WO2023073099A1 - Method to improve phagocytosis - Google Patents

Abstract

The present invention relates to the improvement of phagocytosis. In this study, the inventors observed that HRV16 infected macrophages show defective phagolysosome biogenesis, leading to an inability to efficiently clear bacteria. Membrane trafficking is regulated by small GTPases of the Rab, Arf and Arf-related (Arl) families, which recruit and activate the fusion machineries on specific compartments. They report here that the ARL5b protein (Houghton et al., 2012) expression is upregulated by the rhinovirus in target cells. They further demonstrated that the depletion of ARL5b in HRV16-challenged macrophages prevents the viral induced changes in the endocytic machinery and improves bacterial clearance. Therefore, they provide the first direct evidence that ARL5b is targeted by HRV16 and controls endocytic dynamics in infected cells. These results offer a new connection between viral factories and a comprehensive insight into a key molecular mechanism used by HRV16 to hijack and disrupt macrophage phagocytic and antimicrobial function. Thus the invention relates to an inhibitor of ARL5b for use in the improvement of phagocytosis in a subject in need thereof.

Description

METHOD TO IMPROVE PHAGOCYTOSIS

FIELD OF THE INVENTION:

The present invention relates to an inhibitor of ARL5b for use in the improvement of phagocytosis in a subject in need thereof.

BACKGROUND OF THE INVENTION:

Human rhinovirus (HRV) is a member of the Picornaviradae family and is a small, nonenveloped virus with a single stranded, positive sense RNA genome within an icosahedral capsid [Jacobs et al., 2013], HRV is known to productively infect epithelial cells [Arruda 1995, Gern 1996, Papi 1999, Sajjan 2008], but there is evidence that HRV can infect monocytes and macrophages (Gern 1997, Laza-Stanca 2006 jubrail 2020) and that a direct interplay occurs between epithelial cells and monocytes/macrophages (Zhou 2017). HRV is routinely associated with upper respiratory tract infections. However, in patients with chronic inflammatory diseases such as chronic obstructive pulmonary disease (COPD) or asthma, the virus can reach the lower respiratory tract and cause disease exacerbations (Gern 1997, Wilkinson 2006, Wilkinson 2017). In addition, for a subset of patients with COPD, HRV infection can precede a secondary bacterial infection and it has been reported that HRV induces a phagocytic defect in macrophages leading to a failure to effectively respond to secondary targets [Bellinghausen et al., 2016; Jubrail J, 2017; Oliver et al., 2008; Finney 2018, Jubrail 2018, 2019],

Macrophages are professional phagocytes that internalize material and cell debris through surface receptors (Canton et al., 2013; Flannagan et al., 2012; Niedergang and Grinstein, 2018). Following ligand and receptor interactions, signaling cascades induce strong, local and transient actin polymerization in addition to remodeling of the plasma membrane, leading to the formation of a closed compartment called the phagosome. The phagosome then matures into a highly degradative compartment termed the phagolysosome through fusion and fission steps with the endocytic machinery (Fairn and Grinstein, 2012).

The phagosomes initially acquire early endocytic markers such as Early Endosomal Marker 1 (EEA1) and starts to accumulate v-ATPase components to initiate acidification, as well as the NADPH oxidase activity to generate reactive oxygen species (ROS). The phagosomes then move along microtubules to fuse with late endosomes and finally lysosomes in the juxtanuclear region. Late endocytic markers such as CD63 are transiently recruited on phagosomes before lysosome-associated protein 1 and 2 (LAMP1 and LAMP2). Through fusion with lysosomes, the phagolysosomes acquire luminal proteases and other hydrolytic enzymes that participate in the degradation of the internalized material (Depierre, Jacquelin and Niedergang, 2021). These combined activities promote the degradation of the internalized bacteria.

Exacerbations due to viral infections are thought to rely in part on macrophages defective in their ability to clear bacteria. The inventors have recently revealed how human rhinovirus alters the uptake capacities of macrophages (Jubrail et al., 2020), but whether the degradative capacities of the virus-treated macrophages is altered in these conditions is unclear (Jubrail J, 2017).

SUMMARY OF THE INVENTION:

In this study, the inventors observed that HRV16 infected macrophages show defective phagolysosome biogenesis, leading to an inability to efficiently clear bacteria. Membrane trafficking is regulated by small GTPases of the Rab, Arf and Arf-related (Ari) families, which recruit and activate the fusion machineries on specific compartments. They report here that the ARL5b protein (Houghton et al., 2012) expression is upregulated by the rhinovirus in target cells. They further demonstrated that the depletion of ARL5b in HRV16-challenged macrophages prevents the viral induced changes in the endocytic machinery and improves bacterial clearance. Therefore, they provide the first direct evidence that ARL5b is targeted by HRV16 and controls endocytic dynamics in infected cells. These results offer a new connection between viral factories and a comprehensive insight into a key molecular mechanism used by HRV16 to hijack and disrupt macrophage phagocytic and antimicrobial function.

Thus, the invention relates to an inhibitor of ARL5b for use in the improvement of phagocytosis in a subject in need thereof.

Particularly, the invention is defined by its claims.

DETAILED DESCRIPTION OF THE INVENTION:

Accordingly, the present invention relates to an inhibitor of ARL5b for use in the improvement of phagocytosis in a subject in need thereof.

As used herein, the term “ARL5b” for “ADP-ribosylation factor-like protein 5B” has its general meaning in the art and denotes a major regulator for retrograde transport and transport between the Golgi apparatus and endosomes. Its UniProtKB/Swiss-Prot accession number is Q96KC2. As used herein, the term “inhibitor of ARL5b” denotes all molecules which inhibit the activity and the expression of ARL5b. The term “inhibitor of ARL5b” also denotes inhibitors of the expression of the gene coding for ARL5b (Entrez Gene number: 221079).

