerial department 25 for initial appointments, that is, "excluding cases of renewal of the person in the same position or appointment to a similar type of position". In the National-level Civil Service, the gender quota applied to first appointments among about 3600 senior executive positions (including Government appointments) 26 . Finally, the law provided for financial penalties to administrations that would not comply. This policy tool thus appeared as a negative incentive: sanctions would be applied in case of non-compliance with the goal [START_REF] Ingram | Improving Implementation Through Framing Smarter Statutes[END_REF]Schneider, 1990 cited in Engeli andMazur 2018 114-15). However, even negative incentives do not necessarily have a binding character, depending on if and how they are implemented. A closer look at implementation is therefore needed in order to better characterize this quota. The first striking element in the implementation of the Sauvadet quota is that the implementing decree was adopted quickly, unlike what is often the case for gender equality legislation. The Sauvadet implementation decree was adopted less than 2 months after the law was passed. It provided a precise list of the positions included in the perimeter of the law, and set the amount of the fine to 30.000€ for each "missing unit" (that is, for each woman -or man -missing in order to fill the quota) in 2013, 60.000€ in 2015 and 90.000€ in 2018. The decree therefore confirmed the coercive potential of the law by giving more substance to the idea of sanctions in case of noncompliance.

Moreover, the decree specified a mechanism of accountability and monitoring. By April, 30 th of each year, administrative agencies were required to deliver the list of initial appointments from the previous year to their assessors and to the ministry for Civil Service, as well as the sex ratio of these appointments and, if applicable, the amount of the fine due. A "Senior executive mission" hosted by the General Secretariat of the Government (SGG in French) was to be set up to collect the information for each ministerial department and take steps to "favor the attainment of this goal". The Civil Service and administration general management (Direction générale de l'administration et de la fonction publique, DGAFP) was to circulate each year a public report documenting the sex ratio for new appointments and, if applicable, the amount of the fine due.

Arguably, the existence of precise amounts of fines and the definition of a mechanism of monitoring and accountability could favor a strong implementation, leading to a policy situation of full coercion. The profile of the organizations in charge of implementation (SGG and DGAFP), however, could introduce doubts in this respect. Indeed, after being promoted by feminist advocates, quotas were integrated as part of administrative routines. Their implementation and the supervision thereof were assigned to Civil Service administrative services, far from state feminist institutions. Neither the women's rights bureau, nor the parliamentary Delegations women's rights, nor networks of women's higher civil servants, were formally included in the policy implementation process. Initially conceived as a tool to fight against gender inequalities, quotas thus became an administrative tool "just like any other", implemented by "laypeople in the field of equality" [START_REF] Perrier | L'objectif d'égalité des sexes dans la mise en oeuvre des politiques d'emploi à Berlin. De la diffusion professionnelle aux difficiles réappropriations profanes[END_REF], whose mission is defined in terms of legal compliance rather than in terms of feminist advocacy.

A feminist implementation by mainstream bureaucratic actors

The fact that quota monitoring was not devoted to Women's policy structures did not impede a strong implementation. Each actor followed the letter of the law. A procedure was defined to facilitate the declaration of nominations, with a spreadsheet made available on the Civil Service ministry website (see image below). Men and women were counted, and at the end of 2016, for the first time since the introduction of the quota, two ministers -the Department of Justice and the department of Defense -were imposed financial penalties for non-complying with the 30% quota. How can we account for such a diligent implementation on the part of state actors not endowed with a feminist mission? The format of the policy instrument at stake certainly helped: as argued earlier, the quota is a very simplistic and rather straightforward translation of the ideal of equality. The margin of interpretation is limited. Hence the characteristics of the implementing actors seem less likely to influence the outcome. While an absence of implementation could have been a possible outcome, the instrument was implemented as planned. Moreover, the DGAFP's involvement in the implementation process was not limited to monitoring the gender breakdown of new senior executive appointments. In its yearly reports on the Sauvadet quota implementation, the DGAFP developed more qualitative comments, pressing in favor of a more coercive implementation process. In its 2016 report, it pointed to two main limits of the legal provision 27 . On the one hand, ministries were expected to voluntarily pay their fines, but no enforcement procedure was provided for in case of a failure to pay, so that "the coercive potential of the sanctions may turn out limited". On the other hand, the DGAFP pointed out that the fines contribute to the state's general budget, when they could be used to fund gender equality policy in the Civil Service. Such comments would typically have been expected on the part of women's policy agencies, and here they were voiced by the DGAFP. In other words, not only did mainstream administrative actors implement the law as planned, but they also pressed in favor of its reinforcement, fueling a policy feedback effect [START_REF] Pierson | When Effect Becomes Cause: Policy Feedback and Political Change[END_REF]. In GEPP terms (see Engeli and Mazur, this issue), implementation was marked by high substantive empowerment without any clear evidence of descriptive empowerment. Does this mean that feminist advocacy had no influence on the process? Such a conclusion should be nuanced in several ways. First, this success in the mainstreaming of gender equality concerns in policy implementation can be analyzed as the result of the longterm diffusion of feminist ideas in non-feminist circles (Albenga et al, 2015): beyond feminist advocacy structures, people working in mainstream bureaucratic settings such as the DGAFP in the 2010s had been socialized to some extent to feminist ideas, through higher education for the youngest (via the diffusion of gender studies programs at university level), through the mass media or through peer influences. Moreover, the adoption of the Sauvadet law coincided with an upsurge in feminist mobilizing in civil society, which increased this diffusion of feminist ideas in mainstream settings (notably through increased discussions of feminist ideas in the media).

Against the backdrop of the 2012 presidential campaign, feminist groups pressed in favor of the creation of a strong Women's policy agency, a new "Ministry of women's rights" (Ministère des droits des femmes), in reference to the Roudy episode in the 1980s. François Hollande had committed to this prior to his election. Once elected, he appointed Najat Vallaud-Belkacem as minister for Women's rights, at a high rank and with all the more visibility since Vallaud-Belkacem was also the government spokesperson, and soon became very present and vocal in the media. Her position as Government spokesperson helped reinforce the legitimacy of women's rights as a policy issue, which she also made sure to embed more firmly in the different ministerial departments, with the appointment of senior executives in charge of gender equality (hauts fonctionnaires à l'égalité) in each ministry.

