Understanding the immunopathogenesis of autoimmune diseases by animal studies using gene modulation: A comprehensive review

Journal Pre-proof Understanding the immunopathogenesis of autoimmune diseases by animal studies using gene therapy: A comprehensive review

Keum Hwa Lee, Byung Soo Ahn, Dohyeon Cha, Won Woo Jang, Eugene Choi, Soohyun Park, Jun Hyeong Park, Junseok Oh, Da Eun Jung, Heeryun Park, Ju Ha Park, Youngsong Suh, Dongwan Jin, Siyeon Lee, Yong-Hwan Jang, Tehwook Yoon, Min-Kyu Park, Yoonje Seong, Jihoon Pyo, Sunmo Yang, Youngin Kwon, Hyunjean Jung, Chae Kwang Lim, Jun Beom Hong, Yeoeun Park, Eunjin Choi, Jae Il Shin, Andreas Kronbichler PII:

S1568-9972(20)30012-4

DOI:

https://doi.org/10.1016/j.autrev.2020.102469

Reference:

AUTREV 102469

To appear in:

Autoimmunity Reviews

Received date:

16 September 2019

Accepted date:

20 September 2019

Please cite this article as: K.H. Lee, B.S. Ahn, D. Cha, et al., Understanding the immunopathogenesis of autoimmune diseases by animal studies using gene therapy: A comprehensive review, Autoimmunity Reviews(2020), https://doi.org/10.1016/ j.autrev.2020.102469

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© 2020 Published by Elsevier.

Journal Pre-proof

Review article Understanding the Immunopathogenesis of Autoimmune Diseases by Animal Studies Using Gene Therapy: A Comprehensive Review

Keum Hwa Lee1, Byung Soo Ahn2, Dohyeon Cha2, Won Woo Jang2, Eugene Choi2, Soohyun Park2, Jun Hyeong Park2, Junseok Oh2, Da Eun Jung2, Heeryun Park2, Ju Ha Park2, Youngsong Suh2, Dongwan Jin2, Siyeon Lee2, Yong-Hwan Jang2, Tehwook Yoon2, Min-Kyu Park2, Yoonje Seong2, Jihoon Pyo2, Sunmo Yang2, Youngin Kwon2, Hyunjean Jung2, Chae Kwang Lim2, Jun Beom Hong2, Yeoeun Park2, Eunjin Choi2,

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Jae Il Shin1 and Andreas Kronbichler3

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1. Department of Pediatrics, Yonsei University College of Medicine, Seoul, Republic of Korea.

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2. Yonsei University College of Medicine, Seoul, Republic of Korea.

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Innsbruck, Austria

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3. Department of Internal Medicine IV (Nephrology and Hypertension), Medical University Innsbruck,

Corresponding Author: Jae Il Shin, M.D., Ph.D. Address: 50 Yonsei-ro, Seodaemun-gu, C.P.O. Box 8044, Department of Pediatrics, Yonsei University College of Medicine, Seoul 03722, Republic of Korea Tel: +82-2-2228-2050; Fax: +82-2-393-9118; E-mail: [email protected] *The authors disclose no financial or non-financial conflicts of interest.

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Journal Pre-proof Abstract Autoimmune diseases are clinical syndromes that result from pathogenic inflammatory responses driven by inadequate immune activation such as T- and B-cells. Although the exact mechanisms of autoimmune diseases are still elusive, genetic factors also play an important role in the pathogenesis. Recently, with the advancement of understanding of the immunological and molecular basis of autoimmune diseases, gene therapy has become a potential approach for the tailored treatment of autoimmune disorders. Gene therapy can be applied to regulate the levels of interleukins (IL), tumor necrosis factor (TNF), cytotoxic T-

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lymphocyte–associated antigen 4 (CTLA-4), interferon-γ and other inflammatory cytokines by inhibiting

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these cytokine expressions using siRNA or by inhibiting cytokine signaling using small molecules. In addition, gene therapy delivering anti-inflammatory cytokines or cytokine antagonists showed effectiveness

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in regulating autoimmunity. In this review, we summarize the potential target genes for immunomodulation

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and gene therapy in autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosus

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(SLE), inflammatory bowel diseases (IBD) and multiple sclerosis (MS). This article will give a new perspective on understanding immunopathogenesis of autoimmune diseases not only in animals but also in

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human. Emerging approaches to investigate cytokine regulation through gene therapy may be a potential

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approach for the tailored immunomodulation of some autoimmune diseases near in the future. Keywords: Immunopathogenesis; Autoimmune Diseases; Gene Therapy; Immunomodulation treatment

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Journal Pre-proof 1. Introduction Autoimmune diseases are caused by autoantibodies which are generated due to over-activation of the immune system potentiated by inadequate activation of T- and B cells [1, 2]. These autoimmune diseases can show autotoxic effects in various organs such as brain, lung, pancreas, endocrine organ, gastrointestinal tract, kidney, bone and skin [3]. Mechanisms leading to autoimmune diseases comprise genetic, epigenetic, molecular, and cellular factors that result in pathogenic inflammatory responses which are driven by selfantigen-specific T-cells [4]. These include genetic or acquired defects of immune regulatory pathways,

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molecular similarity to viral or bacterial protein, and impaired clearance of apoptotic cell materials [5].

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Although several medications such as analgesics, non-steroidal anti-inflammatory drugs, diseasemodifying anti-rheumatic drugs, biological agents, and glucocorticoids are effective in some autoimmune

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diseases [6, 7], the proportion of patients achieving long lasting remission by the current management is still

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low. Reduction of symptoms and improvement of the quality of life are among the unmet needs of the

individual patient is highly desirable [9].