Binding to ARL5b and inhibition of the biological activity of ARL5b may be determined by any competing assays well known in the art. For example, the assay may consist in determining the ability of the agent to be tested as inhibitor of ARL5b to bind to ARL5b. The binding ability is reflected by the Kd measurement. The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M). KD values for binding biomolecules can be determined using methods well established in the art. In specific embodiments, an inhibitor of ARL5b that binds to ARL5b is intended to refer to an inhibitor that binds to human ARL5b with a KD of IpM or less, lOOnM or less, lOnM or less, or 3nM or less. Then a competitive assay may be settled to determine the ability of the agent to inhibit biological activity of ARL5b. The functional assays may be envisaged such evaluating bacterial clearance. In order to test the functionality of a putative inhibitor of ARL5b a test is necessary. For that purpose, to identify inhibitor of ARL5b a bacterial clearance test could be used, with live bacteria (see for example Jubrail Jamil et al, EMBO Rep 2020).

As used herein, the term “subject” denotes a mammal. Typically, a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with inflammatory diseases. Typically, a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with like chronic obstructive pulmonary disease (COPD) or asthma.

Thus, the invention also relates to an inhibitor of ARL5b for use in the improvement of phagocytosis in a subject afflicted with an inflammatory disease. In other word, the invention also related to an inhibitor of ARL5b for use in the treatment of an inflammatory disease. Particularly, the inflammatory diseases are chronic obstructive pulmonary disease (COPD) or asthma.

Particularly, the invention relates to an inhibitor of ARL5b for use in the treatment of chronic obstructive pulmonary disease (COPD) or asthma.

More particularly, the invention relates to an inhibitor of ARL5b for use in the treatment of chronic obstructive pulmonary disease (COPD) or asthma induced by a respiratory virus. As used herein, the term “asthma” has its general meaning in the art and refers to a condition of the airways of the lung. Inflammation and tightening of the muscles around the small airways causes asthma symptoms such as cough, wheeze, shortness of breath and chest tightness. Asthma exacerbation, also called an asthma attack, is defined as a worsening asthma symptoms and lung function, i.e as a respiratory attack that requires emergency treatment. Respiratory infections are the main culprits of asthma exacerbations, and rhinovirus being the most common agent.

As used herein, the term “respiratory virus” has its general meaning in the art and refers to a viruses inducing upper and lower respiratory tract infections. These respiratory viruses include members of the Pneumoviridae family, including human respiratory syncytial virus (hRSV) type A and B, and human metapneumovirus (hMPV) type A and B; members of the Paramyxoviridae family, including parainfluenza virus type 3 (PIV-3) and measles virus; and members of the Coronaviridae family, including endemic human coronaviruses (HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKUl); severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle-East respiratory syndrome coronavirus (MERS-CoV).

Particularly, the respiratory virus is the human rhinovirus (HRV) or the Human respiratory syncytial virus (HRSV).

More particularly, the invention relates to an inhibitor of ARL5b for use in the treatment of chronic obstructive pulmonary disease (COPD) or asthma exacerbations induced by a respiratory virus.

As used herein, the term "Chronic obstructive pulmonary disease (COPD)" has its general meaning in the art and is a type of obstructive lung disease characterized by long-term breathing problems and poor airflow. The main symptoms include shortness of breath and cough with sputum production. COPD is a progressive disease, meaning it typically worsens over time. Eventually, everyday activities such as walking or getting dressed become difficult. Chronic bronchitis and emphysema are older terms used for different types of COPD. The term "chronic bronchitis" is still used to define a productive cough that is present for at least three months each year for two years. Those with such a cough are at a greater risk of developing COPD. The term "emphysema" is also used for the abnormal presence of air or other gas within tissues.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

In some embodiments, the inhibitor of ARL5b according to the invention targets specifically macrophages.

In other words, in some embodiments, the inhibitor of ARL5b according to the invention is designed to target macrophages. For example, antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence may be under the control of a macrophage-specific promoters.

Macrophage-specific promoters are well known in the art (e.g see for example Kang W S, et al. Gene Ther. 2014; Ellett F, et al. Blood. 2017; Walton E M, et al. PLoS One. 2015; Luo Y-L, et al. ACS Nano. 2018; Greaves D R, et al. Int J Hemato. 2002 ; He W, et al. Hum Gene Ther. 2006)

In some embodiment, the inhibitor of ARL5b is combined with a macrophage-targeted drug delivery system. Macrophage-targeted drug delivery systems and methods for designing and producing drug delivery system to target macrophage are well known in the art (e.g. see for example Hu G, et al. Front. Immunol. 2019; Mukhtar M, et al. Expert Opin Drug Deliv. 2020; He W, et al. Advanced Drug Delivery Reviews. 2019; Chono S. Yakugaku Zasshi. 2007).

In one embodiment, the inhibitor of ARL5b according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).

The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.

In another embodiment, the antibody according to the invention is a single domain antibody against ARL5b. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb. The term “VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term “complementarity determining region” or “CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH.

The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation.

VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).

In one embodiment, the compound according to the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

Then, for this invention, neutralizing aptamers ARL5b are selected.

In one embodiment, the compound according to the invention is a polypeptide.

In a particular embodiment the polypeptide is an antagonist of ARL5b and is capable to prevent the function of ARL5b. Particularly, the polypeptide can be a mutated ARL5b or a similar protein without the function of ARL5b.

In one embodiment, the polypeptide of the invention may be linked to a cell-penetrating peptide” to allow the penetration of the polypeptide in the cell. The term “cell-penetrating peptides” are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).

The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.

When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.

In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.

A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain. Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.

Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N. J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory halflife of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa).

In addition, to the polymer backbone being important in maintaining circulatory halflife, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.

In another embodiment, the ARL5b inhibitor according to the invention is an inhibitor of ARL5b gene expression.

Small inhibitory RNAs (siRNAs) can also function as inhibitors of N ARL5b expression for use in the present invention. ARL5b gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that ARL5b gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

In a particular embodiment, an siRNA according to the invention can have the following sequences: siARL5b.l (SEQ ID NO: 1): GUUCAUCAUUCUUGUUGUU siARL5b.2 (SEQ ID NO: 2): CUCAUGAGGAUUUACGGAA Ribozymes can also function as inhibitors of ARL5b gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of ARL5B mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays. Both antisense oligonucleotides and ribozymes useful as inhibitors of ARL5b gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing Notch receptors or Notch ligands. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991).

