A Novel Therapeutic Modality Using CRISPR-Engineered Dendritic Cells to Treat Allergies

Biomaterials 273 (2021) 120798

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Biomaterials

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A novel therapeutic modality using CRISPR-engineered dendritic cells to treat allergies

Byoungjae Kim a,b,1, Young Eun Lee c,d,1, Ji Woo Yeon a, Ga-Yeon Go c, Junhyoung Byun a, Kijeong Lee a, Hyomin K. Lee e, Junho K. Hur f,g, Mihue Jang c,h,*, Tae Hoon Kim a,** a Department of Otorhinolaryngology-Head & Neck Surgery, College of Medicine, Korea University, Seoul, 02841, Republic of Korea b Neuroscience Research Institute, Korea University, College of Medicine, Seoul, 02841, Republic of Korea c Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seongbuk-Gu, Seoul, 02792, Republic of Korea d Department of Life Sciences, College of Life Sciences and Biotechnology, Korea University, Seoul, 02841, Republic of Korea e Department of Medicine, Major in Medical Genetics, Graduate School, Hanyang University, Seoul, 04763, Republic of Korea f Department of Genetics, College of Medicine, Hanyang University, Seoul, 04763, Republic of Korea g Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, 04763, Republic of Korea h KHU-KIST Department of Converging Science and Technology, Kyung Hee University, Seoul 02447, Republic of Korea

ARTICLE INFO ABSTRACT

Keywords: Despite the important roles of dendritic cells (DCs) in airway allergies, current therapeutic strategies such as Dendritic cells drugs, allergen immunotherapy and biologics haven’t been targeted at them. In this study, we established a CRISPR promising DC-based therapeutic approach for the alleviation of allergic rhinitis (AR)-associated allergic re Vacuolar protein sorting 37 actions, using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated targeted gene Adoptive cell therapies disruption. RNA sequencing analysis revealed upregulation of vacuolar protein sorting 37 B (VPS37B) in AR- Allergies derived DCs, indicating a novel molecular target. Following antigen presentation, VPS37A and VPS37B enabled endocytosis of the mannose receptor, which recognizes the house dust mite (HDM) allergen Der p 1. DCs with targeted disruption of VPS37A/B alleviated Th2 cytokine production when co-cultured in vitro with allo + geneic naïve CD4 T cell from patients with AR. Furthermore, nasal administration of Vps37a/b-disrupted bone marrow DCs to a mouse model of AR resulted in strongly reduced AR-related symptoms. Thus, this novel mo dality using genetically engineered DCs can provide an effective therapeutic and preventative strategy for allergic diseases.

1. Introduction Th2-driven immunity. DCs serve as sentinels on the mucosal surfaces, recognizing different antigens using various pattern recognition re Allergic airway diseases, including asthma, allergic rhinitis, and ceptors (PRRs), such as Toll-like receptors and C-type lectin receptors atopic dermatitis, are caused by antigen-specific inflammation charac (CLRs) [6,7]. For example, DC CLRs like dendritic cell-specific intra terized by a T helper type 2 (Th2) immune response and consequent cellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) and synthesis of allergen-specific immunoglobulin (Ig)E and Th2 cytokines mannose receptor (MR) recognize and internalize glycoallergens. such as interleukin (IL)4, IL5, and IL13 [1–3]. As antigen presenting cells Interestingly, DCs derived from patients with house dust mite (HDM) residing in the airway lumen, dendritic cells (DCs) play key roles in allergies have higher MR expression than DCs from healthy controls, and allergic inflammation [3,4]. DCs form a physical network with airway these patients have increased numbers of MR-expressing myeloid DCs as epithelial cells (AECs) that plays important roles in modulating in well [8–10]. MR-deficient DCs fail to induce Th2 cell differentiation, flammatoryresponses to allergens [5]. Allergen recognition and uptake suggesting a role for MR in allergen-induced Th2 differentiation [10]. by DCs is a crucial step in antigen presentation, which results in Conversely, in response to HDM Der p 1 allergen, DC-SIGN activates Th1