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treatment of a majority of autoimmune diseases [8]. Therefore, identification of optimal measures to treat an

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Although the exact mechanisms of autoimmune diseases are still elusive, genetic factors also play an

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important role in the pathogenesis. Multiple genes have been reported to be associated with autoimmunity and they have a variable implication in one’s individual risk [10]. Major regulatory elements of T-cells have been reported to be associated with genetic variants that contribute to the risk of autoimmune disease in human [11]. There is also strong statistical evidence that 180 genetic loci are associated with more than 12 autoimmune diseases, indicating that there is a relationship between autoimmune diseases and genes [12]. A number of rare and common variants that contribute to the pathophysiology and the risk of some diseases have been uncovered by large international genomic consortia [13] and there are also familial autoimmune diseases caused by mendelian genetic variant with rare minor allele frequency such as hereditary C1q deficiency, autoimmune lymphoproliferative syndrome (ALPS), immune dysregulation, polyendocrinopathy, 3

Journal Pre-proof enteropathy, and X-linked (IPEX) [14]. Recently, with the advancement of our understanding of the immunological and molecular basis of autoimmune diseases, gene therapy has become a promising approach for the tailored treatment of autoimmune diseases. The goal of gene therapy is to regulate the level of interleukins (IL), tumor necrosis factor (TNF), cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4), interferon-γ and other inflammatory cytokines, thus leading to reduced infiltration of lymphocytes to the affected sites. In this review, we highlight studies on potential target genes for immunomodulation and gene therapy in animal models of

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autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), inflammatory bowel diseases (IBD) and multiple sclerosis (MS). This article will give a new perspective on

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understanding immunopathogenesis of autoimmune diseases not only in animals but also in human.

2.1 TNF-α, IL-1 related gene therapy

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2. Animal models of gene therapy in rheumatoid arthritis (RA)

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There are various cytokines that are known to play major roles in the pathogenesis of RA which led to

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numerous attempts investigating the relationship between cytokine levels and RA activity (Table 1). Khoury et al. showed that targeting major cytokines such as IL-1β, IL-6 and IL-18 at the same time using intravenous injection of short interfering ribonucleic acid (siRNAs) lipoplex (also known as ‘triple therapy’) not only reduced the severity of established collagen-induced arthritis (CIA) but also prevented arthritis in a murine in vivo model, which showed that triple therapy could be the possible alternative to currently employed measures [15]. Kim et al. evaluated the efficacy of direct intramuscular injection of plasmid deoxyribonucleic acid (DNA), which contains complementary DNA (cDNA) for interleukin-1 receptor antagonist (IL-1Ra), to the bovine type II collagen immunized DBA/1 murine model [16]. Successful prevention of moderate to severe 4

Journal Pre-proof CIA, which was evaluated by inflammation, swelling and deformities of the paws, was achieved (P<0.05). In addition, significant reduction of cartilage erosion in knee joints was observed (P<0.05), accompanied by by a lowering in IL-1β expression (P<0.01) [16]. The preventive effect of murine CIA has been proven with with intramuscular gene therapy injecting plasmid DNA, which contains cDNA for IL-1Ra [58]. This finding suggests that gene therapy modulating the cytokine profile might have efficacy to suppress the inflammatory pathology of arthritis [16]. Smeets et al. showed the relationship between CIA and neutralized IL-18 using an adenoviral vector containing the murine IL-18-binding protein isoform c gene (AdCMVIL-

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18BPc) [17]. IL-18BPc-treated mice showed successful reduction of CIA incidence and severity [17]. Moreover, treatment showed the exclusive reduction of IgG2a antibodies to native human type II collagen

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antibodies (IgG2a anti-CII Abs), while IgG1 anti-CII Abs remained elevated, which can possibly lead to a

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better understanding of IL-18 related inflammation [17]. The same group showed a difference between two

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modes of IL-1 inhibition, soluble IL-1 receptor accessory protein (sIL-1RAcP) and IL-1Ra in arthritis [18].

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Both modes successfully reduced the clinical manifestation of CIA, but only IL-1Ra was able to suppress the lymphocyte proliferation [18]. Courties et al. demonstrated that gene silencing in cluster of

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differentiation 11b+ (CD11b+) cells through intravenous injection of anti-calcium-dependent phospholipase

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A2α (anti-cPLA2α) siRNA sequence lipoplex decreased both the severity and incidence of CIA [19]. The authors showed local reduction of TNF-a secretion and T helper 1 (Th1) suppression which could indicate one possible basic mechanism between cPLA2α and RA [19]. Unlike the observed Th1 suppression, Th17 suppression seemed to be an unpromising approach [19]. Ko et al. highlighted the relationship between cytotoxic T lymphocyte antigen-4 and IgG fusion protein (CTLA-Ig), which is used in the management of RA, and regulatory T-cell (Treg) [20]. The results showed that CTLA-Ig reduced the CIA severity [20]. It is hypothesized that these results are achieved by the mechanism of CTLA-Ig stimulating dendritic cells (DCs) to develop tolerogenic property and increase the CD4+CD25+ forkhead box P3 (Foxp3)+ Treg population depending on TGF-β level [20]. In RA, protein tyrosine phosphatase non-receptor type 22 (PTPN22) could 5

Journal Pre-proof be one of the possible target genes of clustered regularly interspaced short palindromic repeats (CRISPR), because inhibition of PTPN22 reduces pro-inflammatory cytokines and inflammatory mediators and showed anti-arthritic effects [21]. Zheng et al. demonstrated the anti-arthritic effect of pinitol, which is a known anti-diabetic agent isolated from Sutherlandia frutescens leaves, by showing the significant of pro-inflammatory cytokines (TNF-α, IL-1β , IL-6) and inflammatory mediators (cyclooxygenase (COX), IL-1β) [21]. Other experiment using a docking study of pinitol with PTPN22 showed excellent interaction with each other. Pinitol inhibits PTPN22 gene effectively and showed anti-arthritic effect [21]. Komano et

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al. suggested that the complex of siRNA and the wrapsome (siRNA-TNF-α/WS) was effective in potential therapeutic effects in RA by silencing the expression of inflammatory cytokines produced by macrophages

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and neutrophils [22].