While marginal, these positions helped diffuse feminist ideas in the different ministerial departments. In parallel, both Women's policy agencies and the more informal feminist networks described in the previous section contributed to monitoring the implementation of the Sauvadet quota by favoring the maintenance of a perceived risk of shaming in case of non-compliance. This context favored the reinforcement of gender equality policy in the public sector, and even the reinforcement of the quota itself. The Government signed with public employees unions a "protocol agreement on equal employment in the Civil Service" in 2013, and a new "Charter for the promotion of equality and the fight against discriminations in the Civil Service" was adopted that same year. The 2014 law on "real equality between women and men" reinforced the provisions of the Sauvadet law: it increased to 50% the minimum share of people of each sex in the executive boards of public institutions (extending to the public boards the principle of the Copé-Zimmermann law), and set for 2017 (instead of 2018) the obligation of at least 40% of appointees of each sex for new appointments to the highest positions. Finally, the 2018 protocol agreement on equal employment in the Civil Service created a separate gender employment equality fund, meant to receive the fines paid by noncompliant administrations 28 . The new fund was to fuel other gender equality initiatives in the public administration, as advised by the DGAFP report mentioned above.

A restrictive yet coercive tool with incremental effects

The Sauvadet law introduced a coercive policy tool, with financial sanctions in case of non-compliance, but restricted it to a limited number of positions: out of about 3600 positions in the State Civil Service workforce within the perimeter of the law, only a few hundreds were filled with first appointments each year. The quota applied to 641 initial appointments in 2013, and 587 in 2017.

Between 2013 and 2016, first appointments to senior executive positions within the perimeter of the Sauvadet quota were systematically filled with 30% to 33% of women. With the exception of the departments for Justice and Defense, all ministerial departments complied with the legal obligation during the four first years of its implementation. Yet, at the end of 2017, the first year of implementation of the 40% quota, the rate was only 36%, with 6 out of 11 ministerial departments being in non-compliance. The ministry of Economic and Financial Affairs (Bercy) alone, with the lowest rate (26%), had to pay a contribution of 1.710.000€ (out of a total of 2.340.000€) 29 .

A mitigated quantitative effect: a smoothed curve It is not so easy to assess the extent to which the Sauvadet quota has led to an increased share of women among senior executive positions of the State Civil Service. Since the perimeter of application does not match any pre-existing administrative and statistical category, it is impossible to have a precise assessment of the gender breakdown in these positions prior to the implementation of the law. It is therefore difficult to assess the quantitative impact of the measure. Available figures since the early 2000s point to the hypothesis of a limited impact: rather than a turning point increasing the pace of feminization of senior executive positions, the Sauvadet quota led to securing and smoothing the curve of feminization. According to Civil Service reports between 2015 and 2017, the share of women among senior executive positions within the perimeter of the Sauvadet quota went from 26% to 28%. This demonstrates the limitations of a policy that only targets the flux (first appointments) rather than the stock (all the positions).

Moreover, if one looks at statistical categories that had been, until the Sauvadet law, used as indicators of the presence of women in the administrative elite (concerning around 1.900 senior executive positions included in the perimeter of the law), reports show a very limited impact of the Sauvadet law on the rate of women. As shown in the graph below, and mentioned earlier, the share of women in senior executive positions started increasing as of the middle of the 2000s, and particularly as of 2007, when Sarkozy came to office and hastened the pace of new public management reforms. In the years preceding the Sauvadet law, women promoting gender equality at the highest level of the bureaucracy had already managed to frame feminization as a key marker of the administration's "modernization" policy. The share of women in all senior executive positions increased by 1.8 points (from 21,9% to 23,7%) between 2007 and 2012 -prior to the Sauvadet law -, and by 2.1 points (from 25,1% to 27,2%) between 2013 and 2016 (the last year with available figures). This comparison of pre-and post-Sauvadet law levels of feminization suggests a limited immediate impact of the quota: its main effect was to secure and smooth an underway pattern of feminization of senior executive positions, rather than to accelerate its pace. The gradual dimension of the Sauvadet quota (rising from 20% in 2013 to 40% in 2017, and applying to first appointments only) partly accounts for this mitigated quantitative impact five years after the law was passed. While it does not entail an immediate, sharp increase in the gender ratio among senior executive positions, it allows for an incremental increase. In order to increase the potential impact of this policy tool, networks of higher executive women in the Civil Service started lobbying in favor of a new law by 2017, demanding more coercive measures, notably increasing the perimeter of the positions considered and increasing the fines 30 . The context, however, was not favorable. The implementation of the quota at the level of 40% for the first time in 2017 reactivated a strong, albeit invisible, hostility towards the measure: "we are still in a hostile environment", argued Admod president 31 . These oppositions remained very discreet: seldom publicly stated, they took the form of unobtrusive resistance on the part of men in the higher ranks of the public administration, as informally reported by several women's network's members. This resistance had concrete effects, translating into a regulatory backlash. First, following its poor results in 2017 (26%), the minister of Economic and Financial Affairs, in the hope of improving compliance without having to appoint more women, signed a decree reducing by one-third the perimeter of positions concerned by the Sauvadet quota on December, 27 th , 2018. Second, more significantly, the 2019 law on the public service 32 introduced several technical modifications that significantly weakened the law's impact. It notably legalized the established practice of the "rounding down" in setting the number of new women's appointments necessary to achieve 40%. Given the low numbers at stake, this rule leads to allow the appointment of less than 40% of women. For example, in the case of 9 new appointments to fill, at least 3,6 persons of the underrepresented sex would have to be appointed, theoretically; yet, in practice, the "rounding down" rule authorizes to appoint only 3 women (33%), rather than 4 (44%).

Beyond numbers: gender transformation through symbolic impact Beyond the limited quantitative impact of the Sauvadet quota, one should also stress its intrinsic limitations as a gender equality policy tool. Targeting only a small number of elite women, the quota mainly represents an opportunity for women who cumulate a series of resources (notably in terms of social class and education). It does not act upon the organizational mechanisms that produce the glass ceiling [START_REF] Rouban | L'accès des femmes aux postes dirigeants de l'Etat[END_REF][START_REF] Jacquemart | Femmes hautes fonctionnaires en France[END_REF][START_REF] Marry | Le plafond de verre et l'État -La construction des inégalités de genre dans la fonction publique[END_REF]. These limitations reflect the elitist profile of the feminist actors who have been advocating in favor of gender quotas in decision-making sites since the 2000s.

Nevertheless, the Sauvadet quota did not leave the gender regime unchanged (Connell, 2006;Acker, 2009) of the French Civil Service. Indeed, gender transformation does not boil down to quantitative measures: the symbolic dimension of the tool must be taken into account, and the implementation of the quota also paved the way for significant gender transformation in this respect. Firstly, the Sauvadet law and concomitant "soft" gender equality reforms (labels, charters…) turned the question of the glass ceiling in the Civil Service into a public problem [START_REF] Gusfield | The Culture of Public Problems. Drinking-Driving and the Symbolic Order[END_REF]. For example, the fines imposed to the departments of Justice and Defense in 2017 received extensive media coverage, stressing the shocking persistence of gender inequality. Secondly, the introduction of gender equality policies within the bureaucracy favored the denunciation of gender inequality. For sure, women's capacity to be vocal on these issues depends on their professional position (often correlated with their upper-class background and/or education in the most prestigious training tracks such as the ENA). However, the emergence of gender equality claims among women bureaucrats reflected the new legitimacy of a gendered perspective on careers within the public administration, challenging a long tradition of a gender-blind universalism [START_REF] Marry | Le plafond de verre et l'État -La construction des inégalités de genre dans la fonction publique[END_REF]. Thirdly, the Sauvadet quota and other gender equality policies also impacted men.