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wildtype adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigenencoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and mi croencap sul ati on .

In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter may be specific for macrophages. Thus, in preferred embodiments, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a macrophage-specific promoter. For example, a specific expression in myeoloid cells (monocytes, macrophages) may be obtained through the promoter of the lysozyme 2 gene (Lyz2) is suitable. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.

In a further aspect, the present invention relates to an inhibitor of ARL5b according to the invention in combination with one or more anti-COPD compound for use in the treatment of COPD in a subject in need thereof.

The term “anti-COPD” has its general meaning in the art and refers to compounds and therapeutic active agent used which can be used to treat the symptoms and the progression of the disease. Anti-COPD compounds can be bronchodilators like P2 agonists and anticholinergics or corticosteroids.

In a further aspect, the present invention relates to an inhibitor of ARL5b according to the invention in combination with one or more anti-asthma compound for use in the treatment of asthma in a subject in need thereof.

The term “anti-asthma” has its general meaning in the art and refers to compounds and therapeutic active agent used which can be used to treat asthma. Anti-asthma compounds can be beta2-adrenoceptor agonists like salbutamol, anticholinergic like ipratropium bromide or adrenergic agonists like epinephrine.

In some embodiments, the inhibitor of ARL5b of the present invention is administered sequentially or concomitantly with one or more therapeutic active agent.

In one embodiment, said additional active compounds may be contained in the same composition or administrated separately.

Typically an inhibitor of ARL5b according to the invention as described above are administered to the subject in a therapeutically effective amount.

By a "therapeutically effective amount" of the inhibitor of ARL5b of the present invention as above described is meant a sufficient amount of the inhibitor of ARL5b for treating COPOD and/or asthma at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the inhibitor of ARL5b of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific inhibitor of ARL5b n employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific inhibitor of ARL5b n employed; the duration of the treatment; drugs used in combination or coincidental with the specific inhibitor of ARL5b employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the inhibitor of ARL5b at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the inhibitor of ARL5b of the present invention for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the inhibitor of ARL5b of the present invention, preferably from 1 mg to about 100 mg of the inhibitor of ARL5b of the present invention. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

In a particular embodiment, the inhibitor of ARL5b according to the invention may be used in a concentration between 0.01 pM and 20 pM, particularly, the inhibitor of ARL5b of the invention may be used in a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 pM.

In some embodiments, the inhibitor of ARL5b is administered to the respiratory tract (e.g. lungs).

According to the invention, the inhibitor of ARL5b of the present invention is administered to the subject in the form of a pharmaceutical composition. Thus, the invention also relates to a therapeutic composition comprising an inhibitor of ARL5b for use in the improvement of phagocytosis in a subject in need thereof.

Typically, the inhibitor of ARL5b of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising the inhibitor of ARL5b of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The inhibitor of ARL5b of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized agent of the present inventions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the inhibitor of ARL5b of the present invention plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media, which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The pharmaceutical composition of the invention may conveniently be administered by any method that allows administration to the respiratory tract (e.g. lungs). For example, nasal drops can be instilled in the nasal cavity by tilting the head back sufficiently and apply the drops into the nares. The drops may also be inhaled through the nose. Alternatively, a liquid preparation may be placed into an appropriate device so that it may be aerosolized for inhalation through the nasal or buccal cavity. For administration by inhalation the compositions may be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant. Administered spray and drops can be a single dose or multiple doses. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable diluent is dictated by the amount of inhibitor ofARL5b with which it is to be combined, the route of administration and other well- known variables. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In another embodiment, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of COPD and/or asthma in a subject in need thereof.

In a further aspect, the present invention relates to a method for improving phagocytosis in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of an inhibitor of ARL5b.

The invention also provides kits comprising the inhibitor of ARL5b of the invention.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: hMDMs were treated with siRNA against luciferase (siLuc, control) or ARL5b (siARL5b.l and siARL5b.2) and then challenged with HRV16. (A) Cells were lysed to analyse the protein content by Western blot. Representative immunoblot with anti-ARL5b and anti-GAPDH as a loading control. (B) Quantification of the ARL5b band intensity relative to GAPDH and normalized to control (siLuc) of each condition. n=4, **p<0.01 , ****p<0.0001 One sample t test. (C) Quantification of the intensity of EEA1 staining in 60 random cells, **** p<0.0001 One Way Anova with Bonferonni Post Test vs siLuciferase. (D) CD63 surface staining n=4, ****p<0.0001 One Way Anova with Bonferonni Post Test vs MI. (E) Intracellular NTHi survival following ARL5b depletion and HRV16 challenge, n=3 *p<0.05, **p<0.01 Two Way Anova with Dunnett’s Post Test vs MI or vs siLuciferase HRV16 (for siARL5b).

EXAMPLE:

Material & Methods

Antibodies and reagents

The following primary antibodies were used: purified rabbit anti- SRBCs (IGN Biochemicals), mouse anti-tubulin alpha (clone DM1A, Sigma, T9026), mouse anti-human EEA1 (BD Transduction Laboratories, 610456), mouse monoclonal CD63 (clone TS63, Eurobio) and mouse anti -human LAMP1 (clone H4A3, BD Bioscience). DAPI was from Sigma (D9542). Secondary antibodies were: Alexa Fluor 488, Cy3/5-labelled F(ab’)2 anti-mouse or rabbit IgG; horseradish peroxidase (HRP)-labelled anti-mouse and anti-rabbit IgG (Jackson Immunoresearch). siRNA sequences were: 5’GUU CAU CAU UCU UGU UGU U3’ (siARL5b.l, SEQ ID NO: 1) and 5’CUC AUG AGG AUU UAC GGA A3’ (siARL5b.2, SEQ I DNO: 2), 5’CGU ACG CGG AAU ACU UCG A3’ (siLuciferase, SEQ ID NO: 3).