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2.2 Anti-angiogenesis and RA

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It is generally accepted that neovascularization, characterized by the formation of new blood vessels, plays a

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pivotal role in the progress of RA (Table 1). Vascular endothelial growth factor (VEGF) protein is important for the formation of blood vessels. For the neovascularization, RA patients express increased VEGF protein

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levels in serum and synovial fluid compared to healthy controls [23-25]. This could lead to the hypothesis that blocking VEGF expression may have a therapeutic benefit in RA. Afuwape et al. showed that disease activity of CIA could be suppressed by using adenoviral-delivered VEGF receptor which inhibits VEGF activity [26]. They injected adenovirus overexpressing the soluble VEGF receptor, soluble fms-like tyrosine kinase-1 (sFlt-1), which was intravenously administered via the tail of DBA/1 mice with CIA. Adenoviruses expressing human soluble VEGF receptor 1 (AdvsFlt-1)-treated arthritic mice showed significant reduction in the clinical RA score, and this was due reduced paw swelling, synovial inflammation and bone destruction in a dose-dependent manner [26]. 6

Journal Pre-proof Thrombospondin 1 (TSP-1) is an endogenous angiogenesis inhibitor and activates transforming growth factor -β (TGF-β), which acts as an immunosuppressive and anti-inflammatory cytokine. These mechanisms highlight the potential usefulness of TSP-1 in the treatment of RA [27, 28]. Jou et al. demonstrated that intraarticular injection of adenoviral vectors encoding TSP-1 (AdTSP-1) can improve the clinical course of CIA [29]. AdTSP-1 showed both therapeutic and prophylactic effects on rat CIA with respect to clinical, histologic and radiographic aspects [29].

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2.3 Extracellular matrix degradation and RA

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Destruction of the cartilage is one of the main pathological features of RA [30]. Irreversible cartilage

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degradation means a death of chondrocyte and matrix metalloproteinases (MMPs)-mediated cartilage matrix

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destruction [31, 32]. MMPs are secreted by chondrocytes which are stimulated by IL-1, synovial fibroblasts

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(SF), and macrophages and increased in rheumatoid synovial tissue [33-35]. And there are endogenous inhibitors of MMPs, named tissue inhibitors of metalloproteinase (TIMPs), maintaining the balance of

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action exerted by MMPs [36].

RA patients have an increased expression of MMPs and TIMPs [37-43]. But still there is an imbalance

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between both with a higher expression of MMPs compared TIMPs, and this is one of the mechanisms leading to cartilage degradation in RA [44]. Lann et al. showed that overexpression of TIMP-1 and TIMP-3 by gene transfer of adenoviral (Ad) vectors has a protective effect on the cartilage in vitro and in the severe combined immunodeficiency (SCID) mouse model [44]. The reduction of both invasiveness and proliferation was observed particularly in the Ad vector expressing human TIMP (AdTIMP)-3 [44]. These results imply that TIMP overexpression may be used as a target of gene therapy [44]. IL-13 is abundantly found in the synovial fluid of RA patients and is known to have anti-inflammatory effects [45, 46]. Nabbe et al. demonstrated that intra-articular gene transfer of IL-13 reduces chondrocyte 7

Journal Pre-proof death and MMP-mediated cartilage degradation during immune-complex-mediated arthritis (ICA) [47]. They injected recombinant adenovirus encoding for IL-13 into the knee joint of the mouse and found decreased cartilage destruction despite enhanced joint inflammation [47]. A full list of experiments is highlighted in Table 1.

2.4 Other gene therapies There are a number of gene therapies with a potential implication in the management of RA (Table 1). Li et

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al. demonstrated that silencing microRNA-223 (miR-223), which is highly expressed in the synovium of RA

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patients and ankle joints of the CIA mouse model, can down-regulate the severity of arthritis in a murine

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model [48]. In addition, up-regulation of nuclear factor-1A (NF-1A) levels and down-regulation of

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macrophage colony stimulating factor receptor (M-CSFR) levels were shown in the synovium of mice after

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silencing miR-223 [48]. Song et al. showed the possibility of vaccine-therapy using pcDNA-CCOL2A1 tolerizing DNA vaccine [49]. The vaccine successfully induced tolerance against CIA which was

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accompanied by an up-regulation of CD4+ CD24+ Treg cells [49]. Lam et al. revealed that down-regulation of TNF superfamily member B cell-activating factor (BAFF) using lentivirus expressing specific small

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hairpin RNA (shRNA) could suppress the development of CIA [50]. Moreover, shRNA administered into the CIA murine model showed suppression of the plasma cell and Th17 cell population, which highlights the role of BAFF in Th17 cell regulation [50].

3. Animal models of gene therapy in systemic lupus erythematous (SLE)

3.1 Anti-inflammatory cytokine targeted therapies Inflammatory responses are controlled by a balance between stimulatory cytokines and anti-inflammatory 8

Journal Pre-proof factors [51, 52]. Injection of cytokine-related genes into mice leads to an increase in the amount of the respective cytokine in circulation [53]. Furthermore, intramuscular injections of plasmid cDNAs generating TGF-β significantly increased survival rates, while injecting plasmid cDNAs inducing IL-2 decreased the survival rate in the murphy roths large (MRL)/lpr/lpr mice with SLE [54]. In contrast, Huggings et al. found that orally administered Salmonella transfected with plasmids of IL-2 significantly suppressed anti-double stranded DNA (anti-dsDNA) antibody levels and reduced glomerulonephritis as well as vasculitis in mice with SLE [55]. However, the effect of orally administered

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plasmids with TGF-β on SLE disease activity was not significant [55] (Table 2).

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3.2 Blockade therapies

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Blocking the costimulatory signal of T cells is one way to prevent T cell activation, which is effective in

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reducing the immune response [56]. Takiguchi et al. demonstrated that soluble CTLA4/IgG molecules (fusion protein that combines mouse CTLA4 and the Fc portion of human IgG1), capable to bind to B7.1

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(CD80) or B7.2 (CD86), reduced autoantibody-related disease in MRL/lpr/lpr mice when administered systemically [57]. Development of a recombinant adenovirus vector incorporating the CTLA4/IgG gene

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allowed for production of high serum levels of CTLA4/IgG, which reduced production of autoantibodies and maintained renal function [58].