While in the first years of its implementation, the quota had a limited impact on men's odds to access higher bureaucratic positions, the new visibility of gender equality goals in the Civil Service had a symbolic effect on men: as the most conservative attitudes toward the gender equality agenda were delegitimized, men holding executive positions were to redefine their professional identities in relation to gender equality norms [START_REF] Bereni | Diriger comme un homme moderne. Les élites masculines de l'administration française face à la norme d'égalité des sexes[END_REF].

Conclusion

At first, by introducing a minimum quota of 40% of persons of each sex in new appointments to the highest administrative positions, the Sauvadet law appeared like a turning point, away from the "symbolic policy" [START_REF] Mazur | Gender Bias and the State: Symbolic Reform at Work in Fifth Republic France[END_REF] that had been promoted by the French state so far. Analyzing the details of this policy tool and its implementation, however, leads to a more complex assessment.

A closer look at the immediate effects of the measure points to the idea of a limited gender transformation: first, because the increase in the share of women in senior executive positions had in fact started prior to the enactment of the law, and secondly, because of the limitation of the quota's perimeter to "first appointments", rather than the whole stock of senior executive positions. This very technical feature, however, has endowed this tool with a potential to foster incremental social change. In other words, its effect was limited at first, but it is likely to increase over the years, as appointments made since 2012 represent a larger share of the stock of senior executives. The gender quota's relative strength also derives from the continuous involvement of state feminist actors who, in the years following the adoption of the law, kept lobbying to maintain its feminist framing. In this respect, a few years after its introduction, this reform cannot be categorized as a "policy failure", unlike other cases of gender quota reforms [START_REF] Verge | The Contentious Politics of Policy Failure: the Case of Corporate Board Gender Quotas in Spain[END_REF].

The persistence of this positive gender transformation, as shown by the first years of implementation, will depend upon a close monitoring of the process by women's networks and feminists within the state apparatus, even though Women's policy agencies are not formally part of the implementation mechanism. Indeed, as illustrated by the 2018 decree adopted by the ministry of Economic and Financial Affairs (Bercy) to limit its own obligations towards the law and by the technical modifications introduced in 2019, legal backpedaling always is a threat. On the other hand, the government's endorsement of a coercive provision is likely to have a symbolic effect. It fuels a representation of inequalities as illegitimate, while legitimizing their denunciation and the state's intervention to correct them. This strong gender equality statement, however, also runs the risk of diffusing an idea of equality as "already in place". The evolution of both the legislation and its implementation in the future years will confirm whether the quota has a transformative potential.

From a more theoretical perspective, this case study reveals the limit of focusing on the genesis and effects of a single reform to assess the transformation of gender relations. Indeed, as we have seen in this case, the same factors (mainly institutional feminist mobilization in the aftermath of the parity laws, and the broader transformations of the women's cause field (Bereni, 2019)) that help explain the adoption of the quota also influenced the feminization of the higher levels of the public administration prior to its adoption, thus conversely limiting its direct measured impact. Moreover, the adoption of the Sauvadet law cannot be explained short of the broader context of the diffusion of the quota as a gender policy tool in other sectors, through other legal reforms [START_REF] Lépinard | From breaking the rule to making the rules: the adoption, entrenchment, and diffusion of gender quotas in France[END_REF]. Finally, the fact that the Sauvadet quota was simultaneously reinforced (for example, by the creation of the equal employment fund in 2018) and weakened (for example, by the Bercy decree of December, 2018 and the 2019 law on public service) in the aftermath of its adoption points to the ongoing political struggles around this policy tool. 2 Gender quotas were only a part of the Sauvadet law, which is better known for introducing a new tenure system for government employees previously on precarious contracts. 3 The French Civil Service is divided into three categories: State, hospitals, and local administrations. Our study only concerns the State branch. Within the State Civil Service, out of 2 300 000 employees ("fonctionnnaires" as and "contractuels"), there are about 10 000 "executive positions" (DGAFP, Rapport annuel sur l'état de la fonction publique, 2018) and about 3600 senior executive positions (DGAFP, Bilan du dispositif des nominations équilibrées sur les emplois supérieurs et dirigeants de la fonction publique, 2017). les femmes et les hommes dans la fonction publique. 2018, p.9.

29 DGAFP, Bilan du dispositif des nominations équilibrées sur les emplois supérieurs et dirigeants de la fonction publique, 2018, p. 22-23, p.25. 30 See for example the joint press release by 9 organizations on June, 1st, 2018. 31 Entretien avec Nathalie Pilhes, AEF Info, 14 février 2018.

32 LOI n° 2019-828 du 6 août 2019 de transformation de la fonction publique

  Image: "Formulaire de déclaration des nominations équilibrées FPE (année 2016)", Ministère de l'action et des comptes publics, "Les nominations équilibrées dans l'encadrement supérieur de la fonction publique -08/12/2017", https://www.fonction-publique.gouv.fr/nominations-equilibrees-dans-lencadrement-superieur-dela-fonction-publique , webpage accessed on 21/02/2019.

  Sources: DGAFP, Rapport annuel sur l'état de la fonction publique, from 2008 to 2018. The category "Senior executive positions" includes here around 2.000 of the senior executive positions in the State Civil Service ("Emplois à décision du gouvernement", senior executive positions in central government and Senior executive positions in decentralized administration).

Notes1

  We wish to thank Amy Mazur, Robert Elgie, the members of the GEPP team, and the anonymous reviewers from French politics for their comments on previous version of this paper. This research was supported by a public grant overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program LIEPP (reference: ANR-11-LABX-0091, ANR-11-IDEX-0005-02) and the Université de Paris IdEx ANR-18-IDEX-0001. We dedicate this article to the memory of Robert Elgie.

A decree adopted on 30 April 2012 established the list of eligible positions, such as ambassadors or prefects.

DGAFP, Bilan du dispositif des nominations équilibrées sur les emplois supérieurs et dirigeants de la fonction publique, 2017, p.15-16.