Cell Culture

Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy donors (Etablissement Frangais du Sang Ile-de-France, Site Trinite, Inserm agreement #15/EFS/012 and #18ZEFS/030 ensuring that all donors gave a written informed consent and providing anonymized samples) by density gradient sedimentation using Ficoll- Plaque (GE Healthcare). This was followed by adhesion on plastic at 37°C for 2 h and culture in the presence of adhesion medium (RPMI 1640 (Life Technologies) supplemented with 100 pg/ml streptomycin/penicillin and 2 mM L-glutamine (Invitrogen/Gibco)). Then, the adhered cells were washed once with warm adhesion medium and left to rest in macrophage medium (adhesion medium supplemented with 10% FCS (Eurobio). The next day, cultures were washed with adhesion medium and then supplemented every 2 days with fresh macrophage medium. The adherent monocytes were left to differentiate into macrophages as described previously (Jubrail et al., 2020) and used after 10 days. HeLa Ohio cells were purchased from the European Collection of Authenticated Cell Cultures (ECACC) and were cultured in DMEM GlutaMax containing 25 mM D-glucose (Life Technologies) supplemented with 10% FCS, 100 pg/ml streptomycin/penicillin and 2 mM L- glutamine. They were passaged every 3 days.

Bacterial strains and culture

NTHi strain RdKW20 (Bishop-Hurley et al., 2005; Domenech et al., 2016) and Moraxella catarrhalis strain 25293 (Blakeway et al., 2014) were purchased from the American Type Culture Collection (ATCC). Staphylococcus aureus strain 160201753001 and Pseudomonas aeruginosa strain 160601067201 from blood culture were provided by Professor Claire Poyart (Cochin Hospital). NTHi, S. aureus and P. aeruginosa were cultured on chocolate agar plates and M. catarrhalis was cultured on brain-heart infusion (BHI) agar plates. Plates were incubated for 24 h at 37°C until colonies appeared. All strains were grown in LB medium but for NTHi, this was also supplemented with 10 pg/ml hemin and 1 pg/ml nicotinamide adenine dinucleotide (NAD).

Human rhinovirus production

Human Rhinovirus 16 (HRV16) (VR-283, strain 11757, lot 62342987) was purchased from the ATCC and stocks were produced by infecting HeLa Ohio cells in virus medium (DMEM GlutaMax containing 25 mM D-glucose supplemented with 10% FCS and 2 mM L- glutamine) as described previously (Bennett et al., 2012). Briefly, HeLa Ohio cells were grown to 80% confluence and infected with 5 ml HRV16 or control media for 1 h at room temperature with agitation. The remaining solution was made to 10 ml and the cells with HRV16 left for 48 h to allow for 90% CPE to develop. Supernatants were then clarified by centrifugation and filtration (Bennett et al., 2012) and 1 ml stocks were produced and stored at -80°C. To UV inactivate HRV16 it was treated with UV light (1000mJ/cm2) for 20 minutes. Inactivation was confirmed by adding the inactivated virus to HeLa Ohio cells and checking for CPE.

Quantification of the tissue culture infective dose 50 (TCID50) of HRV16

HeLa Ohio cells were cultivated in 96 well plates at 1 x 105 cells/well for 24 h. HRV16 was diluted 10-fold from undiluted to 10-9 in virus medium 50 pl of each dilution was added to the cells in 8 replicate wells. 50 pl of virus medium was added to 2 groups of control wells in 8 replicate wells per group. Cultures were incubated for 4 days at 37°C until CPE was observed in 50% of wells. TCID50 was calculated using the Spearman-Karber formula as previously outlined (Bennett et al., 2012).

HRV16 and bacterial infection of human macrophages Macrophages were washed once in PBS and rested in virus medium. HRV16, HRV16UV or MI supernatants were added to the macrophages and placed at room temperature for 1 h with agitation to achieve 1 x 10⁷ TCIDso/ml. Cultures were then washed with virus medium and rested in macrophage medium overnight.

NTHi, M. catarrhalis, S. aureus or P. aeruginosa were grown until mid-log growth phase, centrifuged at 1692 x g for 5 min and re-suspended in 1 ml phagocytosis medium (RPMI 1640 supplemented with 2 mM L-glutamine). Bacteria were added to macrophages pre-treated with HRV16, HRV16UV or mock infected (MI) to achieve a multiplicity of infection (MOI) of 10/cell or 40/cell (HRV16 only). Cultures were then centrifuged at 602 x g for 2 min and placed at 37°C, 5% CO2 for 1 h. Cultures were then washed with PBS and treated with 100 pg/ml gentamicin (NTHi, S. aureus, P. aeruginosa) or 20 pg/ml (M. catarrhalis) for 20 min. The 1 h cultures were washed and lysed in saponin as previously described (Jubrail et al., 2016) and colony forming units (CFU) estimated using the Miles-Misra technique (Miles et al., 1938). The remaining cultures were left in 2 pg/ml gentamicin for 0.5 h, 3.5 h or 24 h and treated in the same manner to determine intracellular CFU.

Measurement of phagolysosome activity by flow cytometry

Macrophages were infected with HRV16 or controls as described above. After overnight rest cultures were washed with PBS and challenged with either DQ-BSA or H2DCFDA- Oxyburst IgG-opsonized carboxylate beads (kind gift from Dr David Russell, Cornell University, USA) for up to 2 h (Podinovskaia et al, 2013; Dumas et al, 2015). At each time point, cultures were washed with phagocytosis medium and fixed in 4% paraformaldehyde (Sigma-Aldrich) on ice for 45 min. They were then treated with 0.05 M NH4C1/PBS1X for 7 min and detached. Analysis was performed using the BD Fortessa through the APC (calibrator) and Alexa Fluor 488 (sensor) channels acquiring 10,000 events per sample.