Serum interferon-γ (IFN-γ) level positively correlates with SLE activity and deletions of IFN-γ or IFN-γ receptor result in a significant decrease of disease severity [59-61]. Lawson et al. showed that intramuscular injections of plasmids with cDNA producing IFN-γ receptor/Fc could not only delay the progression of SLE but also improve the status of MRL/lpr/lpr mice [62]. Injections increased the survival rate and decreased occurrence of glomerulonephritis [62]. Synthetic oligonucleotides (ODNs) can block the induction of pro-inflammatory cytokines [63]. Dong et al. 9

Journal Pre-proof demonstrated that repetitive administration of synthetic ODNs is postponed the onset and exacerbation of glomerulonephritis, while levels of IL-12, IFN-γ, and anti-dsDNA autoantibodies are reduced in BWF1 mice [63]. On the other hand, patients with SLE have immune complexes including endogenous hypomethylated CpG rich DNA and anti-DNA [64]. Hasegawa et al. suggested that synthetic ODNs containing CpG motifs (CpG-ODN) may induce production of IL-6, which correlates with the occurrence of glomerulonephritis and results in increased urine protein-to-creatinine levels [65] (Table 2).

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3.3 Other gene therapies

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Christensen et al. reported that the lack of toll-like receptor 7 (TLR7) results in failure to produce antibodies

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in response to RNA-containing antigen [66]. In mice lacking TLR7, disease activity is alleviated and low serum IgG is found. A contrasting outcome was reported when mice lacked TLR9, which lead to aggravation

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of SLE, increased IFN-α and serum IgG [66]. In spite of analogous signaling pathways and tissue expression,

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TLR7 and TLR9 have contrary function on inflammatory and regulatory reactions [66] (Table 2).

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4. Animal models of gene therapy in inflammatory bowel diseases (IBD) 4.1 Anti-inflammatory cytokines

In line with other autoimmune disorders, changes in the immune response play a pivotal role in inflammatory bowel disease (IBD) (Table 3). Cytokines play a critical role in IBD and determine T cell differentiation [67]. Several studies proposed that IL-10 can prevent and attenuate IBD. In rats treated with Ad IL-10, one day prior to the induction of colitis, a prevention of colitis was observed [68]. Ad vector encoding murine IL-10 (AdvmuIL-10) injected into 4–week-old IL-10-/- mice induced no signs of colitis throughout the 10-week experiment, while AdvmuIL-10 injected into 10-week-old IL-10-/- mice with established colitis had improved clinical scores compared to control mice [69]. AdvmuIL-10 treatment in 10

Journal Pre-proof mice with trinitrobenzenesulfonic acid (TNBS)-induced colitis prevented severe body weight loss and diminished local or systemic inflammations [70]. The positive effects on severity of TNBS-induced colitis were associated with a modulation of IFN-γ and IL-6 expression [70]. Ad IL-10 gene transfection reduced IBD disease activity, associated with preserved body weight, colonic morphology, a lowered histopathology score, and suppression of mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) in dextran sulfate sodium (DSS) colitis.[71] Other studies suggest that IL-4, IL-22, IL-35 can attenuate IBD severity. Xiong et al. demonstrated that IL-

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4 and IL-10 injections dramatically inhibited colon tissue damage, weight loss, and disease activity index [98]. Also, IL-4, IL-10 injections significantly blocked IFN-γ, IL-6, and TNF-α [72]. Injections of

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adenovirus 5 vector expressing interleukin-4 (Ad5IL-4) dramatically reduced injury in TNBS-colitis [73].

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Also, Ad5IL-4 gene transfer inhibited nitric oxide (NO) synthesis in colon [73]. Sugimoto et al. proved that

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local IL-22 gene delivery into colonic mucosa rapidly attenuated T helper cell-2 (Th-2)-mediated colitis [74].

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IL-35 treatment reduced Th-1, Th-17 dependent inflammatory responses in the colon [75]. IL-6 transsignaling leads to anti-apoptotic features and worsens IBD [76]. Anti-IL-6R antibody induced apoptosis of

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lamina propria T cells [76].

The balance between the expression of retinoic acid receptor-related orphan receptor-γt (ROR-γt) and TGF-

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β is known to determine the differentiation of naïve CD4+ T cells to Th17 or Treg cells, which plays a crucial role in the pathogenesis of IBD [77-79]. Withers et al. showed that transient inhibition of ROR-γt is capable to reduce Th17 cells without evoking ROR-γt dependent group 3 innate lymphoid cells (ILC3) in both a murine model and tissue from a Crohn’s disease patient in-vitro [78]. Fantini et al. suggested in-vitro TGF-β induced Treg cells could suppress T-cell mediated intestinal inflammation [79]. Peroxisome proliferator-activated receptor γ (PPARγ), a repressor of inflammatory activation, is found to be decreased in chronic intestinal inflammation thereby lowering the efficacy of a therapy targeting PPARγ ligand [80]. The delivery of the PPARγ gene via adenovirus vector (Ad-PPARγ) in addition to current ligand 11

Journal Pre-proof therapy showed a marked decrease in inflammatory cytokine expressions and tissue inflammation itself in a murine model [80]. The above mentioned three factors could suppress IBD progression, whereas one study found a geneANGPT2- responsible for IBD progression [81]. Leukocyte infiltration and angiogenesis which are critical steps to IBD progression can be decreased by targeting Ang-2, yet accompanying defects in lymphatic vascular integrity could possibly worsen the symptoms of IBD [81].

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4.2 Antimicrobial peptides

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Antimicrobial peptides such as human defensin-5 (HD5) and cathelicidin antimicrobial peptide (CAMP) are known to attenuate IBD. Salzman et al. suggests that human enteric α-defensins can help to attenuate IBD in

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mice [82]. CAMP also attenuated IBD by reduction of disease symptoms, disease activity index, colonic

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mucosal damage, inhibiting shortening of colon and reduced number of apoptotic cells [83] (Table 3).

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4.3 The endoplasmic-reticulum (ER) stress in Crohn’s Disease Polymorphism in autophagy-related protein 16-like1 (ATG16L1) in the intestinal epithelium is known to be

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a predisposing factor of Crohn’s disease (CD) [84]. In Atg16l1 knockout mice, transmural colitis resembling CD was induced via impaired clearance of endoplasmic reticulum (ER) stress sensor (IRE1α) and defective autophagy in intestinal epithelial cells (IECs) [84] (Table 3).