Ministère de l'action et des comptes publics, Accord relatif à l'égalité professionnelle entre


PhD Benjamin Caulier
email: benjamin.caulier@gmail.com

Gaëlle Stofleth

Dalil Hannani

Mélanie Guidetti

Véronique Josserand

David Laurin

Jadwiga Chroboczek

Pascal Mossuz

Dominique Plantaz

Evaluation of the human type-3 adenoviral dodecahedron as a vector to target acute myeloid leukemia

Keywords: Adenoviral dodecahedron, virus-like particles, acute myeloid leukemia, leukemic stem cells, delivery vector

Introduction

Acute myeloid leukemia (AML) has one of the lowest survival rates among childhood cancers with a 5-year prognostic survival of 60 to 75%. [START_REF] Rasche | Successes and challenges in the treatment of pediatric acute myeloid leukemia: a retrospective analysis of the AML-BFM trials from 1987 to 2012[END_REF][START_REF] Creutzig | Diagnosis and management of acute myeloid leukemia in children and adolescents: recommendations from an international expert panel[END_REF] In adults, AML is the most common leukemia and patients under 60 years of age have a 5-year prognostic survival of 30% to 40% and this dramatically decreases below 15% above 60 years of age. [START_REF] Döhner | Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet[END_REF] For decades, AML was classified according to cell morphology using the French-British-American (FAB) classification (AML M0 to M7, i.e: M3 acute promyelocytic leukemia). Since then, multiple revisions from the World Health Organization (WHO) led to 4 new classes: AML with recurrent genetic abnormalities, AML with myelodysplasia-related changes, therapy-related AML and AML not otherwise specified. [START_REF] Arber | The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia[END_REF] Although tremendous progress has been achieved over the past 40 years in the understanding of AML physiopathology, treatment has not substantially changed. Indeed, cytarabine and anthracyclines drugs, both impeding DNA replication, remain the gold standard of intensive chemotherapy treatment used in combination with hematopoietic stem cells (HSCs) transplantation but are associated with severe off-target toxicities and long-term sequelae, especially in children and elderly patients. [START_REF] Creutzig | Diagnosis and management of acute myeloid leukemia in children and adolescents: recommendations from an international expert panel[END_REF][START_REF] Döhner | Acute Myeloid Leukemia[END_REF][START_REF] Mcgowan | Anthracycline Chemotherapy and Cardiotoxicity[END_REF][START_REF] Leung | Late Effects of Treatment in Survivors of Childhood Acute Myeloid Leukemia[END_REF] The specific targeting of AML cells is thus a challenge of importance to enhance overall chemotherapy tolerability as well as for the delivery of new therapies. [START_REF] Briot | Advances in treatment formulations for acute myeloid leukemia[END_REF] So far, many synthetic or bioinspired drug carriers are currently in development and trials but only few of them are in use with the most represented being liposomes and polymeric nanoparticles. [START_REF] Briot | Advances in treatment formulations for acute myeloid leukemia[END_REF][START_REF] Torchilin | Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery[END_REF] These carriers improve bioavailability, toxicity and may ameliorate remission. For example, Vyxeos, a liposomal formulation of cytarabine and daunorubicin in a 5:1 molar ratio was approved by the FDA in 2017 and the EMA in 2018 for the treatment of adults with newly diagnosed high-risk secondary AML. [START_REF] Krauss | FDA Approval Summary: (Daunorubicin and Cytarabine) Liposome for Injection for the Treatment of Adults with High-Risk Acute Myeloid Leukemia[END_REF][START_REF]Vyxeos liposomal (previously known as Vyxeos)[END_REF] Among bio-inspired delivery systems, virus-like particles (VLPs) are stable, self-assembled homogeneous nanoparticles, composed of virus capsid or envelope proteins. [START_REF] Yoo | Bio-inspired, bioengineered and biomimetic drug delivery carriers[END_REF] They are easily producible, carry no genetic material and represent a versatile tool for biomedical applications including cargo delivery for therapy. [START_REF] Koudelka | Virus-Based Nanoparticles as Versatile Nanomachines[END_REF] The capsid surface of the human serotype-3 adenovirus (Ad3) is composed of 3 structural proteins: the hexon, the fiber and the penton base (Pb). The hexon is the major building block of adenoviruses capsid but also the most immunogenic protein responsible for the bulk of neutralizing antibody production [START_REF] Sumida | Neutralizing antibodies to adenovirus serotype 5 vaccine vectors are directed primarily against the adenovirus hexon protein[END_REF] , especially against the hypervariable loops that differ among adenovirus serotypes. [START_REF] Fausther-Bovendo | Pre-existing immunity against Ad vectors: humoral, cellular, and innate response, what's important?[END_REF] The Ad3 fiber directs targeting properties through its specific attachment to cellular receptors such as ubiquitous complement regulatory protein CD46 [START_REF] Gaggar | CD46 is a cellular receptor for group B adenoviruses[END_REF] , epithelial desmoglein-2 [START_REF] Wang | Desmoglein 2 is a receptor for adenovirus serotypes 3, 7, 11 and 14[END_REF] and costimulatory molecules CD80/86 [START_REF] Short | Adenovirus serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors[END_REF] . The Ad3 Pb is meanwhile responsible for the entry through the interaction of its RGD (Arg-Gly-Asp) motifs within α V β 3 and α V β 5 integrins present on cell membranes, which subsequently triggers endocytosis. [START_REF] Wickham | Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment[END_REF][START_REF] Cuzange | The penton base of human adenovirus type 3 has the RGD motif[END_REF][START_REF] Xiong | Crystal Structure of the Extracellular Segment of Integrin alpha Vbeta 3 in Complex with an Arg-Gly-Asp Ligand[END_REF] In addition, an important feature of Ad3 (but not Ad2 or Ad5) is its ability to produce dodecahedral VLPs during infection, consisting of fiber and Pb. [START_REF] Fender | Synthesis, cellular localization, and quantification of penton-dodecahedron in serotype 3 adenovirus-infected cells[END_REF] Otherwise, the recombinant expression of Ad3 Pb alone is sufficient to give rise to fibreless dodecahedron (Dd, commonly referred as Base (Bs)-Dd). [START_REF] Fender | Adenovirus dodecahedron, a new vector for human gene transfer[END_REF][START_REF] Szolajska | The Structural Basis for the Integrity of Adenovirus Ad3 Dodecahedron[END_REF] This simplified VLP is ~30 nm in diameter and is exclusively composed of 12 copies of Ad3 Pb. Similarly to the Ad3 of origin, Dd shares the entry mechanism connecting Pbs to α V integrins. [START_REF] Fender | Adenovirus dodecahedron cell attachment and entry are mediated by heparan sulfate and integrins and vary along the cell cycle[END_REF] Therefore, this VLP has been efficiently used in the delivery of proteins [START_REF] Garcel | Protein transduction into human cells by adenovirus dodecahedron using WW domains as universal adaptors[END_REF][START_REF] Sumarheni | The Use of Adenovirus Dodecahedron in the Delivery of an Enzymatic Activity in the Cell[END_REF] , therapeutic compounds [START_REF] Zochowska | Adenovirus Dodecahedron, as a Drug Delivery Vector[END_REF] and mRNA analogs. [START_REF] Zochowska | Virus-like particle-mediated intracellular delivery of mRNA cap analog with in vivo activity against hepatocellular carcinoma[END_REF] Recently, a study on human healthy blood demonstrated Dd natural affinity for leukocytes, ~45-fold higher than for red blood cells (RBCs) or platelets. [START_REF] Jedynak | Leukocytes and drug-resistant cancer cells are targets for intracellular delivery by adenoviral dodecahedron[END_REF] We postulate that Dd can represent a promising vector for AML cells. We thus characterized its ability to target leukemic cells in vitro with attention for vector dose, subcellular localization, intracellular vector maintenance, endocytosis pathway and toxicity. We compared the internalization among peripheral blood mononuclear cells (PBMCs) in healthy and patient settings with a focus on cell type targeting. Further, we assessed AML and acute lymphoid leukemia (ALL) blasts targeting as well as the addressing of AML leukemic stem cells (LSCs) ex vivo. Finally, we evaluated the in vivo biodistribution of Dd following IV injection, especially in peripheral blood and bone marrow.