FcR-mediated phagocytosis and phagosome staining

Macrophages were challenged with IgG-opsonized SRBC for up to 60 min. SRBCs were washed in PBS/BSA 0.1% and opsonized for 30 min with rotation in rabbit-IgG anti-SRBCs. They were further washed, re-suspended in phagocytosis medium and added to macrophages to give approximately 10 SRBCs per cell. The plates were centrifuged at room temperature at 502 x g for 2 min and then placed at 37°C for various time points. At each time point, cells were washed with room temperature phagocytosis medium and fixed in 4% paraformaldehyde (PF A) at room temperature for 15 min and then treated with 0.05 M NH4C1/PBS1X for 10 min.

Microscopy Cultures were washed in PBSlX/2% FCS and external SRBCs were labelled for 30 min with F(ab’)2 anti-rabbit IgG Alexa Fluor 488 in PBS1X /2% FCS. Cells were then washed with PBS1X /2%FCS and re-fixed in 4% paraformaldehyde (PF A) for 15 min at room temperature and then treated with 0.05 M NH4C1/PBS1X for 10 min before being permeabilized in PBS1X/2%FCS/O.O5% saponin (permeabilization buffer). Recruitment of markers around phagosomes was then detected using either anti-human EEA1 (BD), anti-CD63 (Eurobio) or anti -human LAMP1 (BD) in permeabilization buffer for 45 min. After washing, cultures were stained with Cy3-labelled F(ab’)2 anti-mouse IgG (to detect markers) or Cy5-labelled F(ab’)2 anti-rabbit IgG (to detect intracellular SRBCs) in the same buffer for 30 min. After washing in permeabilization buffer cells were stained with DAPI for 5 min and mounted using Fluormount G (Interchim). To quantify phagocytosis, the numbers of internalized and bound SRBCs were counted in 30 cells randomly chosen on the coverslips. The phagocytic index, i.e. the mean number of internalized SRBCs per cell, was calculated. The index obtained for virus-treated cells was expressed as a percentage of the index obtained for control cells. The index of association, which corresponds to the number of bound and internalized SRBCs per cell, was also calculated. To determine the number of internalized SRBCs with specific recruitment of EEA1, CD63 or LAMP1, the internalized SRBCs were counted and scored as positive or negative for each marker. The percentage of positive phagosomes was then calculated. Image acquisition was performed on an inverted wide-field microscope (Leica DMI6000) with lOOx (1.4 NA) objective and a MicroMAX camera (Princeton Instruments). Z series of images were taken at 0.3 pm increments.

To analyse EEA1 or CD63 staining after HRV16 challenge and overnight rest, cultures were washed in PBSlX/2% FCS/0.05% saponin (permeabilization buffer) and labelled for 45 minutes with anti-EEAl or anti-CD63 in the same buffer. Cells were then washed with permeabilization buffer and incubated with Cy3 -labelled F(ab’)2 anti -mouse IgG in the same buffer for 30 min. After washing, cells were stained with DAPI for 5 min and mounted using Fluormount G (Interchim). Images were acquired on an inverted wide-field microscope (Leica DMI6000) with lOOx (1.4 NA) objective and a MicroMAX camera (Princeton Instruments) acquiring 30 random cells per coverslip. Z series of images were taken at 0.3 pm increments.

Quantification of EEA1 and CD63 intensity and number in macrophages

To quantify the intensity of staining of EEA1 and CD63 in macrophages and the total number of vesicles in macrophages, a macro was developed that quantified the two parameters on a per cell basis. Quantification was performed using Imaged 64bit software on entire 16-bit Z stacks. In order to properly quantify the number and intensity of single endosomes, Z stack images of the entire thickness of cells were acquired. On a duplicate stack, after z-max projection, the number of cells to be quantified in the field of view was manually selected (all cells completely within the field were included).

For each selected cell a freehand selection was made to fix the cell boundaries as well as the center of the nucleus. After a denoising process, a top-hat filter was applied, and a mask created by automatic threshold (Otsu algorithm). The number of endosomes and their intensity were calculated. The results were reported as a summary for each cell in ImageJ, transferred to Graphpad Prism and plotted.

Western blots

Macrophages were lysed with lysis buffer (20 mM Tris HC1, pH 7.5, 150 mM NaCl, 0.5% NP-40, 50 mM NaF and 1 mM sodium orthovanadate supplemented with complete protease inhibitor cocktail (Roche Diagnostic)) for 15 min. Lysates were centrifuged at 16,100 x g for 10 min at 4°C. The supernatants were removed and stored at -20°C and an equal amount of protein (BCA dosage kit, Pierce) was analyzed by SDS-PAGE. Proteins were transferred onto a poly vinylidene difluoride (PVDF) membrane (Millipore) at 4°C for 100 min and incubated in blocking solution (0.1% Tween-20 supplemented with 5% milk or BSA in TBS IX) for 2 h. Blots were rinsed with TBS/0.1% Tween-20 and primary antibodies were incubated in the blocking solution overnight or for 2 h as required. The membrane was further washed and incubated with HRP-coupled secondary antibodies in blocking buffer for 45 min. Detection was performed using ECL Dura Substrate (GE Healthcare) and bands imaged by Fusion (Vilber Lourmat) and quantified in ImageJ.

CD63 surface staining by flow cytometry

Macrophages were infected with HRV16 or MI control as described above. After overnight rest, cultures were washed with PBS and stained with mouse monoclonal anti-CD63 on ice for 45 min in PBSlX/2% FCS. They were washed with PBSlX/2% FCS and stained with Alexa Fluor 488-labelled F(ab’)2 anti-mouse IgG for 30 min in PBSlX/2% FCS on ice. They were then fixed in 4% paraformaldehyde on ice for 45 min, treated with 0.05M NH4Cl/PBSlX for 10 min and then analyzed by BD Fortessa using the Alexa Fluor 488 channel acquiring 10,000 events per sample.

Next generation RNA sequencing hMDMs were challenged with HRV 16, HRV 16UV or mock infected and after overnight rest were lysed for total RNA using Trizol (Sigma). Lysed samples were then stored at -80°C until processing. RNA was extracted using the Qiagen RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions.