* Corresponding author. Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seongbuk-Gu, Seoul, 02792, Republic of Korea. ** Corresponding author. E-mail addresses: [email protected] (M. Jang), [email protected] (T.H. Kim). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biomaterials.2021.120798 Received 8 September 2020; Received in revised form 29 March 2021; Accepted 30 March 2021 Available online 10 April 2021 0142-9612/© 2021 Elsevier Ltd. All rights reserved. B. Kim et al. Biomaterials 273 (2021) 120798 differentiation [11,12]. DC-SIGN-deficient DCs facilitate Th2-mediated 2.2. RNA sequencing and data processing immunity, displaying an increase in IL4-producing cells in an in vitro autologous DC-T cell co-culture system [13]. Moreover, Der p 1-sensiti Total RNAs from allergic and non-allergic DC samples (n = 3 each) zation of monocyte-derived DCs (MDDCs) results in enhanced endocy were isolated and processed for quality control. Whole transcriptome tosis of DC-SIGN [14]. Therefore, depending on the expression and library preparation and sequencing were performed by Macrogen, Inc. endocytic behavior of PRRs, allergen-sensitized DCs can induce either a (Korea). The total RNA concentration was measured using the Quant-iT Th1 or a Th2 immune response. The molecules participating in these RiboGreen RNA Assay Kit (Invitrogen, USA), and its integrity was responses may be attractive targets for therapeutic or preventative verified by RNA ScreenTape assay on an Agilent 2200 TapeStation strategies for allergic diseases. system (Agilent Technologies, USA). Only high-quality samples with Clustered regularly interspaced short palindromic repeats (CRISPR) RNA integrity numbers >7.0 were used for RNA library construction. technology, which is based on a microbial defense system, has produced Total RNA (1 μg) was used as input material for library construction. incredible advances in diverse fields [15]. CRISPR-mediated DNA After removal of ribosomal RNA, the RNA fragments were reverse cleavages induce DNA break repair via the non-homologous end joining transcribed using random primers, and 100-nt paired-end sequencing and/or homology-direct repair pathways [16,17], enabling selective was performed using an Illumina HiSeq 2500 System. The libraries were and efficient gene editing in human cells by manipulating genes of in quantified using quantitative polymerase chain reaction (qPCR) ac terest in a site-specificmanner [18–20]. Additionally, the simplicity and cording to the qPCR Quantification Protocol Guide (Kapa Biosystems, flexibility of CRISPR technology has revolutionized the field of gene Wilmington, MA, USA) on an Agilent Technologies 2100 Bioanalyzer therapeutics [21]. Thus, CRISPR-mediated gene engineering offers great (Agilent Technologies). opportunities for the discovery of promising therapeutic targets and the The raw reads from the sequencer were preprocessed to remove low development of novel clinical strategies. However, despite its excellent quality reads and adapter sequences, and the processed reads were ability to manipulate DNA, several issues need to be resolved for in vivo aligned to Homo sapiens genome assembly GRCh38 (hg38) using HISAT CRISPR-mediated gene therapy, including off-target effects, safety, v2.0.5. The reference genome sequence of H. sapiens genome assembly immunogenicity, and targeted delivery, especially for clinical applica GRCh37 (hg19) and its annotation data were downloaded from the tions [21,22]. Alternatively, ex vivo cell-based therapies using CRISPR University of California Santa Cruz table browser (http://genome.ucsc. technology can offer clinically distinct benefits, and are currently edu). After alignment, StringTie v1.3.3 b was used to assemble aligned favored for clinical use. Here, we have used CRISPR technology to reads into transcripts and estimate their abundance, which provided the develop a powerful potential DC-based therapy for allergic treatment by relative abundance estimates as fragments per kilobase of exon per manipulating a therapeutic molecular target on DCs, resulting in the million mapped reads (FPKM) of the transcripts and genes expressed in inhibition of allergen-specific Th2 immunity. each sample. Normalized FPKM values were used for comparative analysis of DEGs between samples. Functional gene set analysis was 2. Materials and methods performed using Gene Ontology Consortium Enrichment analysis (htt p://geneontology.org/page/go-enrichment-analysis) and GSEA anal 2.1. Isolation of myeloid DCs from blood ysis (http://software.broadinstitute.org/gsea/index.jsp).

The venous blood of patients with AR and healthy controls was 2.3. Western blotting collected directly into BD Vacutainer CPT Cell Preparation Tubes (BD Biosciences, USA) containing 0.1 M sodium citrate, and human (h) DC protein extracts were used for western blotting. Briefly, equal PBMCs were isolated following the manufacturer’s instructions. Briefly, amounts of total protein were resolved using 8% sodium dodecyl sulfate- immediately after blood collection, the tubes were centrifuged at polyacrylamide gel electrophoresis and transferred to nitrocellulose 1500×g for 20 min at room temperature (RT) in a swinging-bucket rotor. membranes. Membranes were blocked with Tris-buffered saline con Subsequently, the hPBMC layer was gently transferred to a 15-mL taining 0.1% Tween 20 and 5% skim milk and incubated overnight at ◦ conical tube and washed with phosphate-buffered saline (PBS) by 4 C with anti-VPS37A (LS-C409401, LSBio Inc., Seattle, WA, USA), anti- centrifugation at 300×g for 15 min. Precipitated hPBMCs were resus VPS37B (PA5-58299, Thermo Scientific), anti-SNX9 (PA5-56734, pended in Roswell Park Memorial Institute (RPMI) 1640 medium Thermo Scientific), anti-MR (ab64693, Abcam, Cambridge, UK), anti- (Welgene, Korea) containing 10% fetal bovine serum (FBS), 100 U ml 1 DC-SIGN (MA5-15746, Thermo Scientific), anti-β-actin (sc-47778, penicillin/100 μg ml 1 streptomycin, and 2 mM glutamine, and either Santa Cruz Biotechnology), and anti-glyceraldehyde 3-phosphate de cultured for subsequent experiments or frozen and stored in liquid ni hydrogenase (GAPDH; sc-47724, Santa Cruz Biotechnology) antibodies. trogen. Prior to blood collection, the protocol and informed consent Next, membranes were incubated with the appropriate anti-mouse (115- form were approved by the Institutional Review Board for Human 036-003, Jackson ImmunoResearch Laboratories Inc., West Grove, PA, Studies at Korea University Hospital (2018AN0061), and all participants USA) or anti-rabbit (711-036-152, Jackson ImmunoResearch Labora provided written informed consent. tories Inc.) horseradish peroxidase-conjugated secondary antibodies. The hPBMCs were washed with staining buffer, centrifuged at 300×g Proteins were detected using an ECL detection kit (SuperSignal™ for 5 min, then stained with BDCA-3/CD141-PE Mouse IgG1 (Clone WestPico PLUS chemiluminescent substrate, Thermo Scientific), and 501,733) and CD11c-CFS Mouse IgG1 (Clone ICRF 3.9) from the Human visualized on a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, Myeloid Dendritic Cell Multi-Color Flow Kit (R&D Systems, USA). Cells USA). were incubated with each antibody or a corresponding isotype control antibody for 45 min at RT in the dark. After washing with the supplied 2.4. Targeted deep sequencing analysis staining buffer, cells were sorted on a BD FACSAria III Cell Sorter (BD Biosciences) and analyzed using BD FACSDiva software (BD Bio The efficiencyof genome editing at the target locus was analyzed by sciences). Viable cells were detected using forward and side light scat targeted high-throughput sequencing analyses, as previously described tering, and cell debris was excluded from the analysis. DC subsets with minor modifications [23]. In summary, DNA sequences including positive for BDCA-3 and CD11c were gated, sorted, and either lysed with the on-target sites were amplified from genomic DNA by 25 cycles of QIAzol Lysis Reagent (Qiagen, Germany) for mRNA sequencing or nested PCR using Phusion High-Fidelity DNA Polymerase (Thermo extracted with 5 × Laemmli sample buffer containing 5% β-mercap Fisher Scientific, USA). During amplification, unique indices and toethanol for western blotting. adaptor sequences were added to each amplicon, followed by deep sequencing using an Illumina MiniSeq System. Deep-sequencing data