4.4 miRNA in ulcerative colitis There are two studies on genes which when activated were found to be inducing ulcerative colitis (UC) development. Inhibition of GATA3-with DNAzyme hgd40-and miRNA214-with miRNA214 inhibitor-each have shown reduced colitis severity and even hindered progression to colorectal cancer in case of 12

Journal Pre-proof miRNA214 inhibitor [85, 86] (Table 3).

5. Animal models of gene therapy or immunotherapy in multiple sclerosis (MS) 5.1 IL-17, IFN-γ related therapies In multiple sclerosis (MS), several studies suggested potential immunotherapies related to IL-17 and IFN-γ (Table 4). First, Yan et al. blocked IL-17 signaling in astrocytes of experimental autoimmune encephalomyelitis (EAE) mice by knocking down Act1 which is a major and common transcription factor of

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the IL-17 pathway [87]. As a result, infiltration of inflammatory cells was decreased in the central nervous

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system (CNS) and disease progression was inhibited [87]. Another study investigated DNA vaccination against EAE and found decreased Th-17 cell responses, including IL-17 and IL-21 responses, and the effect

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disappeared when IFN-β was silenced [88]. When EAE mice were treated with the IFN-γ-producing vector,

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and apoptosis of CNS-infiltrating cells [89].

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most recovered form EAE and this was associated with increased TNF receptor 1 (TNFR1) mRNA levels

In the following section, we discuss pivotal proteins associated with MS, which might act as potential

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targets of gene therapy. Doi et al. demonstrated that nuclear receptor subfamily 4 group A member 2

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(NR4A2), an orphan nuclear receptor, is an important transcription factor causing inflammatory reactions in MS and EAE, suggesting that NR4A2 can be a promising therapeutic target [90]. NR4A2 is significantly upregulated in T cells of MS patients and causes excessive expression of IL-17 and IFN-γ [120]. In contrast, according to the study of Makar et al., when brain-derived neurotrophic factor (BDNF) was delivered to EAE mice, disease onset was significantly delayed and disease severity was decreased by reducing the levels of the pro-inflammatory cytokines IFN-γ and TNF-α [91]. Moreover, it was found that BDNF increased the cytokines IL-4, IL-10, and IL-11 which play a role in anti-inflammation [91]. Unlike NR4A2, BDNF can be utilized for the treatment of MS by overexpression [91, 92].

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Journal Pre-proof 5.2 Treg cell related therapies Two studies independently suggested that increased levels of IL-4 and myelin oligodendrocyte glycoprotein (MOG) 40-55 can suppress MS (Table 4). Butti et al. have found that the anti-inflammatory cytokine IL-4 might display a promising therapy in the management of MS [92]. EAE mice treated with an IL-4expressing vector administered into the CSF recovered from the disease. This was explained by an increase of the chemokine levels of chemokine ligands 1 (CCL1), CCL17, and CCL22 by IL-4, which recruit regulatory T cells [92]. In another study, Eixarch et al. demonstrated that transfer of bone marrow cells expressing autoantigen MOG40-55 into EAE mice caused tolerance and improvement of the disease through

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the participation of Treg cells [93].

5.3 Other therapies

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There are several studies dealing with β1-integrin, glatiramer acetate, IL-10, and others in MS (Table 4).

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Bauer et al. suggested that the primary action of natalizumab is interference with T cell extravasation via inhibiting α4β1 integrins [94]. Natalizumab, a humanized monoclonal antibody against the α4 subunit of

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integrin heterodimers α4β1 and α4β7, is an effective treatment for relapsing-remitting MS [95]. One study

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found that upon deletion of β1-integrin gene in T cells of EAE mice β1-deficient T cell blasts did not accumulate in the CNS [94].

The following three studies focus on glatiramer acetate (GA). Burger et al. demonstrated that GA enhances monocytic production of the secreted IL-1 receptor antagonist (sIL-1Ra) while it diminished that of IL-1β in chronic inflammation [96]. Arnon et al. suggested that the effect of GA was not restricted to antiinflammation, but involved neuroprotection and neurogenesis [97]. In another study, Pul et al. examined whether GA has also direct effects on microglia in vitro [98]. GA promoted the phagocytosis and increased the secretion of IL-10 while decreasing that of TNF-α [98].

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Journal Pre-proof Zhu et al. suggested that sFlt-1 might be a potential therapeutic target in MS [99]. VEGF expression is known to be enhanced in EAE and MS [100]. By transferring the sFlt-1 gene into the brain of EAE mice, CNS autoimmune inflammation was inhibited and EAE severity was ameliorated [99]. Sloane et al. showed that IL-10 gene therapy is effective in a rat model of MOG-EAE. This therapy reversed paralysis, ameliorated worsening sensitivity to touch, prevented allodynia, reversed body weight loss, and suppressed activation of CNS glia [101]. Chen et al. suggested that genetically engineered, self-reactive T cells producing latent TGF-β are a useful

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approach to treat autoimmune diseases [102]. Less TGF-β1 is produced by T cells from MS patients than by normal T cells [103]. Myelin basic protein (MBP)-specific cloned T cells infected with a recombinant

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retrovirus producing latent TGF-β1 were injected to EAE mice and the development of EAE was

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ameliorated [102].

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Park et al. suggested that consideration of pathologic mechanisms implicated in the respective autoimmune

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disease is crucial for performing immunotherapy [104]. In an elegant approach to account for different disease states, the seven-amino acid truncated (7ND) gene therapy was administered to three types of EAE

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rats [104]. 7ND gene therapy was ineffective during the first attack of biphasic EAE and acute EAE,

6. Conclusion

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whereas it was effective in the relapse of biphasic EAE and chronic EAE [104].