RNA integrity was analysed on the Fragment Analyzer platform (AATI, IA, USA) using standard sensitivity RNA kit. RNA was then diluted to 20 ng/ul and used as input to create mRNA libraries using TruSeq Stranded mRNA kit (Illumina, CA, USA) with dual indexing following standard instructions. Libraries were validated on the Fragment Analyzer platform (AATI, IA, USA) using standard sensitivity NGS fragment analysis kit and the concentration was determined using Quant-iT dsDNA High Sensitivity assay kit on the Qubit fluorometer (Thermo Fisher, MA, USA). Sample libraries were pooled in equimolar concentrations and diluted and denatured according to Illumina guidelines. Sequencing was performed using High Output 2 x 76 bp kit on an Illumina NextSeq500.

RNA sequencing fastq files were processed using bcbio-nextgen (version 0.9.9) where reads were mapped to the human genome build hg38 (GRCh38.78) using hisat2 (version 2.0.4) yielding between 17.3-38.3 million mapped reads (average 25.5 million) with a mapping frequency ranging between 86-95% (average 90%) per sample. Sequence quality was evaluated by inspection of phred scores, per N base content, per sequence GC content, duplication levels, genomic distribution of mapped reads, and gene coverage. Gene level quantifications, counts and transcript per million (TPM), were generated with featurecounts (version 1.4.4) and sailfish 5version 0.10.1), respectively, all within bcbio. Differential gene expression was assessed in R (version 3.3.1) with DESEq2, using raw counts as input. Genes were considered significantly differentially expressed if they had a FDR<0.05 using a Benjamini -Hochberg method for multiple testing correction. Array Studio version 10 (OmicSoft, Cary, NC) was used for further data analysis. qPCR

Macrophages were infected with HRV16 or MI control as described above. After overnight rest, cultures were washed with PBS and RNA was extracted as previously described (Chomczynski and Sacchi, 1987). Briefly, hMDMs were washed with PBS at room temperature and lysed using Trizol reagent (ThermoFischer Scientific). Proteins (organic phase) and RNA and DNA (aqueous phase) were separated using chloroform for 2 min at room temperature followed by 15 min centrifugation at 4°C at 12,000 x g. The aqueous phase was collected and isopropanol was added to precipitate RNA and incubated for 10 min at room temperature. Samples were centrifuged for 20 min at 4°C at 15,000 x g and the pellet of RNA washed with 75% ethanol and centrifuged for a further 5 min at 4°C at 10,000 x g. The pellet was dried at room temperature and resuspended in pure water and warmed at 55°C for 5 min. The total amount of RNA was quantified using nanodrop. For reverse transcription Ipg of mRNA was retro-transcribed into DNA using SuperScript II Reverse Transcriptase (ThermoFischer Scientific). qPCR was performed using the LightCycler 480 SYBR Green I Master (Roche) with specific oligos to detect ARL5b with 18S RNA as control (Table 1). siRNA Treatment

Macrophages at day 7 were washed twice with macrophage medium and kept in macrophage medium at 37°C. The siRNA solution was prepared in OptiMEM medium (GlutaMAX supplemented, Gibco), containing lipofectamine RNAiMAX reagent (Invitrogen) and siRNA at a final concentration of 240 nM. siRNA was added to each well and cultures left for 24 h at 37°C before being infected with HRV16 or MI control and processed for FcR phagocytosis, flow cytometry or bacterial clearance as outlined above.

Statistics

Statistical tests were performed using Graphpad prism version 6 software. All statistical tests are listed in the figure legends and significance was determined if p<0.05.

Results

Human rhinovirus 16 impairs human macrophage ability to clear respiratory bacteria

We first set out to determine if macrophages could clear internalized bacteria after HRV16 challenge. We challenged human monocyte-derived macrophages (hMDMs) with HRV16, HRV16 inactivated by a UV treatment (HRV16UV) or mock infected medium (MI) for 1 h at room temperature followed by overnight rest. The next day, we exposed them to non- typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis, Staphylococcus aureus or Pseudomonas aeruginosa and monitored bacterial internalization at 1 h after washing and incubation with antibiotics to kill extracellular bacteria. We then maintained cultures in low dose antibiotic and measured intracellular bacterial survival over 24 h (data not shown). In control conditions, challenge of hMDMs with NTHi, M. catarrhalis or P. aeruginosa resulted in approximately 80% clearance after 24 h (data not shown), while challenge with S. aureus lead to approximately 40% clearance after 24 h (data not shown). In contrast, hMDMs challenged with HRV16 were significantly impaired in their ability to clear the four bacteria over 24 h compared to hMDMs challenged with HRV16UV or mock infected, resulting in approximately 20% clearance of data not shown).

Taken together, these results demonstrate that hMDMs challenged with HRV16 are impaired in intracellular bacterial clearance and killing, suggesting modifications of phagolysosome activity and/or generation. Human macrophages challenged with rhinovirus 16 show reduced hydrolytic activity and reactive oxygen species production

We next wanted to determine if the inability of macrophages challenged with HRV16 to clear internalized bacteria could be related to phagolysosome activity. For this, we analyzed the late steps of phagosome maturation and the luminal content of phagosomes using 3 pm beads decorated with IgG to target the Fc receptors (FcR). These beads were coupled to a fluorophore sensitive to the hydrolytic activity (DQ-BSA beads) or to the oxidative burst and the presence of reactive oxygen species (ROS) produced in the phagolysosome (dichlorodihydrofluorescein diacetate (H2DCFDA)-OxyBURST beads) as well as a calibration fluorophore (Podinovskaia et al., 2013b; Yates and Russell, 2008) (data not shown). Human macrophages pre-exposed to HRV16 or not were incubated with the beads for up to 120 min and analyzed by flow cytometry, focusing on the population associated with beads. The hydrolytic activity was detected as early as 30 min after bead contact and increased with time till 120 min in control conditions (cells treated with the UV-inactivated HRV16 or mock infected) (data not shown). hMDMs challenged with HRV16 showed the same initial detection of the phagosomal hydrolytic activity at 30 min and a gradual increase over 120 min, but from 60 min there was significantly less hydrolytic activity compared to control infected macrophages (data not shown). The oxidative burst was detected as soon as 10 min in control conditions with a peak at 30 min and a decline by 120 min (data not shown). In hMDMs challenged with HRV16, the detection of ROS in phagosomes was reduced at each time point, which was significant at 20 and 30 min compared to mock infected macrophages. As the peak in production was still at 30 min, we can infer from the data that HRV16 challenge of macrophages lead to a decrease in ROS production rather than a delay (data not shown). These results demonstrate that there is an impairment of the phagolysosome activity in HRV16 challenged hMDMs.