2 B. Kim et al. Biomaterials 273 (2021) 120798 consisting of paired-end reads harboring the target sites were analyzed DCs for confocal microscopic imaging analysis, cells were incubated ◦ using Cas-Analyzer (CRISPR RGEN tools) [24]. Indels located 3 bp up with each primary antibody (1:1000) overnight at 4 C, followed by stream of the protospacer adjacent motif (PAM) were considered mu incubation with an Alexa Fluor 488 secondary antibody (Invitrogen, tations induced by CRISPR genome editing. Primers used in the study are 1:1000 dilution) for 1 h. Cells were analyzed by confocal microscopy on listed in Table S1 (Supporting Information). a Zeiss LSM 700 Inverted Confocal microscope (Carl Zeiss).

2.5. Off-target analysis by nest generation sequencing 2.10. Co-culture of DCs and T cells

Genomic DNA extracted from wild type and Cas9 RNP-treated sam THP-1 cells were cultured in RPMI 1640 containing GM-CSF and IL4 ples were subjected to whole exome sequencing (WES), which was (100 ng mL 1 and 100 ng mL 1, respectively) for 6 d to form imDCs. performed at a sequencing depth of 30X to 40X using an Illumina TruSeq After CRISPR/Cas9 gene editing, imDCs were matured by treatment X Ten Sequencer (Macrogen, South Korea). The WES data were mapped with Der p 1 (2 μg mL 1; Indoor Biotechnologies, Charlottesville, VA, to a reference genome (hg19) by Bowtie 2. The indel rates at the putative USA) for 48 h. off-target sites were analyzed by using Cas-Offinder and Integrative Human naïve T cells were isolated from hPBMCs with the EasySep™ + Genomics Viewer [25–27]. The sequences at on- and off-target sites Human Naïve CD4 T Cell Isolation Kit (STEMCELL Technologies). The were inspected for sequence variants, and rates of insertions and de hPBMC suspension was mixed with 50 μL of EasySep™ biotinylated anti- + letions (indels) were analyzed by determining the mutant vs wild type CD45RO antibody and 50 μL of EasySep™ Human naïve CD4 T cell sequence ratios. isolation cocktail. After incubation for 5 min at RT, the cell suspension was mixed with 50 μL of EasySep™ Dextran RapidSpheres™ and incu 2.6. In vitro generation of THP-1 MDDCs bated for 5 min at RT. The cell suspension tube was magnetized for 5 + min, and then human naïve CD4 T cells were collected and cultured in The human monocytic leukemia cell line THP-1 was obtained from RPMI 1640 medium. the American Type Culture Collection (USA). For imDC generation, To examine the effects of VPS37 k/o DCs on T cell differentiation, 5 + THP-1 monocytes (2 × 105 cells mL 1) were incubated in RPMI 1640 × 104 mDCs were co-cultured with 2.5 × 105 allogeneic CD4 naïve T medium supplemented with 10% FBS and treated with rhIL4 (100 ng cells for 4 d in 250 μL culture medium in 96-well plates. After attach mL 1) and rhGM-CSF (100 ng 1mL) for 5 d. The medium was replaced ment, half the medium was replaced with IL2 (100 U mL 1, PeproTech)- every 2 d [28]. For mDC generation, THP-1-derived imDCs were containing medium. After incubating for 2 d, the T cells in each well resuspended in serum-free culture medium containing rhIL4 (200 ng were divided into two wells, and 125 μL of culture medium (containing mL 1), rhGM-CSF (100 ng mL 1), rh tumor necrosis factor α (20 ng 50 U mL 1 IL2) was added to each. Every other day, half the medium mL 1), and ionomycin (200 ng mL 1) for 2 d. was replaced with fresh medium containing IL2. On day 10, T cells were stimulated with medium containing anti-CD28 antibody (10 μg mL 1, 2.7. Generation of genetically engineered DCs via Cas9 RNP treatment BioLegend) in wells coated with CD3 antibody (10 μg mL 1, BioLegend) for 24 h. Each time the medium was refreshed, the used medium was ◦ For in vitro generation of VPS37-disrupted mDCs, THP-1 monocyte- collected and stored at 80 C for cytokine measurements by ELISA. derived imDCs were treated with Cas9 RNPs targeting VPS37A/B for 24 h. Briefly,sequence-specific crRNAs were hybridized with tracrRNAs in 2.11. Adoptive transfer of Vps37 k/o BMDCs in a mouse model of AR IDT duplex buffer (Integrated DNA Technologies, USA) according to the manufacturer’s instructions. Hybridized RNAs were mixed with spCas9 Female BALB/c mice (5 weeks old) were purchased from Orient Bio ◦ protein at a 1:5 w/w ratio in IDT buffer for 30 min at 4 C. Next, THP-1- Inc. (Seongnam, Korea) and housed in specificpathogen-free conditions. derived imDCs were incubated with 178 pM of Cas9 RNPs and quickly The animal experiment was approved by the Institutional Animal Care treated with a Neon electroporator (Invitrogen, USA) in one 20-ms pulse and Use Committee of Korea University (korea-2018-0038). AR was at 1600 mV. The electroporated cells were incubated for 24 h, then induced using OVA (Sigma-Aldrich). The mice were sensitized with 25 matured for 2 d as described above. To generate Vps37-disrupted mouse μg of OVA emulsified with 25 μL of adjuvant (ImjectTM Alum, Thermo BMDCs, 178 pM of Cas9 RNPs were incubated with BMDCs for 24 h prior Scientific)in 125 μL of PBS by intraperitoneal injection on days 0, 7, and to their use in experiments. 14. Mice were divided into three groups (n = 4 each), which were treated with negative control, Vps37a k/o, and Vps37b k/o BMDCs, 2.8. Analysis of human MMDCs respectively. As performed previously [29], the mice were treated by adoptive transfer of 1 × 106 BMDCs into the nasal cavity on days 16 and Monocytes were isolated from hPBMCs with the EasySep™ Human 18 and then intranasally administered 500 μg of OVA in 50 μL of PBS on CD14 positive selection kit II (STEMCELL Technologies, Vancouver, BC, days 21–27. Mice were sacrificedand samples were collected on day 28. Canada). The hPBMC suspension was mixed with 100 μL of EasySep™ Human CD14 positive selection cocktail. After incubation for 10 min at 2.12. Symptom score RT, the cell suspension was mixed with 100 μL of EasySep™ Dextran RapidSpheres™ and incubated for 3 min at RT. After placing the cell A blinded observer recorded the frequencies of nasal rubbing and suspension tube into the magnet (STEMCELL Technologies) for 3 min, sneezing in mice treated with Vps37 k/o and negative control BMDCs for the monocytes were collected and cultured in RPMI 1640 containing 15 min on day 28. GM-CSF and IL4 (50 ng mL 1 and 40 ng mL 1, respectively; PeproTech, Rocky Hill, NJ, USA) for 6 d to produce immature MDDCs. These were 2.13. Quantitative real-time PCR of mouse tissues matured by incubation with Der p 1 (2 μg mL 1, Indoor Biotechnologies, Charlottesville, VA, USA) for 24 h, with or without the protein transport Mouse lung lobes and nasal mucosa were lysed with QIAzol Lysis inhibitors, monensin and filipin (2 and 4 μM, respectively; both from Reagent (Qiagen) and incubated for 5 min at RT. The lysed suspensions Sigma-Aldrich). were mixed with chloroform, briefly shaken, and then incubated for 3 ◦ min at RT. After centrifugation at 14,000 rpm for 15 min at 4 C, the 2.9. Immunofluorescence RNA was enriched in the upper transparent layer. This layer was mixed with isopropanol by inversion and incubated for 5 min on ice. After ◦ To monitor the localization of VPS37A/B or MR on VPS37-disrupted centrifugation at 14,000 rpm for 10 min at 4 C, precipitates were