Results

Dd efficiently vectorizes human leukemic cell lines

Dodecahedral VLPs were produced upon recombinant Ad3 Pbs expression in baculovirusinfected insect cells and purified by a two-step chromatography yielding soluble and stable nanoparticles (Figure S1).

In this study, we first evaluated the vectorization of leukemic cell lines during 1 hour with a labeled Dd (Dd-AF647). A significant increase of the percentage of Dd-AF647-positive cells was observed between 0.1 nM and 1 nM with a mean of 22±18% and 94±9%, respectively (Figure 1A). At 5 nM Dd, that percentage was over 98% for all cell lines. In addition, the relative amount of vector in cells (MFI) rose concomitantly with increasing concentration above 0.1 nM. The MFI signal increased over incubation time (Figure 1B), until 24 hours (P <.05) and showed only a slow decline over 72 hours indicating a good internal maintenance of the AF647 cargo (Figure 1C).

The vector toxicity was assessed over a week with treatment every 48 hours (Figure 1D).

We used 1 µM of doxorubicin as a positive cell death control (10 nM of doxorubicin had no effect, not shown) since it induced 85.7±7.8% of dead cells in 24 hours (not shown). A metabolic assessment of the viability indicated that 1, 5 and 10 nM Dd had a minor toxic effect with 93.6±7.4%, 84.4±14.3% and 83.1±13.9% of alive cells at day 7, respectively. Higher doses of 25 and 50 nM displayed much more toxicity, with 76.3±11.5% (P <.001) and 55.7±20.1% (P <.0001) of alive cells at day 7.

Noteworthily, the AML cells vectorization featured high intracytoplasmic content of particles often close to the nucleus, that was not assigned to membrane adsorption of Dd (Figure 1E).

Taken together, these results demonstrate Dd as a dose-and time-dependent vector, efficiently targeting leukemic cell lines while displaying moderate toxicity.

The Dd vectorization involves clathrin-mediated energy-dependent endocytosis and strongly correlates with α V β 3 integrin expression in leukemic cells

We investigated the effect of temperature and ATP depletion on the cellular uptake of Dd-AF647 in leukemic cells. The vector accumulation was reduced upon treatment at 4°C, with 87% of reduction in Dd-AF647 MFI compared to 37°C (P <.01; Figure 2A). Similarly, when the cellular ATP pool was depleted by treatment with sodium azide and 2-deoxy-D-glucose, the vectorization was nearly abolished with 96% of reduction (P <.01).

These results indicated that the vectorization is temperature-and energy-dependent.

To clarify which endocytosis pathways is involved in leukemic cells vectorization, we explored the effect of wortmannin, methyl-β-cyclodextrin (MBCD) and chlorpromazine that specifically inhibit micropinocytosis/phagocytosis, caveolae/raft-and clathrin-mediated endocytosis (CME). [START_REF] Araki | A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages[END_REF][START_REF] Nabi | Caveolae/raft-dependent endocytosis[END_REF][START_REF] Wang | Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation[END_REF] When cells were treated with wortmannin or MBCD, Dd-AF647 MFI slightly decreased while the treatment with chlorpromazine significantly reduced Dd uptake of ~92% compared to control (P <.05; Figure 2B). Noteworthily, phagocytic Mono-mac-6 and THP-1 cells as well as the chronic myeloid leukemia K-562 were less efficiently vectorized compared to others. These inhibition results showed that the active transport of Dd is carried out by CME.

Among integrins, α V β 3 is characterized as the entry receptor for Dd. [START_REF] Fender | Adenovirus dodecahedron cell attachment and entry are mediated by heparan sulfate and integrins and vary along the cell cycle[END_REF] Therefore, we quantified its expression along Dd-AF647 internalization on leukemic cells. As expected, the MFI displayed a very close linear correlation between α V β 3 expression and the cellular level of Dd (Figure2C). Conversely, although Dd was shown to interact with heparan sulfates [START_REF] Vivès | Heparan sulfate proteoglycan mediates the selective attachment and internalization of serotype 3 human adenovirus dodecahedron[END_REF] , a competitive inhibition of entry using heparin did not reduce Dd uptake in leukemic cells (Figure S2).

Taken together, these results reveal the leukemic cells Dd-mediated vectorization as a temperature-& energy-dependent phenomenon involving CME in α V β 3 integrin expressing cells.

Dd vectorizes human healthy donor peripheral blood mononuclear cells, targeting preferentially monocytes

PBMCs were purified from 25 healthy donor bloods and treated with Dd-AF647. Monocytes appeared as the best-targeted cells followed by B lymphocytes with 96±4% and 47.7±13.4% of Dd-AF647-positive cells for 1 nM, respectively (P <.0001, Figure 3A, left panel).

Comparatively, granulocytes, T lymphocytes and NK lymphocytes exhibited very close Dd penetration and were less efficiently vectorized. Importantly, T lymphocytes appeared to be the least-vectorized population, with 53.5±6.7% of vector-positive cells at 10 nM (P <.0001, not displayed). Similar results were obtained for MFI except for granulocytes that showed a very low vector accumulation with a fold increase of 7±2 at 10 nM (Figure 3A, right panel).

Thus, Dd vectorizes PBMCs ex vivo, targeting preferentially monocytes.

Dd efficiently vectorizes AML blasts compared to ALL, targets monocytes and all subtypes of AML blasts among PBMCs and is able to enter AML leukemic stem cells

We next analyzed the targeting of 33 leukemic patient PBMCs. Among these samples, 8 were obtained from ALL pediatric patients and 25 from AML patients, among which 84% were adult and 16% were pediatric (Table 1). As expected, Dd preferentially vectorized AML blasts compared to ALL, at 1 and 10 nM, both in terms of Dd-positive cells and MFI (Figure 3B). We thus focused on AML samples (Table 2) and showed that the vector targeted blasts independently of the disease status (diagnosis versus relapse; Figures S3 A-B), the age (pediatric versus adult patients; Figures S3 C-D) or whether the biological sample was purified or not (Figures S3 E-F). These data suggest the Dd as a versatile vector for AML.