Although the pathophysiology of autoimmune diseases has not been elucidated in detail, genetic factors also play an important role in the pathogenesis. There are shared as well as individual genetic traits which are associated with the risk to develop autoimmune diseases [10]. Because disease activity can in part be determined according to activation or suppression of these genes, cytokine gene therapy might be an interesting approach for the treatment. 15

Journal Pre-proof Biologic measures neutralizing specific pro-inflammatory cytokines are already in use in the clinic practice and more are about to come [105]. Downregulating the activity of pro-inflammatory cytokines is also possible either by inhibiting cytokine expression using siRNA or by inhibiting cytokine signaling using small molecules [105]. Furthermore, gene therapy delivering anti-inflammatory cytokines or cytokine antagonists showed effectiveness in regulating autoimmunity [105]. In conclusion, our comprehensive review is the first paper that summarizes autoimmune diseases based on genes and cytokines, although they are organized in animal experiments. This article also can give a new

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perspective on understanding cytokine in autoimmune diseases in human. Because several genes or cytokines are not yet fully studied or explained about the underlying mechanisms of autoimmunity, they may

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be required to confirm the effect by more validation researches. Emerging approaches to investigate

Take home messages

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some autoimmune diseases near in the future.

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cytokine regulation through gene therapy may be a potential approach for the tailored immunomodulation of

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 With multiple genes associated with its pathogenesis, autoimmune disorders have a complicated

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genetic basis, leading to difficulties in selecting optimal individual treatment to cure each patient.  Gene therapy delivering anti-inflammatory cytokine or cytokine antagonists showed effectiveness in regulating autoimmunity in animal models.  Studies suggest that cytokine regulation through gene therapy may be a potential approach for the tailored immunomodulation treatment of autoimmune diseases in the future.

Conflict of interests All authors confirm to have no actual or potential conflict of interests within the three years of beginning the submitted work. 16

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Authorship All authors made substantial contributions to all of the following: (1) conception and design of the study, data acquisition, or analysis and interpretation of data; (2) drafting or critical revision of the article for intellectual content; and (3) final approval of version to be submitted.

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Role of funding source

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No financial support was provided for research conduct and/or preparation of the article.

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Tables Table 1. Animal models using gene therapy in RA Related Cytokines Delivery Vector (Target Gene / Gene of Interest) TNF-α, IL-1 related Gene Therapy

Administration Method

Experimental Models

Results

IL-1, IL-6, IL-18 [15]

siRNA

IV injection

DBA/1, CIA

siRNA silenced the proinflammatory cytokines IL-1, IL-6 and IL-8 in DBA/1 mice with CIA.

IL-1 Ra [16]

Plasmid DNA

DBA/1, CIA

Significantly prevented the onset of CIA, while reducing the expression of IL-1 beta.

IL-18 BPc [17]

Adenoviral vector

DBA/1, CIA

IL-1 Ra [18]

Adenoviral vector

IM injection (thigh and calf) Intra-articular injection (both knees) IV injection

cPLA2α [19]

siRNA

IV injection

DBA/1, CIA

IL-18BPc neutralized IL-18 and reduced inflammation and destruction of bone and cartilage, and provided protection against onset of CIA. IL-1 Ra was systemically delivered before initiation of CIA in DBA/1 mice. IL-1Ra reduced the circulating levels of IgG2a antibodies and inhibited lymphocyte proliferation. CIA was ameliorated. Anti-cPLA2α and siRNA reduced the cPLA2α and proinflammatory cytokine (TNF-α and IFN-γ) levels.

CTLA-4 [20]

IgG fusion protein

Ex-vivo

DBA/1

Increased Treg cells suppressed CD4+IL17+ T cells

TNF-α, IL-1b, IL-6, COX [21] (PTPN22)

Pinitol

Paw injection

Wistar Swiss albino rats

TNF-α [22]

siRNA

IV injection

C57BL/6J, CIA

Pinitol reduced proinflammatory cytokines and inflammatory mediators and therefore resulted in anti-arthritic efficacy. The docking study showed that the structural interaction between pinitol and PTPN22 caused the inhibition of PTPN22. siRNA-TNF-α/WS silenced the expression of inflammatory cytokines by incorporation into CD11b+ cells, including neutrophils and macrophages.

Anti-angiogenic Gene Therapy AdvsFlt-1 [26]

Adenoviral vector

IV injection

DBA/1, CIA

TSP-1[29]

Adenoviral vector

Intra-articular injection (ankles) Extracellular Matrix Degradation targeting Gene Therapy

Sprague-Dawley rat, CIA

TIMP-1, TIMP-3 [44]

Adenoviral vector

Ex-vivo

TIMP1, 3 reduced the invasion of rheumatoid arthritis synovial fibroblasts in the SCID mouse model.

IL-13[47]

Adenoviral vector

Intra-articular Injection (knee joints)

SCID mouse, RA synovial fibroblast and cartilage transplantation C57BI/6 mice, ICA

Lentiviral vector

Intra-peritoneal injection IV injection

Sprague-Dawley rat, CIA Wistar rats

miRNA 223 target sequence suppressed miRNA-223, and reduced the arthritis score, osteoclastogenesis and bone erosions. Induced the shift of Th1 to Th2 cells. TNF-α were down-regulated while IL-10 and TGF-beta were upregulated.

Other Gene Therapy miRNA-223 target sequence[64] CCOL2A1 [49] BAFF[50]

Plasmid DNA

l a

rn

u o

J

f o

DBA/1, CIA

o r p

e

r P

Systemically injected AdvsFlt-1 significantly suppressed the disease activity by reducing synovial neovascularization. adTSP-1 administered mice had lower levels of VEGF and fewer vessels. The treatment significantly reduced the severity of CIA.

IL-13 increased the levels of inflammatory cells in the joint cavity, but lowered the chondrocyte death by two thirds. Furthermore, MMP-mediated cartilage damage was halved.