3 B. Kim et al. Biomaterials 273 (2021) 120798 washed with 75% ethanol in diethylpyrocarbonate-treated (DEPC) coupled receptor (GPCR) signaling, and inflammatorycytokines (Fig. 1c, ◦ water and centrifuged at 14,000 rpm for 5 min at 4 C. The RNA- d and Figure S2, Supporting Information). Prostaglandins (PGs) are a containing precipitates were dried for 5 min, resuspended in DEPC family of lipid compounds that affect allergic inflammation through water, and used as a template for cDNA synthesis using cDNA Synthesis GPCR-mediated signaling [35]. Among major bioactive prostanoids, Master Mix (GenDEPOT, Houston, TX, USA). PGE2 plays key roles in DC maturation and migration, acting through The prepared cDNA was amplifiedand quantifiedusing SYBR green the GPCRs E prostanoid (EP) 2 receptor and EP4 receptor [36,37]. master mix (QuantiNova SYBR Green PCR Kit, Qiagen) with the Indeed, genes involved in the PGE-EP signaling axis were enriched in ′ following primers: mouse IL4, forward 5 -TCA ACC CCC AGC TAG TTG patients with AR (Fig. 1c). Moreover, GSEA revealed upregulation of ′ ′ ′ TC-3 and reverse 5 -TGT TCT TCG TTG CTG TGA GG-3 ; mouse IL5, proinflammatory cytokines such as IL6 and IL1 family members and ′ ′ ′ forward 5 -GAA GTG TGG CGA GGA GAG AC-3 and reverse 5 -GCA CAG chemokines such as CCL2 and C-X-C motif chemokine ligand 10, indi ′ ′ TTT TGT GGG GTT TT-3 ; mouse IL13, forward 5 -ATG GCG CTC TGG cating chronic allergic responses in patients with AR (Fig. 1d) [29, ′ ′ GTG ACT GCA GTC CTG-3 and reverse 5 -TTA GAA GGG GCC GTG GCG 38–41]. Intriguingly, Kyoto Encyclopedia of Genes and Genomes ′ ′ AAA CAG TTG-3 ; mouse IFNγ, forward 5 -ACT GGC AAA AGG ATG GTG (KEGG) pathway and Gene Ontology (GO) enrichment analysis revealed ′ ′ ′ AC-3 and reverse 5 -TGA GCT CAT TGA ATG CTT GG-3 ; mouse GAPDH, that the DEGs were involved in endocytosis and protein binding, ′ ′ ′ forward 5 -CAT CCA CTG GTG CTG CCA AGG CTG T-3 and reverse 5 - respectively (Fig. 1e and Figure S3, Supporting Information). As ′ ACA ACC TGG TCC TCA GTG TAG CCC A-3 . Reactions were performed mentioned earlier, an allergen sensitization step involving antigen using a real-time thermal cycler system (Thermal Cycler Dice Real Time recognition and uptake is crucial for allergen-specific DC-driven Th2 System, TP800/TP860, Takara, Kusatsu, Japan) with 40 cycles of immunity. Thus, we selectively focused on genes related to the endocytic ◦ ◦ denaturation at 95 C for 15 s and annealing/extension at 60 C for 45 s. pathway as DC-based therapeutic targets. Among potential target can ΔΔ Data were analyzed using the 2 Ct method. didates, sorting nexin 9 (SNX9), a member of the SNX family, and vacuolar protein sorting 37 B (VPS37B), a component of endosomal 2.14. Cytokine and IgE ELISAs sorting complex required for transport-I (ESCRT-I), were highly expressed in AR-derived DCs (Fig. 1f). ELISA kits for IL4 (Human Instant ELISA™ Kit, eBioscience, Vienna, Austria), IL5 (Human ELISA kit, Thermo Fisher Scientific,Waltham, MA, 3.2. Upregulation of VPS37A/B affects endocytosis in AR-derived DCs USA), and IL13 and IFN-γ (Human LEGEND MAX™ ELISA Kit, Bio Legend, Inc.) were used, to measure cytokine levels, according to the We next tested the expression of SNX9 and VPS37 in AR-derived DCs manufacturers’ instructions. Mouse serum was prepared by centrifuging (Fig. 2a). Western blot analysis revealed strong upregulation of both the the blood, and the Mouse IgE ELISA kit (BD Biosciences) and Mouse OVA VPS37A and VPS37B isoforms in AR-derived DCs, while only marginal specific IgE ELISA kit (LEGEND MAX™, BioLegend, Inc.) were used to increases were observed in SNX9. Additionally, VPS37A and VPS37B measure total and OVA-specific serum IgE levels, respectively. were highly expressed in the nasal tissues of patients with AR (Fig. 2b and Figure S4, Supporting Information). 