Lymphocytes, granulocytes and monocytes (LGM) were also monitored for Dd entry during vectorization of AML blasts. There was 60.5±30.5% of Dd-AF647-positive cells for AML blasts at 1 nM compared to 18±16% (P <.0001), 28±23% (P <.0001) and 81.7±19.9% (P <.01) for LGM, respectively (Figure 3C, left panel). A ten-fold concentrated dose of vector reduced these differences among populations. Quantitatively, data became statistically significant with the use of 10 nM Dd, with 313±301-fold increase of MFI compared to 54±47 (P <.0001), 29±22 (P <.0001) and 608±427 (P <.0001) for LGM (Figure 3C, right panel). It is thus noteworthy that Dd vectorizes massively AML blasts and monocytes compared to lymphocytes and granulocytes.

To go further, we analyzed the vectorization of AML subtypes according to cell morphological classification. All types of AML were vectorized with AML M4 displaying 802±322-fold increase of MFI at 10 nM compared to 351±263 (P <.05), 151±202 (P <.01), 143±154 (P <.01) and 130±192 (P <.001) for secondary AML, AML M0-1-2, M3 and M5, respectively (Figures 3D). Furthermore, we analyzed Dd uptake according to the 2016-revised WHO AML classification. [START_REF] Arber | The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia[END_REF] There were no significant differences in Dd vectorization between these new AML subtypes nor for 9 AML classified with recurrent genetic abnormalities regarding flt3, npm1 and idh1/2 classical mutations (Figure S4 A-B). These results mean that the Dd vectorization is efficient in AML regardless of the morphological and the WHO classification.

A key feature of AML physiopathology is the presence of renewable LSCs particularly those found in granulocyte-macrophage progenitors (GMP-like LSCs). [START_REF] Goardon | Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia[END_REF][START_REF] Thomas | Biology and relevance of human acute myeloid leukemia stem cells[END_REF] To explore the uptake of this particular subpopulation, whole marrow samples were treated with Dd before being enriched in CD34-positive cells. Overall, a similar number of Dd-AF647-positive cells and MFI was observed for total stem cells, myeloid precursors and GMP-like LSCs (Figure 3E).

Together, these results confirm the potential of Dd to vectorize AML blasts. Importantly, the vector is able to penetrate LSCs.

Dd reaches peripheral monocytes and bone marrow precursors in vivo

Finally, an in vivo biodistribution study was conducted to confirm Dd vectorization properties.

Mice were IV injected with Dd-AF647 and sacrificed at 1-, 5-and 12-hours post-injection. Ex vivo fluorescence imaging was performed on isolated organs (Figure 4A). An important uptake was observed in the kidney at 1 and 5 hours and in the liver at the 3 time points while only a weak signal arose from other organs including heart, brain, skin, and spleen.

Additionally, peripheral blood and bone marrow were analyzed. Although we used a small injected amount of Dd, a qualitative analysis of targeted cells could be performed.

Leukocytes were the major vectorized population with regard to platelets and RBCs at 1 hour and the signal decreased rapidly over 12 hours (Figure 4B). The study of leukocytes subsets revealed that monocytes were also preferentially targeted in vivo (Figure 4C).

Bone marrow hematopoietic stem and progenitor cells (HSPCs) showed interesting results

(Figure 4D). The best-targeted population was the multipotent progenitors (MPPs) with a 3 time points-mean of 3.42±1.51% of vector-positive cells, compared to 0.85±0.63%, 0.62±0.41%, 0.97±0.72% and 0.55±0.36% for the common-lymphoid progenitors (CLPs), the common-myeloid progenitors (CMPs), the GMPs and the megakaryocyte-erythrocyte progenitors (MEPs), respectively (P <.001). HSCs displayed a mean of 1.43±1.13% of Dd-AF647-positive cells that was lower than for MPPs (P <.01). Comparisons between CLPs, CMPs, GMPs and MEPs were difficult as these populations showed similar but lower percentage of Dd penetration. Thus, all bone marrow-resident HSPCs were reached by Dd upon IV injection with MPPs accumulating more Dd than other cells Taken together, these results recapitulate ex vivo observations and further demonstrate Dd ability to vectorize peripheral leukocytes in vivo, especially monocytes. Importantly, Dd reaches the bone marrow where it vectorizes progenitors, which is an important property for AML treatment delivery and targeting of LSCs.

Discussion

The targeted delivery of AML cells is crucial for the improvement of chemotherapy tolerability by reducing unspecific off-target effects and subsequent acute and chronic toxicities mediated by intensive treatment. [START_REF] Döhner | Acute Myeloid Leukemia[END_REF][START_REF] Mcgowan | Anthracycline Chemotherapy and Cardiotoxicity[END_REF][START_REF] Briot | Advances in treatment formulations for acute myeloid leukemia[END_REF] Furthermore, myeloid delivery systems are scarce and urgently needed for the avenue of new therapies. In this study, we fully evaluated the use of a non-infectious VLP as a vector to target AML cells since it presents a natural affinity for leukocytes. [START_REF] Jedynak | Leukocytes and drug-resistant cancer cells are targets for intracellular delivery by adenoviral dodecahedron[END_REF] Dd highlights several advantageous properties to represent an interesting delivery system in AML:

The vector is only composed of Ad3 Pbs, and 12 copies are sufficient to trigger the assembly of a dodecahedral VLP that is massively produced upon recombinant expression. [START_REF] Fender | Adenovirus dodecahedron, a new vector for human gene transfer[END_REF][START_REF] Szolajska | The Structural Basis for the Integrity of Adenovirus Ad3 Dodecahedron[END_REF] In addition, the vector is easily purifiable using size-exclusion followed by anion-exchange chromatography. Hence, it could be efficiently manufactured. Comparatively to the virus of origin, Dd is devoid of genome, hexon and fiber proteins thus simpler and non-infectious.

Although pre-existing or de novo generation of neutralizing anti-Ad3 antibodies could mitigate efficient drug vectorization, it has been shown that the hexon was the main target of preexisting antibodies as well as adenovirus specific CD4+ and CD8+ T cells. [START_REF] Sumida | Neutralizing antibodies to adenovirus serotype 5 vaccine vectors are directed primarily against the adenovirus hexon protein[END_REF][START_REF] Serangeli | Ex vivo detection of adenovirus specific CD4+ Tcell responses to HLA-DR-epitopes of the Hexon protein show a contracted specificity of T(HELPER) cells following stem cell transplantation[END_REF][START_REF] Leen | Identification of hexon-specific CD4 and CD8 T-cell epitopes for vaccine and immunotherapy[END_REF] Moreover, Zochowska et al. [START_REF] Zochowska | Virus-like particle-mediated intracellular delivery of mRNA cap analog with in vivo activity against hepatocellular carcinoma[END_REF] showed only a slow and moderate build-up of anti-Dd antibodies following 5 IV injections in rats. This suggest a limited vector neutralization risk at therapy initiation and overtime.