Human macrophages treated with Human rhinovirus 16 exhibit defective phagosome maturation

To better identify where the phagosome maturation is arrested, hMDMs were allowed to internalize IgG-opsonised sheep red blood cells (SRBCs) for various time points before staining them for the early endosomal marker EEA1 (data not shown), the late endosomal marker CD63 (data not shown) and the late endosome/ lysosomal marker LAMP1 (data not shown F). We observed thatEEAl was acquired on phagosomes at 10 and 15 min (data not shown) in HRV16-treated cells as well as in control mock-infected cells. This marker was progressively lost from phagosomes in control conditions, but not in HRV16-treated macrophages, where it was still present at 60 min (data not shown). The late endosomal marker CD63 was enriched on phagosomes at 30 min before being lost at 60 min in control conditions (data not shown). By contrast, it remained associated with the phagosomes at the same level at 30 and 60 min in HRV16-treated macrophages (data not shown). We then monitored the late endosomal/ lysosomal marker LAMP1, which was strongly recruited on phagosomes in control cells at 30 and 60 min, but significantly less on the phagosomes of HRV16-treated macrophages (data not shown).

Together, these results indicate that the phagosome maturation is arrested in HRV16- treated macrophages after the acquisition of the endosomal markers EEA1 and CD63, with a defective recruitment of the late endosomal/ lysosomal marker LAMP1 and a perturbed biogenesis of phagolysosomes.

Human rhinovirus 16 impairs the expression of EEA1 and CD63 in macrophages

Next, we investigated whether HRV16 challenge of hMDMs would impair the localization or the expression of endocytic markers, which could secondarily lead to a defective recruitment around phagosomes. For this, after HRV16 challenge or mock infection, we first stained hMDMs for EEA1. We observed that the EEA1 staining was uniformly distributed throughout the cytosol in mock-infected hMDMs (data not shown). By contrast, HRV16 challenged hMDMs exhibited a more intense EEA1 staining throughout the cell (data not shown). We then quantified the fluorescence intensities using a macro that allowed us to get a measure of EEA1 staining. This quantification confirmed that, relative to mock-infected hMDMs, the intensity of EEA1 staining in HRV16 challenged hMDMs was significantly higher (data not shown). Additionally, there was no change in the number of EE Al -positive vesicles in HRV16 challenged hMDMs (data not shown), but western blotting demonstrated that the total amount of EEA1 protein was increased in HRV16 challenged hMDMs (data not shown). These results demonstrate that HRV16 increases the amount of EEA1 in macrophages.

We then analyzed the expression pattern of CD63 in HRV16 challenged hMDMs. We observed that HRV16 challenged hMDMs had a more heterogeneous localization of CD63 than mock-infected cells (data not shown), with increased staining accumulated at the periphery and close to the plasma membrane (data not shown). Image quantification revealed that CD63- associated fluorescence intensity was significantly higher in HRV16 challenged hMDMs compared to mock-infected cells (data not shown). Furthermore, the number of CD63 positive late endosomes was significantly greater in HRV16 challenged hMDMs compared to mock- infected hMDMs (data not shown). In addition, the surface expression of CD63 was assessed by flow cytometry. In HRV16 challenged hMDMs, the surface level of CD63 was two times higher than in mock-infected macrophages (data not shown). These results show that HRV16 alters the expression profile and distribution of CD63 in HRV16-infected macrophages.

Taken together, these results demonstrate that HRV16 alters the expression and localization of endosomal compartments in macrophages, which could contribute to the perturbation in their recruitment on phagosomes.

Transcriptomic analysis of Human rhinovirus 16-treated macrophages reveals that the expression of ARL5b is upregulated

To allow us to identify potential host cell candidates that HRV16 could affect in macrophages to drive the perturbations we observed, we undertook an RNA sequencing approach on HRV16 treated hMDMs versus HRV16UV treated hMDMs and hMDMs treated with mock medium (mock infection). We identified 2067 differentially expressed genes (DEGs) induced by HRV16 and 2471 induced by HRV16UV as compared to mock infected hMDMs with a common overlap of 1510 genes (false discovery rate (FDR) <0.05) (data not shown). When we compared the fold-changes induced by HRV16 or HRV16UV, we identified a group of genes where the responses to the two treatments differed greatly (data not shown). A total number of 160 genes showed a larger variation from the normal spread than that of normal variation (data not shown). Of these 160 genes, two gene clusters (cluster 2 and 3) where the response was unique to HRV16 and a further two gene clusters (cluster 1 and 4) where the response was unique to HRV16UV were identified (data not shown). We re-analyzed the genes in clusters 2 and 3 and identified 33 genes expressed higher in HRV16 challenged hMDMs than in control conditions. Among these 33 genes, we focused on the GTPase ARL5b, which was consistently and significantly upregulated in HRV16 challenged hMDMs (data not shown). The role of ARL5b in macrophages remains unknown, but this small GTP binding protein was reported to control anterograde and retrograde trafficking from and towards the Golgi apparatus (Houghton et al., 2012). We therefore decided to investigate further its function in HRV16- treated macrophages.