2.15. Statistical analysis Four different ESCRT machineries (ESCRT -0, –I, –II, and –III) participate in diverse biological events by catalyzing a topologically Statistical analyses were carried out using SPSS for Windows (version distinct membrane-remodeling process [42]. ESCRT complexes recog 16.0.0; SPSS, Chicago, IL, USA). Data in graphs are expressed as the nize ubiquitinated surface cargoes on the early endosome, mediating means ± SE of at least three independent experiments. The significance endosomal sorting into multivesicular bodies (MVBs) [43]. Importantly, of differences among groups was determined by one-way analysis of in DCs, MVBs serve as major histocompatibility complex (MHC) class II variance (ANOVA) followed by Tukey’s multiple comparison test. Dif compartments for antigens. ESCRT-1 is a heterodimer consisting of ferences with p-values <0.05 (*) or <0.01 (**) were considered tumor susceptibility 101, VPS28, VPS37, and multivesicular body sub significant. unit 12 A [44]. Thus, upregulation of VPS37 in AR-derived DCs suggests a role in endocytic antigen uptake for these surface receptors. 3. Results In response to allergen exposure, DCs trigger the maturation process by accumulating antigens through receptor-mediated endocytosis. Thus, 3.1. Selection of therapeutic molecular targets in AR-derived DCs we evaluated the effects of endocytosis on the DC maturation process through in vitro generation of human MDDCs [28]. First, we examined To identify potential therapeutic targets for the treatment of airway mature DCs (mDCs) after treatment with monensin and filipin, which + allergies, we first harvested CD11c /blood dendritic cell antigen 3 are chemical inhibitors of clathrin- and cholesterol-dependent endocy + (BDCA-3) DCs from the peripheral blood mononuclear cells (PBMCs) of tosis, respectively [45,46]. As expected, treatment with monensin and patients with HDM-associated AR and healthy donors (Figure S1, Sup filipin caused noticeable phenotypic defects in the DC maturation pro porting Information). Based on phenotypic, functional, and develop cess (Fig. 2c). Furthermore, endocytic inhibitor-treated mDCs had mental criteria, many different DC subsets have been classified[ 30,31]. decreased VPS37A and VPS37B expression compared to untreated cells Allergen challenge induces the recruitment of circulating blood DCs into (Fig. 2d), suggesting that VPS37A and VPS37B are closely related to allergen-responsive mucosal tissues, consequently increasing the num allergen internalization by endocytic receptors. Thus, we determined ber of DCs in the nasal mucosa [32]. The major nasal DC population is that VPS37A and VPS37B were promising therapeutic targets for + + CD11c /BDCA-1 [33]; however, due to difficulties in isolating suffi DC-targeted AR treatment. + + cient DCs for the analysis, we used CD11c /BDCA-3 DCs to identify potential therapeutic targets as reported in our previous paper [34]. We 3.3. In vitro generation of VPS37-disrupted mDCs for AR treatment performed next generation sequencing to compare RNA expression patterns in DCs from patients with AR and healthy donors. Whole To disrupt the VPS37 genes of mDCs, type II CRISPR RNA-guided transcriptome sequencing (WTS) revealed 168 differentially expressed endonuclease Cas9 (Cas9) ribonucleoproteins (RNPs) targeting genes (DEGs) with fold change (FC) values ≥ 2 and p-values < 0.05 VPS37A (VPS37A k/o) and VPS37B (VPS37B k/o) were transfected into (Fig. 1a and b). Of the 168 DEGs, 96 and 72 were up- and downregulated THP-1-derived immature DCs (imDCs), and the VPS37-disrupted imDCs in patients with AR compared to healthy controls (Fig. 1b). Remarkably, were matured for 48 h (Fig. 3 and Figure S5, Supporting Information). gene set enrichment analysis (GSEA) of the WTS data identified airway Flow cytometry analysis exhibited 81 and 74% decreases in the allergic response genes associated with receptor regulation, G-protein expression of VPS37A and VPS37B, respectively, after CRISPR (Fig. 3a