The Ad3 infects epithelial and hematopoietic cells using macropinocytosis as a major entry pathway. [START_REF] Amstutz | Subversion of CtBP1-controlled macropinocytosis by human adenovirus serotype 3[END_REF] Comparatively, the endocytic targeting of Dd mainly involved energy-dependent CME in tested leukemic cells. This behavior might be thus related to the simpler vector composition of only Pbs. The use of a fibreless Dd also restrains undesired cell tropism by reducing the number of attachment receptors. [START_REF] Gaggar | CD46 is a cellular receptor for group B adenoviruses[END_REF][START_REF] Wang | Desmoglein 2 is a receptor for adenovirus serotypes 3, 7, 11 and 14[END_REF][START_REF] Short | Adenovirus serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors[END_REF] In this sense, it has been shown that the Ad3 enters platelets and granulocytes using fiber-mediated binding of desmoglein-2 [START_REF] Wang | Desmoglein 2 is a receptor for adenovirus serotypes 3, 7, 11 and 14[END_REF] while, in this study, fibreless Dd showed only a slight internalization in these cells. Importantly, Dd keeps the ability to trigger specific endocytosis through the interaction of Pb RGD motifs with α V β 3 integrins. [START_REF] Cuzange | The penton base of human adenovirus type 3 has the RGD motif[END_REF][START_REF] Fender | Adenovirus dodecahedron cell attachment and entry are mediated by heparan sulfate and integrins and vary along the cell cycle[END_REF] This feature confers, at least, a relative selectivity toward α V integrins expression and especially α V β 3 such as for highly expressing leukemic cell lines as well as monocytes [START_REF] Weerasinghe | A role for the alphavbeta3 integrin in the transmigration of monocytes[END_REF][START_REF] Brilha | Monocyte Adhesion, Migration, and Extracellular Matrix Breakdown Are Regulated by Integrin αVβ3 in Mycobacterium tuberculosis Infection[END_REF] , AML cells [START_REF] Al-Asadi | A molecular signature of dormancy in CD34+CD38-acute myeloid leukaemia cells[END_REF][START_REF] Miller | In Vivo RNAi Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling[END_REF][START_REF] Johansen | The Possible Importance of β3 Integrins for Leukemogenesis and Chemoresistance in Acute Myeloid Leukemia[END_REF] and bone marrow progenitors [START_REF] Umemoto | Integrin αvβ3 enhances the suppressive effect of interferon-γ on hematopoietic stem cells[END_REF][START_REF] Kräter | Bone marrow niche-mimetics modulate HSPC function via integrin signaling[END_REF][START_REF] Wang | Lineage marker expression on mouse hematopoietic stem cells[END_REF] that were efficiently targeted. However, dedicated mechanistic studies could deeper characterized this α V β 3correlated, CME-mediated endocytosis in AML cells, since partial entrapment in lysosomes has been described and that could mitigate vectorized molecules effectiveness. [START_REF] Sumarheni | The Use of Adenovirus Dodecahedron in the Delivery of an Enzymatic Activity in the Cell[END_REF] Besides, it was shown that Dd preferentially penetrates leukocytes than platelets or RBCs in healthy donor blood. [START_REF] Jedynak | Leukocytes and drug-resistant cancer cells are targets for intracellular delivery by adenoviral dodecahedron[END_REF] That distinct vectorization efficiency was confirmed and we further revealed monocytes as well as AML blasts to be better vectorized than ALL blasts, lymphocytes (T, B and NK) and granulocytes. Additionally, Dd slightly spread outside the bloodstream with most of filtration occurring in 12 hours, which support a rapid clearance after IV injection. It is noteworthy that Dd entered the heart only weakly, which is promising for AML to circumvent the cardiotoxicity induced by anthracyclines. [START_REF] Mcgowan | Anthracycline Chemotherapy and Cardiotoxicity[END_REF] The limited vector spreading is of great importance to allow optimal leukocytes targeting while reducing systemic toxicity. Altogether, these features strengthen the use of Dd to target leukemic cells compared to others carrierswith no selective cell delivery properties -such as liposomes, already evaluated in AML. [START_REF] Krauss | FDA Approval Summary: (Daunorubicin and Cytarabine) Liposome for Injection for the Treatment of Adults with High-Risk Acute Myeloid Leukemia[END_REF][START_REF]Vyxeos liposomal (previously known as Vyxeos)[END_REF] The relevance of LSCs has been thoroughly described in AML [START_REF] Thomas | Biology and relevance of human acute myeloid leukemia stem cells[END_REF] but novel directed systems do not always evaluate the vector ability to reach these cells. The vector presented here is able to target human GMP-like LSCs ex vivo which, along with lymphoid-primed multipotent progenitors LSCs, coexist in 80% of patients. [START_REF] Goardon | Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia[END_REF] Furthermore, upon IV injection in mice, the vector reached the bone marrow and HSPCs, among which MPPs were the best-vectorized.

Since 10 to 15% of AML cases are characterized by a dominant population resembling MPPs [START_REF] Goardon | Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia[END_REF] , that population would be potentially targeted by Dd. These features emphasize the use of Dd to target HSPCs and by extension the place where LSCs reside.

The vectorization of model leukemic cells revealed an efficient targeting at low nanomolar concentration as well as a good intracytoplasmic maintenance of the fluorescent cargo.

These points are of importance since intensive chemotherapy treatment induces effluxmediated multidrug resistance (MDR) mechanisms that greatly contribute to AML relapses. [START_REF] Zhang | Mechanisms of drug resistance in acute myeloid leukemia[END_REF] Of note, the vectorization of AML blasts obtained from relapsed patients was as effective as the vectorization of patient blasts at diagnosis. Thus, the enhanced retaining time of cargo provided with low concentration of Dd could circumvent those hurdles, similarly to doxorubicin in the context of human MDR sarcoma cells. [START_REF] Jedynak | Leukocytes and drug-resistant cancer cells are targets for intracellular delivery by adenoviral dodecahedron[END_REF] Since our data also indicate a moderate toxicity of Dd, its utilization as a vector might be safe in vivo. Hence, Dd and chemotherapy could be associated to enhance the effect of free therapeutic form, thereby reducing off-targets and side effects. Using this strategy, the effective cytotoxic concentration of bleomycin delivered with Dd to human cervical cancer cells was 100 times lower than that of free drug. [START_REF] Zochowska | Adenovirus Dodecahedron, as a Drug Delivery Vector[END_REF] Thus, the vector is effective from very low molecular concentrations, supporting its use as an efficient enhancer for therapeutics in AML cells also via improvement of cell retention while cytotoxicity of Dd remains acceptable.