ARL5b depletion prevents HRV16-mediated endosomal defects and restores bacterial clearance in macrophages To confirm that HRV16 affects ARL5b in macrophages, we first used RT-qPCR to measure its expression in human macrophages after HRV16 challenge. We found that HRV16 induced a significant increase in ARL5b expression in macrophages when compared to mock- infected cells (data not shown). This increase could also be detected at the level of the protein by western blotting (data not shown).

Next, we used an siRNA approach to determine if decreasing ARL5b expression in hMDMs before HRV16 challenge could compensate for the degradative defects we observed previously (data not shown). We first treated hMDMs with two siRNA sequences against ARL5b (siARL5b.l and siARL5b.2) or a control luciferase siRNA (siLuciferase) for 24 h and then challenged them with HRV16 or with mock-infected medium. Immunoblot analysis and quantification demonstrated significant ARL5b reduction for both siRNA sequences in 4 donors (Figure 1A, B). After the overnight rest, we either stained the hMDMs for EEA1, performed flow cytometry to assess the surface expression of CD63 or exposed them to NTHi for 1 h and assessed bacterial clearance over 4 h (Figure 1C-E).

We first observed that hMDMs treated with siLuciferase and then challenged with HRV16 showed a significant increase in the intensity of EE Al staining relative to mock- infected controls (data not shown), in agreement with the results presented previously. By contrast, in hMDMs treated with siARL5b.l or siARL5b.2 and then challenged with HRV16, there was no increase in EEA1 intensity (Figure 1C). Importantly the depletion of ARL5b did not affect the EEA1 intensity in mock-infected hMDMs (Figure 1C). Therefore, ARL5b mediates the HRV16-induced upregulation of EEA1 in human macrophages.

We next monitored the surface expression of CD63 in HRV16 challenged hMDMs after ARL5b depletion. In agreement with our previous results (data not shown), treatment of hMDMs with siLuciferase followed by HRV16 challenge led to a 2-fold increase in surface localized CD63 compared to mock infected hMDMs (data not shown). When we treated hMDMs with siARL5b. l or siARL5b.2 and then challenged them with HRV16 the surface expression of CD63 was reduced back to mock infected levels (Figure ID). These results demonstrate that ARL5b is crucial to mediate the HRV16 - driven mis localization and increase in surface CD63.

Finally, to demonstrate that the upregulation of ARL5b by HRV16 in macrophages has important consequences for viral-bacterial co-infections, we exposed hMDMs to NTHi after ARL5b depletion and HRV16 challenge. As expected based on our observations (data not shown), treatment of hMDMs with siLuciferase and then HRV16 challenge led to impaired clearance of NTHi over 4 h with significant intracellular persistence at 2 and 4 h compared to mock infected hMDMs (Figure IE). Importantly, treatment of hMDMs with siARL5b.l or siARL5b.2 before HRV16 challenge improved bacterial clearance in the hMDMs, as the intracellular bacterial numbers decreased over 4 h in a similar level as in mock-infected hMDMs (Figure IE). These results demonstrate that ARL5b plays a crucial role in HRV16-induced perturbation of the macrophages clearance functions.

Taken together, the results presented revealed that HRV16 hijacks the GTPase ARL5b in human macrophages, driving endosomal perturbations and leading to an inefficient intracellular bacterial clearance, and hence, to bacterial persistence.

Conclusion:

In this study, the inventors show that HRV16 impairs intracellular bacterial clearance in human macrophages, due to a defective process of phagosome maturation and degradation. They reveal that the small GTPase ARL5b is upregulated by the virus and plays a crucial role in this perturbation, by regulating the subcellular localization and recruitment of endocytic compartments, as depletion of ARL5b prevented these viral-induced changes.

They used siRNA-mediated depletion of ARL5b followed by HRV16 infection to determine if this could restore the clearance activity of macrophages. They first found that depletion of ARL5b, but not treatment with a control siRNA, prevented the HRV16-mediated increase in EEA1 intensity as well as the increase in surface CD63 expression. These data revealed that ARL5b regulates the expression and localization of endocytic compartments in virus-treated macrophages. Importantly, depletion of ARL5b restored bacterial clearance in HRV16 infected macrophages, which highlights ARL5b as a central target of HRV16 in macrophages leading to a severe block on bacterial clearance by perturbation of the intracellular endocytic trafficking. In addition, upregulation of ARL5b by the virus could also have important consequences for infection. Indeed, HRV16 was reported to replicate on a network of endomembranes in the proximity of the Golgi apparatus and the endoplasmic reticulum, which is orchestrated by viral and host proteins in epithelial cells (Roulin et al., 2014). This strategy is shared by other enteroviruses (Belov et al 2012, Hsu et al 2010, Spickler et al 2013).

These results therefore contribute to explain how HRV16 promotes bacterial superinfection by targeting a novel host cell factor. They therefore reveal that ARL5b is a major target of HRV16 infection, which opens new avenues for strategies to increase bacterial phagocytosis that would suppress colonisation in the airways. REFERENCES:

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Claims

- 35 - WO 2023/073099 PCT/EP2022/080075 CLAIMS:

1. An inhibitor of ARL5b for use in the improvement of phagocytosis in a subject in need thereof.

2. The inhibitor for use according to claim 1 wherein the subject is afflicted with an inflammatory disease.

3. The inhibitor for use according to claim 2 wherein the inflammatory diseases is a chronic obstructive pulmonary disease (COPD) or asthma.

4. The inhibitor for use according to claim 3 wherein the chronic obstructive pulmonary disease (COPD) or asthma is induced by a respiratory virus.

5. The inhibitor for use according to claim 4 wherein the respiratory virus is the human rhinovirus (HRV) or the Human respiratory syncytial virus (HRSV).

6. The inhibitor for use according to any claims 1 to 5 wherein said inhibitor is a siRNA.

7. The inhibitor for use according to any claim 6 wherein said siRNA has a nucleic acid sequence as set for SEQ ID NO: 1 or SEQ ID NO: 2.

8. A therapeutic composition comprising an inhibitor of ARL5b for use in the improvement of phagocytosis in a subject in need thereof.

9. A method for improving phagocytosis in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of inhibitor of ARL5b.