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Fig. 1. DEGs in AR-derived DCs. CD11c+/BDCA-3+ DCs were isolated from the blood of patients with HDM-associated AR and healthy donors. (a) Heat map of gene expression in AR-derived and control DCs. (b) DEGs that were up- and downregulated in AR-derived DCs compared to control DCs. (c-d) Enriched allergy-related pathways in DCs from pa tients with AR, based on GSEA of the WTS data. NES, normalized enrichment score; FDR, false discovery rate. (e) KEGG pathway enrichment analysis of the DEGs. (f) Expression of endocytic pathway-related genes, including SNX9 and VPS37B, in AR-derived DCs.

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Fig. 2. VPS37A and VPS37B upregula tion is related to endocytosis in AR- derived DCs. (a) Western blot analysis of VPS37A, VPS37B, and SNX9 in con trol and AR-derived DCs. GAPDH was used as a loading control. (b) VPS37A and VPS37B expression in nasal mucosa tissues derived from healthy controls and patients with severe AR. Scale bars: 50 μm. (c) Phenotypic defects in DC maturation after inhibition of endocy tosis in THP-1 MDDCs treated with HDM Der p 1 allergen. Red arrow and black arrow indicate the immature and maturation form, respectively. **p < 0.001 by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test, compared with the mDC/DMSO group. (d) Western blot analysis of VPS37A and VPS37B levels in DCs treated as in C.

and Figure S6, Supporting Information). Consistently, western blotting double k/o, and NC DC samples after Der p 1 treatment. Intriguingly, and confocal microscopy revealed significantreductions in VPS37A and GSEA showed that VPS37-disrupted DCs had downregulation of genes VPS37B expression compared to the NC sample, a negative control in involved in dendritic cell migration, allergy-related chemokine activity, which Cas9 RNPs was complexed with negative crRNA (Fig. 3b and c). and receptor protein maturation (Fig. 4c). Thus, VPS37-disrupted DCs We performed a targeted deep sequencing assay to analyze the inser have defective maturation processes. tion/deletion (indel) frequency at the VPS37A target loci (Fig. 3d and Figure S7, Supporting Information). VPS37A- and double (VPS37A/B)- 3.5. VPS37A and VPS37B help recruit allergens to endosomes for antigen Cas9 RNP-treated DCs gained 51% and 47% indel mutations at the presentation VPS37A loci, respectively, while no indel mutations were detected in NC-treated DCs. We further assessed the frequency of off-target muta We next monitored the behavior of VPS37 proteins during DC tions in Cas9 RNP-treated samples by conducting whole exome maturation by confocal microscopy. After treatment with Der p 1, sequencing (WES) and analyzing the NGS data. WES data revealed that VPS37A and VPS37B had higher expression in mDCs than in imDCs the treatment of VPS37A-Cas9 RNP resulted in 19.54% indel mutations (Fig. 5a). Moreover, both strongly accumulated in intracellular com at the on-target locus, while no indel was detected at any of the putative partments after allergen challenge. Inhibition of endocytic events after off-target loci, up to 4 mismatches (Table S2). antigen uptake by treatment with monensin and filipin resulted in low expression of VPS37A and VPS37B. Dysfunction of endocytic pathways 3.4. Defects in DC maturation in VPS37-disrupted DCs in response to leads to aberrant membrane dynamics, including membrane formation, allergen challenge fusion and scission events, as well as deformed endosomal structures, suggesting impaired recruitment and turnover of the ESCRT machinery Next, we investigated the roles of the VPS37 genes in DC maturation [48]. Additionally, the intracellular compartments labeled with VPS37A upon allergen exposure (Fig. 4a). Notably, defective DC maturation partially overlapped with early endosome antigen 1 (EEA1) and occurred in VPS37A k/o and VPS37B k/o cells, with morphological lysosome-associated membrane glycoprotein 1 (LAMP1), markers of the changes in dendrite formation. In contrast, monocyte-derived mDCs early endosome and lysosome, respectively (Fig. 5b). (NC) formed clear dendrites. In vitro, mDCs upregulate surface markers We also examined the behavior of MR as an allergen recognition such as CD80 and human leukocyte antigen-DR isotype (HLA-DR) after receptor for Der p 1 internalization (Fig. 5c, d and Figure S8, Supporting stimulation with HDM Der p 1 [47]. HLA-DR can therefore serve as a Information). MR is mainly localized on the imDC surface. After Der p 1 maturation marker, and its expression was significantly decreased in challenge, MR expression on mDCs was increased and highly accumu VPS37A k/o and VPS37B k/o cells, suggesting defects in DC maturation lated in intracellular compartments, indicating further antigen presen (Fig. 4b). We performed WTS analysis using VPS37A k/o, VPS37B k/o, tation processing. However, despite this accumulation, disruption of