In conclusion, these results highlight promising features of Dd vector for myeloid and monocytes cell delivery applications as well as for AML therapy-aiming at targeting preferentially blasts and HSPCs. Further studies should now consider a therapeutic form of Dd such as Dd bearing doxorubicin and assess its full potential in AML engrafted mice models in comparison to free-drug doxorubicin or another reference therapy.

Materials & Methods

Dd labelling

Dd was conjugated to AlexaFluor 647 (Dd-AF647) using AF647 Protein Labeling Kit (Molecular Probes) and the structure was verified by nanoparticle tracking analysis on a NanoSight NS300 (Malvern Panalytical).

Dd cell vectorization

Leukemic cell lines, PBMCs, bone marrow mononuclear cells and whole blood/bone marrow were incubated with 0.01 to 10 nM Dd-AF647 for up to 7 days at 37°C. Untreated cells or whole blood/bone marrow were used as controls. After incubation, purified cells were treated with 0.5% Trypsin-EDTA (Gibco) during 5 min at 37°C to remove surface-bound Dd. For LSCs vectorization, CD34 enrichment was performed using CD34 MicroBeads kit (autoMACS Pro, Miltenyi Biotech).

Cytotoxicity assays

The Dd toxicity was assessed on cell lines using a metabolic assessment of cell viability by a chromogenic assay. Cells were quantified using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) on a Varioskan Flash (Thermo Scientific). Doxorubicin (Accord) was used as a positive control for cell death.

Confocal microscopy

Cells were treated with PBS or 10 nM Dd-AF647 for 2h at 37°C. After incubation, cells were fixed with 1x FACS lysing solution (BD Biosciences). Nuclei were stained with DAPI (1:3000; Sigma) and membranes with AlexaFluor 555-conjugated wheat germ agglutinin (WGA; 1:200; Invitrogen) during 10 min at room temperature. Images were acquired after sequential laser excitation using an LSM 710 NLO laser-scanning microscope (Zeiss) onto optical sectioning of 1 µm and analyzed with ImageJ software.

Drug treatment

Cells were pre-incubated during 1h prior to Dd addition in PBS containing the following drugs: 100 nM wortmannin, 30 µM chlorpromazine hydrochloride, 500 µM MBCD, 10 mM 2-deoxy-D-glucose and 10 mM sodium azide (Sigma).

Flow cytometry

Human and mouse antibodies were purchased from BD Biosciences. In all experiments, cells were selected by a viability labelling using Zombie Aqua Fixable Viability Kit (BioLegend).

The percentage of Dd-AF647 positive cells and AF647 mean fluorescence intensity (MFI) were measured for each population. The threshold percent gate was set-up by cell type for background fluorescence of untreated cells. The MFI of Dd-AF647-treated cells was compared to control by dividing (fold increase) or subtracting the MFI of the corresponding untreated cells. For measurement of α V β 3 integrin expression, cells were labeled with antiα V β 3 -PE monoclonal antibody. Data were acquired on FACS Canto II & FACS LSR II (Beckton Dickinson) and analyzed with FlowJo V10 software.

In-vivo biodistribution in blood and bone marrow

The project was authorized by the French ministry of research with the permit number: APAFIS#18922-2019013112124432v4. Twelve 5-weeks old C57Black6 female mice were purchased (Janvier Laboratory) and housed for 1 week before the day of experiment. Mice were injected in the tail vein with 100 µg Dd-AF647 in 200 µL of PBS (n = 9) or with 200 µL of PBS alone (n = 3). Three mice were anesthetized per time point at 1, 5 and 12h postinjection, 500 µL of blood were collected and mice were sacrificed for organs and bone marrow sampling. [START_REF] Liu | Immune Cell Isolation from Mouse Femur Bone Marrow. Bio Protoc 5. for T lymphocytes or CD335+ (NKp46) for NK lymphocytes or CD19+ for B lymphocytes; CD45+ CD11c-MHCII-GR1+ (Ly-6G) for granulocytes and CD45+ CD11[END_REF]

Statistics

Data are reported as the mean values ± standard deviation (SD). Student t-test was used in univariate analysis and Pearson r coefficient was calculated for correlation. One-way or twoway ANOVA tests were performed for multivariate analysis and P-value were conservatively adjusted for multiple comparisons using a Bonferroni correction. P-value <.05 was considered significant and the abbreviations were: n.s., not significant; *, P ≤.05; **, P ≤.01; ***, P ≤.001; ****, P <.0001. Statistics were calculated using GraphPad Prism V7 software. for Dd-AF647. Subpopulations were identified with the following markers: CD45 high and CD3+

Figures Legends

  Figure 1. The adenoviral Dd efficiently vectorizes human leukemic cells,

Figure 2 .

 2 Figure 2. The vectorization is an energy-dependent phenomenon, involving

Figure 3 .

 3 Figure 3. Dd targets preferentially monocytes among PBMCs and blasts from

Figure 4 .

 4 Figure 4. Dd demonstrates limited vector spreading outside the bloodstream

Acknowledgment

This work was funded by Société Française de lutte contre les Cancers et les leucémies de l'Enfant et de l'adolescent, La Ligue contre le Cancer 38, Association Grenobloise pour le Développement d'Etudes et de Recherche en Médecine Infantile, Association Espoir, GEFLUC Grenoble Dauphiné-Savoie and IDEX Univ. Grenoble Alpes. The authors thanks Sylvie Berthier of the Institute of Biology and Pathology (Grenoble) for access and technical assistance with flow cytometers, Alexei Grichine and Mylène Pezet of the Institute for Advanced Biosciences (Grenoble) for technical assistance with confocal microscopes and flow cytometers, Gautier Szymancki, Claire Vettier, Christine Lefebvre and the technicians of the Laboratory of Cellular Hematology (Grenoble), for providing patient samples, Pascal Fender of the Institut de Biologie Structurale (IBS, Grenoble) for helpful advice and discussions on the adenoviral Dd, Marta Jedynak and Ewa Szolajska of the Institute of Biochemistry and Biophysics (Warsaw, Poland) for materials and discussions on the adenoviral Dd, Karine Laulagnier from Remy Sadoul group of the Grenoble Institute of Neuroscience (Grenoble), for access to Nanosight and advice on particle analysis, Daphna Fenel and Guy Schoehn of the electron microscopy (EM) IBS platform (Grenoble). This work used the EM facilities at the Grenoble Instruct-ERIC Center (ISBG; UMS 3518 CNRS CEA-UJF-EMBL) with support from the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05-02) and the Grenoble Alliance for Integrated Structural Cell Biology (GRAL; ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology.

The IBS EM platform is supported by the Rhône-Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale and GIS-IBiSA.

Author Contributions

Conflict of interest

The authors declare no competing interests.

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