Lentivirus

Intra-articular DBA/1 Promoted the expansion of Th17 cells. Injection RA: rheumatoid arthritis, TNF-α: tumor necrosis factor-α, IL: interleukin, siRNA: small interfering ribonucleic acid, IV: intravenous, CIA: collagen induced arthritis, DNA: deoxyribonucleic Acid, IM: intramuscular, IL-18BPc: IL-18 binding protein isoform c, IFN: Interferons, cPLA2α: anti-calcium-dependent phospholipase A2α ,CTLA-4: cytotoxic T lymphocyte antigen-4 and IgG fusion protein, Tregs: regulatory T cells, CD: cluster of differentiation, COX:

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cyclooxygenase, PTPN22: protein tyrosine phosphatase non-receptor type 22, WS: wrapsome, AdvsFlt-1: adenoviruses expressing human soluble VEGF receptor 1, VEGF: Vascular endothelial growth factor, TSP-1: Thrombospondin 1, TIMP: metallopeptidase inhibitor, SCID: severe combined immunodeficiency, MMP: matrix metalloproteinases, miRNA: micro ribonucleic acid, TGF: transforming growth factor, BAFF: B cell-activating factor, Th: helper T cell

f o

l a

e

o r p

r P

n r u

o J

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Journal Pre-proof Table 2. Animal models using gene or immunomodulation therapy in SLE Administration Method

Experimental Models

Results

IM injection(Monthly)

MRL lpr/lpr mice

lL-2 [55]

Salmonella typhimirium (aroAaroD-mutation)

Oral

MRL lpr/lpr mice

Monthly injected TGF-beta1 brought control group of experimental model production of IgG. IL-2 gene therapy showed an effect o restoration of the defective T-lymphoc

IV injection

MRL lpr/lpr mice

IFN-γR/IgG1 [62]

Recombinant adenovirus vector Plasmid

IM injection(with electroporation)

MRL lpr/lpr mice

Synthetic ODN [63]

PBS

Intraperitoneal injection

B/WF1 mice

CpG-ODN [65]

Plasmid

Intraperitoneal injection

B/WF1 mice

Other Gene therapy TLR7 & TLR9 [66] (TLR7 & TLR9 genes)

NA

NA

ro

MRL lpr/lpr mice (lacking TLR7 or TLR9)

A single administration of IV injectio CTLA-4/IgG resulted in complete sup IFN-gamma R/IgG1 plasmid with ele resulted in decreased autoantibody tit renal diseases. It also reduced severity Repeated administration of suppressiv lifespan, delaying the onset and aggra additionally showing a notable decrea and IL-12 in vivo. Treatment with CpG-ODN resulted in and creatinine level, related with seve paralleled aggravation of glomerulone

Absence of TLR7 improved SLE like decreased serum IgG level, while abs more lymphocytes and plasmacytoid

Jo ur

na

lP

re

-p

Blockade Therapy CTLA-4/IgG [58]

of

Related Cytokines Delivery Vector (Target Gene / Gene of Interest) Anti-inflammatory Cytokine Targeted Therapy TGF-β1 [54] Plasmid DNA

SLE: systemic lupus erythematosus, TGF-β1: transforming growth factor-β1, DNA: deoxyribonucleic acid, IM: intramuscular, MRL: murphy roths large, TGF: transforming growth factor, IV: intravenous, RF Ab: rheumatoid factor antibody, IgG: immunoglobulin G, IL: interleukin, CTLA-4: cytotoxic T-lymphocyteassociated antigen 4, IFN-γR: interferon-gamma receptor, IM: intramuscular, IFN: interferons, ODN: oligodeoxynuleotides, CpG-ODN: oligodeoxynucleotides containing CpG motifs, PBS: phosphate buffered saline, mAb: monoclonal antibody, TLR: toll-like receptor, DCs: dendritic cells, NA: no information

23

Journal Pre-proof

s

Delivery Vector

Administration Method

Experimental Models

Results

/

Jo ur

na

lP

re

-p

ro

of

y cytokine

Table 3. Animal models using gene or immunomodulation therapy in IBD

24

Sprague Dawley rat* Adenoviral IL-10 gene therapy in rat was successful in preventing, but canno Journal Pre-proof

Intraperitoneal injection

Adenoviral vector

Intravenous injection

IL-10-/- mice on a C57BL/6 background BALB/c mice

Adenoviral vector

Intraperitoneal injection

C57B6 mice

Recombinant plasmids

Intraperitoneal injection

BALB/C mice

Human type 5 adenovirus Nonmicrobial vector. DNA/lipid complex plasmids

Intraperitoneal injection

Sprague Dawley rat*

Direct local microinjection into colonic mucosa Intraperitoneal injection

C57BL/6 mice

NA

Antibody given by Intraperitoneal injection

Patient-derived colon specimen and BALB/c mice

NA

Oral administration of and cell culture with presence of inhibitor of ROR-γtmediated transcription

C57BL/6 mice and intestinal resection tissue from CD patients

NA

Intraperitoneal injection of In vitro generated Treg cells

BALB/c, SCID, and C57/B6 mice

Adenoviral vector

Intraperitoneal injection

C57BL/6J mice

NA

In vivo

Angiopoietin 2 (-/-) mice

ptides 5 NA

In vivo

Transgenic mouse model

Intrarectal injection of mCRAMP peptide or mCRAMP-expressing plasmids

Cathelicidin (Cnlp) -/- mice

IL-22 gene local delivery is effective in attenuating intestinal inflammation ra potency of IL-22 in treating UC IL-35 administration suppresses Th1 and Th17 cytokines, reducing severity o experimental colitis Anti-IL6R antibody induces T cell apoptosis in lamina propria, thus attenuatin colitis in mice model. IL-6 trans signaling leads to anti-apoptotic feature of T IL-6 blockade resulted in T cell apoptosis ROR-γt transient inhibition enabled selective suppression of Th17 mediated p cytokine releases with intact innate lymphoid cell functions, shown both in m colitis and intestinal samples of IBD patients

FoxP3+ regulatory T cells are induced by TGF-β and is effective in controllin

PPARγ gene therapy successfully attenuated tissue damage and suppressed IC TNF- α expressions in mouse model of colitis Ang-2(-/-) knockout mice allowed for less severe leukocyte infiltration, less i angiogenesis and lymphangiogenesis, and reduced loss of proteins, attenuatin clinical features

re

Cnlp -/- mice showed profound susceptibility to colitis. Cathelicidin peptide s gene therapy effectively attenuated intestinal inflammation