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Fig. 3. In vivo generation of VPS37-disrupted DCs by CRISPR/Cas9. (a-c) Cas9 RNPs targeting VPS37A (VPS37A k/o) and VPS37B (VPS37B k/o) were transfected into THP-1-derived imDCs and the efficacyof gene knockout was evaluated using flowcytometry (a), western blotting (b), and confocal microscopy (c). (d) Targeted deep sequencing analysis of indel mutations in VPS37A loci. The crRNA and PAM sequences are indicated in blue and red letters, respectively.

VPS37 resulted in the degradation of MR. In contrast, DC-SIGN expres Th2 allergic responses after adoptive transfer of mouse Vps37-disrupted sion was increased in VPS37A k/o mDCs. Thus, VPS37A and VPS37B DCs (Fig. 7a and Figure S9, Supporting Information). After OVA sensi play key roles in receptor-mediated endocytosis after allergen exposure, tization, the mice were intranasally challenged twice with mouse DCs enabling further antigen presentation processing in the endosomes. genetically engineered via Cas9 RNP targeting of Vps37a and/or Vps37b. After sacrifice, several AR-related parameters were evaluated in the 3.6. VPS37-disrupted mDCs dampen allergy-induced Th2 immunity mice (Fig. 7b–e). Mice treated with Vps37-disrupted DCs showed a lower frequency of nasal symptoms than NC-treated mice (Fig. 7b). However, During allergen exposure, MR recognizes the Der p 1 allergen and the levels of total and OVA-specific IgE were unchanged between the mediates its internalization, leading to a Th2 allergic response. Thus, we groups (Fig. 7c), indicating that the adoptive transfer of Vps37-disrupted tested the Th2 allergic response in VPS37-disrupted DCs. We established DCs affected nasal immunity only, not systemic immunity. We also an in vitro co-culture system with THP-1 monocyte-derived VPS37-dis evaluated the expression levels of Th1 and Th2 cytokines in mouse nasal + rupted DCs and allogeneic naïve CD4 T cells obtained from patients mucosa and lung tissues (Fig. 7d and e). In particular, IL5 and IL13 were with severe AR (Fig. 6a). Enzyme-linked immunosorbent assays (ELI significantly reduced in Vps37-disrupted DC-treated mice, inhibiting SAs) revealed significantreductions in Th2 cytokines, including IL4, IL5, Th2-mediated allergic inflammation. These results suggest that VPS37- and IL13, during co-culture with VPS37-disrupted DCs (Fig. 6b). Despite disrupted DCs can alleviate allergic reactions, offering a promising a decrease in interferon γ (IFNγ), a representative Th1 cytokine, during cellular therapeutic modality. co-culture with VPS37A-disrupted DCs, VPS37B-disrupted DCs did not affect the Th1 immune response (Fig. 6b). 4. Discussion Consistent to the results from the in vitro co-culture system, an in vivo mouse model of ovalbumin (OVA)-induced AR exhibited relief from In this study, we developed a DC-based immunotherapy for AR-

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Fig. 4. Maturation defects in VPS37-disrupted DCs after HDM Der p 1 exposure. (a) Morphologic phenotypes after allergen treatment. Scale bars: 20 μm. **p < 0.001 by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. (b) Flow cytometry analysis of the expression of the DC maturation marker HLA-DR on VPS37-disrupted mDCs. (c) GSEA of WTS data from VPS37-disrupted and NC DCs. GO means gene ontology. Red and Blue colors in heatmaps indicate high and low relative expression, respectively.

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Fig. 5. VPS37A and VPS37B function in MR endocytosis after allergen chal lenge. (a) Localization of VPS37A and VPS37B after treatment with endocytic inhibitors. White arrows indicate pro tein accumulation in intracellular compartments. (b) Overlap between VPS37 A/B localization and that of the lysosomal marker LAMP1 and the early endosome marker EEA1. (c, d) MR localization and expression were investigated using confocal microscopy imaging (c) and western blotting (d), respectively. DC-SIGN levels were also measured. β-actin was used as a loading control.