Deletion of ATG16L1 in epithelial cells of the intestine caused impaired auto clearance of IRE1α aggregates under ER stress, resulting in CD-like ileitis

Mouse model

GATA3 levels were associated with UC in human. Inhibition of GATA3 by s resulted in reduced levels of inflammatory cytokines and suppressed colitis ac High microRNA214 levels were linked with active UC in human and chemica MiR-214 reduced severity of colitis and risk of colitis-related cancer in mice

na

Intrarectal administration of GATA3blocking DNAzyme Intracolonic administration of MiR-214 inhibitor

Paneth-cell specific HD5 overexpressing transgenic mice is resistant to exper oral Salmonella typhimurium challenge

Mouse model

Patient-derived colon specimens and mouse model

Jo ur

NA

Adenoviral IL-10 gene therapy prevented weight loss and induced decreased IFN-γ levels in a TNBS model of colitis in mice Ad-IL10 therapy resulted in lowering disease severity, histopathologic damag suppressing MAdCAM-1 IL-4 treatment and IL-10 treatment reduced Th1-mediated cytokines includin TNF-α and improved histopathology of colon in TNBS-induced colitis in mic IL-4 is effective in treating acute colitis in rat, resulted in low NO expression

ro

NA

tive colitis NA

experimental colitis. Injecting adenovirus encoding IL-10 prevented experimental colitis in mice.

-p

n’s Disease NA

EBI3−/− mice,IL-27p28−/− mice

lP

plasmids

Intravenous injection

of

Human type 5 adenovirus Adenoviral vector

IBD: inflammatory bowel disease, IL: interleukin, TNBS: trinitrobenzene sulphonic acid, MAdCAM-1:mucosal vascular addressin cell adhesion molecule 1, Th: helper T cell, IFN: interferon, TNF: tumor necrosis factor, NO: nitric oxide, DNA: deoxyribonucleic Acid, IL-6R:interleukin-6 receptor; CD: Crohn’s disease, UC: ulcerative colitis, EBI3: Epstein-Barr virus induced gene 3, ROR: RAR-related orphan receptor, RORC: retinoic acid receptor(RAR)-related orphan receptor C, TGF: transforming growth factor, SCID: severe combined immunodeficiency, PPARγ: peroxisome proliferator-activated receptor γ, ICAM: intercellular adhesion molecule; COX: cyclooxygenase, HD5: human defensin-5; CAMP: human cathelicidin antimicrobial peptide; mCRAMP: mouse cathelicidin-related antimicrobial peptide; FOXP3: forkhead box P3, ER: endoplasmic-reticulum, ANGPT2: angiopoietin 2; ATG16L1: autophagy-related 16-like 1; miRNA: microRNA, NA: no information * Trinitrobenzene sulphonic acid is administered.

25

Journal Pre-proof

Table 4. Animal models using gene therapy or Immunotherapy in MS Related cytokine (Target Gene / Gene of Interest) IL-17 [87] (Act1) IL-17, IL-21, INF-β [88]

Delivery Vector

Experimental Models

Results

Lentivirus

C57BL/6

Knocking down Act1 expression in astrocytes inhibited inflammatory

Plasmid DNA

DA rator LEW.1AV1

IFN-γ [89] (TNFR1) IL-17, IFN-γ [90] (NR4A2) TNF-α, IFN-γ, IL-4, IL-10, IL-11 [91] (BDNF) IL-4, CCL1, CCL17, CCL22 [92]

HSV-1

C57Bl/6

Retrovirus

C57Bl/6

DNA vaccination against EAE decreased IL-17 and IL-21 responses silencing IFN-γ increased the expression of TNFR1 death receptor and induced lymphocytes siRNA targeted for transcription factor NR4A2 increased IL-17 and I

BMSC

SJL/J

HSV-1

C57Bl/6

NA (MOG40–55) [123] β1 integrin [94]

BMSC

C57Bl/6 129Sv/C57Bl/6

GA [96]

Mx1 promoter, or CD4 promoter HSV-1

GA [97]

Plasmid DNA

C57Bl/6

GA [98]

Plasmid DNA

C57Bl/6

Initiation of GA-reactive T-cells and their CNS infiltration, dischargi immunomodulatory cytokine. at the damaged site OPCs have differentiated, proliferated, and survived

VEGF [99] (sFlt-1) IL-10 [101]

Adenovirus

Dark Agouti rat

Anti-VEGF therapy inhibited autoimmune inflammation

TGFβ1 [102]

Retrovirus

7ND [104]

Plasmid DNA

of ro

-p

MOG40-55 induced central tolerance via Treg activation

re

C57Bl/6

IL-4 increased chemokines CCL1, CCL17, and CCL22 which recruit

lP

Plasmid DNA

BDNF decreased pro-inflammatory cytokines TNF-αand IFN-γ and i which play a role in anti-inflammation

Inhibition of α4β1 integrins interferes T cell extravasation

GA enhanced sIL-1Ra production while diminishing the IL-1β produ

LEW.1AV1 rat

IL-10 Inhibited macrophage activation

SJL × BALB/c

Latent TGF-β1 were injected to EAE mice and the development of E

Inhibition of CNS macrophage infiltration was effective only in macr in T cell-dependent MS: multiple sclerosis, IL: interleukin, IFN-γ: interferon-gamma, CNS: central nervous system, EAE: experimental autoimmune encephalomyelitis, DNA: deoxyribonucleic acid, siRNA: small interfering RNA, NR4A2: nuclear receptor subfamily 4 group A member 2, BDNF: brain derived neurotrophic factor, CCL: chemokines chemokine ligands, GA: glatiramer acetate, TNF: tumor necrosis factor, HSV: herpes simplex virus; Tregs: regulatory T cells, MOG: myelin oligodendrocyte glycoprotein, BMSC: bone marrow stem cells, CD: cluster designation, sIL-1Rα: secreted form of IL-1 receptor antagonist, OPCs: oligodendrocyte progenitor cells, VEGF: vascular endothelial growth factor; TGF-β1: transforming growth factor-β1, 7ND: decoy chemokine, NA: no information

Jo ur

na

LEW.1AV1 rat

26