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Fig. 6. VPS37-disrupted mDCs inhibit Th2-mediated cytokine production during allergen challenge. (a) Establishment of an in vitro allogeneic co-culture system + + with VPS37-disrupted mDCs and naïve CD4 T cells. Allogeneic naïve CD4 T cells were obtained from the blood of patients with AR. (b) ELISAs detecting the Th2- induced cytokines IL4, IL5, and IL13 and the Th1-induced cytokine IFNγ produced by T cells after co-culture with VPS37-disrupted mDCs. *p < 0.05 and **p < 0.001 by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. associated allergic reactions. Although several targets have been studied effectiveness of Sipuleucel-T was unsatisfactory [54,55]. However, with the goal of relieving airway allergic responses, antigen processing engineered DC- based therapies are considered a promising approach for molecules have not been previously examined. By analyzing RNA treatment of diseases including cancer, owing to the recently advanced sequencing data from control and AR-derived DCs, we identified the clinical progress and techniques for manipulating the function of DCs antigen-processing molecules VPS37A and VPS37B as therapeutic tar [56]. Besides the adoptive transfer of engineered DCs, in vivo immu gets for AR. We produced VPS37A/B-disrupted DCs using the CRISPR/ nomodulation of DCs is also applicable in multiple approaches. In Cas9 gene editing system, and demonstrated their ability to improved particular, in vivo targeting to DCs can be developed through manipu airway allergic responses in an in vitro co-culture system and in vivo in lation of the DC-specific virus and administration of diverse nano the nasal cavities of AR mice. particles or exosomes [57–59]. Thus, the CRISPR/Cas9-encoding DCs are ideal targets for the development of treatments against plasmids or RNP systems can be effectively delivered into endogenous allergic disease because they control so many aspects of the allergic nasal DC subsets in the future. cascade, including inducing immune responses by processing and pre The antigen presentation process is the result of endosomal traf senting allergens to polarize T cells. High-dose allergen treatment during ficking events, which facilitate the internalization and subsequent allergen-specific immunotherapy (ASIT) increases regulatory T cells degradation of exogenous antigens into peptides for loading onto MHC (Tregs) and regulatory DCs, suppressing allergic responses in patients class-II molecules. In particular, exogenous antigens are internalized with allergies [49,50]. Similar to ASIT, administration of Der p 2 into endosomes via receptor-mediated endocytic pathways for antigen DNA-transfected DCs into C57BL/6 mice induced forkhead box P3 presentation. Among endosomal trafficking machineries, the role of + (FOXP3) Tregs and reduced Th2 responses [51]. Genetic modification ESCRT in the degradation of ubiquitinylated membrane proteins from of OVA-pulsed IL10-transduced BMDCs enhanced the proportion of endosome to lysosomes is well known [60,61]. In this study, we + + + + OVA-specific CD4 CD25 FOXP3 IL10 Tregs [52]. In addition to sup demonstrate that VPS37A and VPS37B are involved in the internaliza pressing immune responses, the pharmacological treatment of DCs with tion of CLR receptors for antigen presentation, and that depletion of a cyclic adenosine monophosphate derivative decreased Th2 responses VPS37A and/or VPS37B causes defects in antigen sensitization. Inter in a human DC-T cell co-culture system, and CD38-overexpressing DCs estingly, however, the actions of CLR receptors in allergen processing inhibited asthma development in an animal model of allergy [34,53]. were dependent on VPS37A/B disruption. In Der p 1-matured DCs, MR However, despite considerable investigation of engineered DC-based was upregulated while DC-SIGN was not. In addition, the expression of allergy treatments, the genetic modulation of allergen processing mol MR was decreased in all VPS37A and/or VPS37B k/o DCs, while the ecules in DCs has not been reported. In addition, the CRISPR/Cas9 expression of DC-SIGN was increased in VPS37A and double k/o DCs, system was used to genetically engineer DCs in this study rather than but not in VPS37B k/o DCs. These differences may be due to the viral infection, which enhances the efficiencyand safety of the method. glycosylation profileof natural Der p 1, which has mannose and fucose Despite the first approval of DC-based therapy (Sipuleucel-T, PRO branches [62], as MR binds to mannose-based oligosaccharides while VENGE) for the treatment of metastatic prostate cancer, the DC-SIGN binds to mannose and fucose-containing glycans [63]. These

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Fig. 7. Alleviated allergic symptoms in an in vivo mouse model of AR after nasal administration of Vps37-disrupted mDCs. (a) Strategy for nasal administration of engineered DCs for AR treatment in an OVA-1-induced mouse model of AR. (b) Frequency of nasal rubbing and sneezing over a 15 min period. (c) Serum con centrations of total and OVA-1-specificIgE. (d) Th2- or Th1-induced cytokines were measured in nasal mucosa and lung tissues by quantitative real time PCR. *p < 0.05 and **p < 0.001 by one-way ANOVA followed by Tukey’s multiple comparison test. data suggest that VPS37A and VPS37B are involved in the Der p while only VPS37A k/o DCs resulted in a reduced Th1 immune response. 1-MR-Th2 immune response, but only VPS37A is involved in the Der p Combining the CLR data with the immune response data, VPS37A ap 1-DC-SIGN-Th1 immune response [11]. Thus, we speculate that Der p 1 pears to regulate both the Th1 and Th2 immune responses while VPS37B may bind mainly to MR, which is recycled through the normal ESCRT affected the Th2 immune response specifically. This indicates that pathway, while DC-SIGN may be alternatively activated in the absence VPS37B is a potential target for allergic diseases. of MR upon VPS37 disruption. In our in vitro co-culture and in vivo animal experiments, VPS37B k/ o DCs inhibited greater Th2 immune responses than VPS37A k/o DCs,

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